Dr Joanna Bandorowicz

Pikula Pr

Jean-Marie Ruysschaert

Pr Xiao Zha

Dr Saida Mebarek

Pr Yuqing

Pr René

Dr Pr Xuexun Fang

Dr Pierre Jurdic

Hong-Wei Li

Pr Xiao

Zha Universite

Claude Bernard -Lyon

M François-Noël

Gilly M Le Professeur

Hamda Ben

Hadid M Le Professeur

Philippe Lalle

M Le Professeur

Germain Gillet

M Alain Helleu

Composantes Sante

J Etienne

La Mme

C Professeure

Burillon

D Bourgeois

THESE EN COTUTELLE Pour obtenir le grade de Docteur délivré par

Bone is a dynamic organ, which consists of an abundant calcified extracellular matrix that makes up the skeleton. The skeleton can support, maintain and protect the vital tissue and organs in our bodies [1]. Bone is also a constantly changing tissue which carries out several metabolic functions, such as: mineral reservoir [2][3][4], cytokine-induced inflammation [5] resorption and formation of bone matrix. Bone cells and their progenitors control bone remodeling. The bone remodeling is a recycling process where the old bone tissue or damaged tissue is removed from the bone surface and the new bone matrix is deposited subsequently.

Three consecutive phases participate in the cycle of remodeling process: resorption, where the bone is resorbed, then the trabecular bone is repaired and the accumulation of old matrix in microarchitecture changes is removed [6].

Bone Resorption and Formation

In bone, there are three major cell types which are osteoclasts, osteoblasts and osteocytes.

Bone resorption is generated by mature osteoclasts which require multiple sequential process [7][8][9], that consists in six steps: firstly, the mature osteoclast adheres to the bone matrix surface; then a special sealing zone is formed; the third step is the activations of the proton and chloride channels of osteoclats, forming a local acidic environment in the bone resorption lacuna (sealing zone); the fourth step is the dissolution of old minerals by acidification;

following by a skeletal organic matrix degradation and the last step is the migration of mature osteoclasts to another resorption site [10]. Bone formation is generated by osteoblasts [11] which are activated by osteoclasts once the bone resorption step was completed [12] (Fig. 1).

Both bone resorption and formation are also regulated by osteocytes which includes osteoclastic resorption in the regulation of bone mass and architecture and osteoblastic mineralization to form primary new bone.

Communications between osteoclast and osteoblast

Osteoblasts (Ob) derive from bone marrow stromal cells (BMSC) which are also known as mesenchymal stem cells (MSC). (Fig. 1). They are responsible for bone inorganic compound synthesis (mainly hydroxyapatite) and the organic matrix generation. In osteoblasts, parathyroid hormone receptor when stimulated by parathyroid hormone (PTH) activates the production of receptor activator of nuclear factor kappa-B ligand (RANKL). Osteoclasts are developed by fusion of monocytes that originate from hematopoietic stem cells.

Hematopoietic cell precursors stimulated by macrophage colony stimulating factor (M-CSF)

give rise to osteoclasts that express RANK receptor. The RANKL/RANK interaction (Fig. 1 red arrow process ) stimulates the differentation of osteoclasts so that they can resorb bone.

Mature osteoclasts are large, multi-nuclear cells. Osteocytes are thought to be one if not the major bone cell type responsible for sensing mechanical strain and orchestrating signals of resorption and formation. Evidence suggests that the primary function of the osteocyte relates to the determination and maintenance of bone structure.

Cathepsin K is necessary for bone resorption by Osteoclast

In healthy skeletal bone, an equal balance of new bone matrix formation and old bone matrix resorption is achieved via the coordinated activity of bone-degrading osteoclasts and bone-forming osteoblasts [13,14]. To degrade bone, osteoclasts must first create a resorption pit. Osteoclasts do this by forming large arrays of multiple F-actin-rich cell-matrix adhesion structures, called podosomes, which create a basolateral sealing zone on the bone surface.

Hematopoietic cell Mesenchymal cell

This allows the osteoclast to control bone degradation by the rapid assembly or disassembly of podosomes in response to intracellular signaling generated via the Rho family of GTPases10 and Src family of kinases [15]. Once the resorption pit is formed, enzymes such as the cysteine protease, cathepsin K, are secreted to facilitate the localized degradation of the underlying bone; cathepsin K is the only mammalian protease having the ability to cleave the helical and non-helical type-I collagen fibers in mineralized bone [16]. Both the acidic microenvironment and activity of cathepsin K contribute to the bone resorption process.

Cathepsin K exhibits about 60% amino acid sequence identity with cathepsin S but is distinct by its tissue expression profile and substrate specificity. Cathepsin K is predominantly found in osteoclasts. To lesser levels, it is also expressed in ovary, heart and skeletal muscle, lung, placenta, testis, small intestine and colon [17]. Cathepsins K are also expressed in hypertrophied chondrocytes. Everts et al. demonstrated that E64 and leupeptin, both inhibitors of papain-like cathepsins, were able to inhibit bone resorption in osteoclast cell assays [18] suggesting the involvement of cysteine protease in osteoclast-mediated bone resorption.

Random sequence analyses of expressed sequence tag (EST) clones estimated that approximately 4% of the cDNA of an osteoclast derives a library encoded cathepsin K and that 98% of the total cysteine protease ESTs were cathepsin K [19]. The importance of cathepsin K in osteoclast-mediated resorption of the bone matrix provides a rationale for the design of inhibitors of cathepsin K as potential drugs for the treatment of diseases of excess bone remodeling such as osteoporosis.

Inhibition of Cathepsin K for the treatment of Osteoporosis and Osteoarthritis

Osteoporosis, the sixth most common disease in the world is a socioeconomic threat [20,21]. Osteoporosis is characterized by bone loss and microstructural deterioration that results in skeletal fragility and an increased risk in bone fractures. Detailed knowledge of bone biology [22] with molecular insights into the communication between bone-forming osteoblasts and bone-resorbing osteoclasts, as well as the signaling networks involved, has led to the identification of several therapeutic targets. Presently, a broad range of drug therapeutics is available to treat bone loss although none of them are able to completely prevent osteoporotic fractures. Besides hormone replacement therapy (HRT), common treatment regimes for osteoporosis include the use of bisphosphonates, calcitonin, calcium, and vitamin D supplements. All present pharmaceutical agents for the treatment of osteoporosis have shortcomings in either their general efficacy, their mode of application, and various side effects. Thus the urge for more effective and safe anti-osteoporotic drugs remains high [23]. Osteoarthritis (OA) is much more prevalent and common in old age. Early signs of OA include progressive loss of proteoglycans, especially aggrecan, from articular cartilage, excessive damage to type II collagen, and general degeneration of the cartilage surface, resulting eventually in a loss of articular cartilage [23,24]. OA affects approximately 80% of people over the age of 65 but it is rare under 45. The number of people with OA is rising as the population ages whereas there is essentially no pharmaceutical intervention for OA available.

Not only the cysteine protease cathepsin K can also cleave the triple helix of types I and II collagens but cathepsin K is also capable of degrading other matrix molecules of hyaline cartilage, such as aggrecan and link protein 11 [25]. This would suggest that up regulation of cathepsin K might contribute to the degradation of cartilage. Therefore, the prevention of cartilage degradation by cathepsin K inhibition may represent a valid strategy for pharmacological intervention in OA [26].

Cathepsin K inhibitors

One of the most promising drug treatments for osteoporosis so far is based on the specific inhibition of the osteoclast protease cathepsin K (CatK) in order to slow down bone resorption [27,28]. CatK is a collagenase and the predominant papain-like cysteine protease expressed in osteoclasts, which has been recognized as an attractive target for antiresorptive osteoporosis therapy [28][29][30]. Considerable effort has been put into developing highly selective and orally applicable CatK inhibitors (CKI) [29]. Four CKIs, Balicatib, Relacatib, Odanacatib (ODN) and MIV-711 have been evaluated as possible drug therapies to prevent bone resorption [31,32]: MIV-711 has been evaluated successfully in a phase I of clinical research for the treatment of osteoarthritis and other bone related disorders. Only ODN has presently reached phase three of clinical research [33,34]. Although developed as antiresorptive agents, several compounds also show lysosomotropic effects [35],

cutaneous adverse effects and anabolic activity [36], which are intrinsically related to the selectivity of inhibitors toward CatK. Therefore, alternative compounds having better selectivity toward CatK may complement the use of CKIs in bone resorption therapy.

Typically, CKIs are mainly derived from peptides or peptidomimetic structures, which generally contain electrophilic entities prone to covalently interact with the cysteine-thiol moiety in enzymes. With the rapid development of powerful and selective inhibitors for CatK, azapeptide nitriles have attracted much attention due to their extremely potent inhibition albeit with a relatively low selectivity [37][38][39][40].

Among these, proteolytically stable azadipeptide nitriles have been developed, [41] having picomolar K i towards the therapeutically relevant cathepsins B, K, L and S with which they form reversible isothiosemicarbazide adducts [42,43]. Recently, we synthesized two series of candidate azanitrile inhibitors that were selected for their inhibition against human CatK activity in vitro [42,43]. One inhibitor (CKI-13, IC 50 = 0.006 nM) from the first group, resulted in a picomolar K i with remarkable selectivity over the cathepsins B and S (Scheme 1

and Table 1).

SCHEME 1 Ο N H N N N Ο N H N N N Ο O CKI-8 CKI-13
Chemical structures of cathepsin K inhibitors CK-8 and CK-13

The other inhibitor (CKI-8, IC 50 = 0.263 nM, an isomer of CKI-13) (Scheme 1 and Table 1) from the second group, led to further improvement in the cathepsin K selectivity.

These azanitriles inhibit cysteine proteases by forming a reversible thioimidate adduct resulting from the nucleophilic attack of a thiol on the carbon-nitrogen triple bond.

Biomarker of bone formation in Osteoblast: Alkaline phosphatases

The osteoblasts are enriched in tissue non-specific alkaline phosphatase (TNAP) which hydrolyzes inorganic pyrophosphate, an inhibitor of hydroxyapatite formation. Crystalline hydroxyapatite [Ca 10 (PO 4 )6(OH) 2 ] is the major mineral component of bone inorganic matrix.

Alkaline phosphatase (AP) (EC 3.1.3.1) catalyzes the hydrolysis of almost any phosphoesters to give inorganic phosphate and the corresponding alcohol [44]. There are four human isozymes, among them three are tissue specific : intestinal (IAP), placental (PLAP) and germ cell (GCAP) and one is tissue non-specific (TNAP). Osteoblasts and chondrocytes are cells which can mineralize and both are enrichied in TNAP. TNAP activity is often used as a biochemical marker in bone [45][46][47]. TNAP is an ubiquitous enzyme expressed at high levels in bone, liver, and kidney [48]. Transgenic mice whose phenotypes show pathologic calcification strongly support the hypothesis that the level of extracellular pyrophosphate (ePP i ), a TNAP substrate, regulates crystal deposition both physiologically and pathologically [START_REF] Hessle | Tissue non-specific alkaline phosphatase and plasma cell membrane glycoprotein-1 are central antagonistic regulators of bone mineralization[END_REF][START_REF] Harmey | Concerted regulation of inorganic pyrophosphate and osteopontin by Akp2, Ennp1 and Ank[END_REF]. ePPi, is an inhibitor of hydroxyapatite (HA) formation. Under physiological conditions HA is formed in bones, while under pathological conditions HA is accumulated in arteries (vascular calcification), in ligaments or tendons (ankylosing spondylitis) as well as in cartilage (osteoarthritis). There are three key enzymes that are central regulators of ePP i , i.e.

TNAP [START_REF] Gollub | The role of alkaline phosphatase in mineralization[END_REF] which hydrolyzes ePP i , the progressive ankylosis enzyme, (a PPi transporter ANK) [START_REF] Wang | Role of the progressive ankylosis gene (ank) in cartilage mineralization[END_REF][START_REF] Zaka | Role of the progressive ankylosis gene in cartilage mineralization[END_REF], contributing to the increase in ePP i level, and the extracellular nucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) which generates ePPi from extracellular nucleotide triphosphates [START_REF] Richette | An update on the epidemiology of calcium pyrophosphate dihydrate crystal deposition disease[END_REF]. TNAP which hydrolyzes ePP i is considered as an enzyme antagonistic to ANK and NPP1. The key role of TNAP in skeletal mineralization has been substantiated in the case of human hypophosphatasia, a rare heritable form of rickets, caused by mutations in the gene encoding TNAP which lead to defective TNAP activity and bone formation [55,[START_REF] Whyte | Hypophosphatasia and the role of alkaline phosphatase in skeletal mineralization[END_REF]. High ePP i concentration persisting especially when TNAP activity is low, can inhibit HA formation leading to rickets or osteomalacia [55,[START_REF] Whyte | Hypophosphatasia and the role of alkaline phosphatase in skeletal mineralization[END_REF]. ePP i deficiency caused by the loss of NPP1 activity can lead to ectopic HA arterial calcification of the kind occurring in generalized arterial calcification of infancy (GACI) [START_REF] Rutsch | Mutations in ENPP1 are associated with "idiopathic" infantile arterial calcification[END_REF][START_REF] Ruf | The mutational spectrum of ENPP1 as arising after the analysis of 23 unrelated patients with generalized arterial calcification of infancy (GACI)[END_REF]. A loss of function ANK, causing ePP i deficiency, was described in the case of craniometaphyseal dysplasia, where excessive growth of craniofacial bones occurs [START_REF] Richette | An update on the epidemiology of calcium pyrophosphate dihydrate crystal deposition disease[END_REF]. High TNAP activity, leading to low ePP i concentration, as observed in the case of vascular calcification and in osteoarthritis cartilage, can lead to HA deposition in arteries or in cartilage respectively. Therefore, TNAP as well as ANK and NPP1 can be considered as attractive drug targets for the treatment of calcification disorders [START_REF] Mebarek | Ankylosing spondylitis, late osteoarthritis, vascular calcification, chondrocalcinosis and pseudo gout: toward a possible drug therapy[END_REF]. The search of inhibitors or activators of ANK, NPP1 or TNAP can lead to therapeutic treatment of mineralization disorders as well to novel molecules for determining their roles in ePP i homeostasis. So far, the screening of TNAP inhibitors has relied on the determination of the recombinant TNAP activity using para-nitrophenylphosphate (p-NPP) [START_REF] Dahl | Discovery and validation of a series of aryl sulfonamides as selective inhibitors of tissuenonspecific alkaline phosphatase (TNAP)[END_REF] as TNAP non-natural substrate. TNAP inhibitors selected under such conditions may act differently on the living cells. Therefore, there is a strong demand for flexible, fast and costeffective strategies to select inhibitors with improved prospects for clinical success [START_REF] Sergienko | High-throughput screening of tissue-nonspecific alkaline phosphatase for identification of effectors with diverse modes of action[END_REF].

State of art of phosphatase activity assays

Most of assays for measuring ePP i are based on coupled enzyme assays monitoring NADPH [START_REF] Lust | A rapid, enzymatic assay for measurement of inorganic pyrophosphate in biological samples[END_REF], NADH [START_REF] Cook | A rapid, enzymatic assay for the measurement of inorganic pyrophosphate in animal tissues[END_REF][START_REF] Sakasegawa | Colorimetric inorganic pyrophosphate assay using a double cycling enzymatic method[END_REF], luciferase [START_REF] Nyrén | Enzymatic method for continuous monitoring of inorganic pyrophosphate synthesis[END_REF][START_REF] Nyrén | Enzymatic method for continuous monitoring of DNA polymerase activity[END_REF][START_REF] Nyrén | Solid phase DNA minisequencing by an enzymatic luminometric inorganic pyrophosphate detection assay[END_REF][START_REF] Nyrén | Inorganic pyrophosphatase-based detection systems. I. Detection and enumeration of cells[END_REF][START_REF] Nyrén | Inorganic pyrophosphatase-based detection systems. II. Detection and quantification of cell lysis and cell-lysing activity[END_REF][START_REF] Ronaghi | Real-time DNA sequencing using detection of pyrophosphate release[END_REF][START_REF] Marques | Optimized chromatographic and bioluminescent methods for inorganic pyrophosphate based on its conversion to ATP by firefly luciferase[END_REF] 2-amino-6-mercapto-7-methylpurine [START_REF] Webb | A continuous spectrometric assay for inorganic phosphate and for measuring phosphate release kinetics in biological systems[END_REF][START_REF] Upson | A spectrophotometric method to measure enzymatic activity in reactions that generate inorganic pyrophosphate[END_REF] or luminol [START_REF] Jansson | Enzymatic chemiluminescence assay for inorganic pyrophosphate[END_REF]. Additional controls are required to take into account competitive activities and to maintain saturated concentrations of substrates for optimal activity measurements. Direct colorimetric methods to measure hydrolytic activity of ePP i were recently developed. Malachite green to detect inorganic phosphate was successfully used as a fully automated high throughput screening assay for pyrophosphate and phosphate release from enzymes [START_REF] Pegan | A Universal, Fully Automated High Throughput Screening Assay for Pyrophosphate and Phosphate Release from Enzymatic Reactions[END_REF]. Zn (II) and Cd (II) coumarin-derivative complexes could serve as a turn on and turn off assay for monitoring pyrophosphate hydrolytic activity of alkaline phosphatase [START_REF] Das | Zn(II) and Cd(II)-based complexes for probing the enzymatic hydrolysis of Na4P2O7 by alkaline phosphatase in physiological conditions[END_REF]. Pyrophosphate anion in an acidic acetonitrile-water solution form yellow ([(P 2 O 7 )Mo 18 O 54 ] 4-) allowing its titration [START_REF] Katano | Assay of enzymes forming AMP+PPi by the pyrophosphate determination based on the formation of 18-molybdopyrophosphate[END_REF]. None of these methods measures protein content, P i or substrate concentrations simultaneously, allowing direct kinetic determinations in cells in a single assay.

So far, IR spectroscopy [START_REF] Jencks | Infrared measurements in aqueous media[END_REF][START_REF] Goormaghtigh | Secondary structure and dosage of soluble and membrane proteins by a reflection[END_REF][START_REF] Kumar | Following enzyme activity with infrared spectroscopy[END_REF] is able to detect protein concentration, secondary structure and to measure enzymatic activity. It has been successfully employed to monitor activities of alkaline phosphatase [START_REF] Wong | FTIR spectroscopic kinetic analysis of alkaline phosphatase under hyperbaric manipulation[END_REF], amidase [START_REF] Pacheco | Measuring enzymatic activity of a recombinant amidase using Fourier transform infrared spectroscopy[END_REF][START_REF] Pacheco | Application of Fourier transform infrared spectroscopy for monitoring hydrolysis and synthesis reactions catalyzed by a recombinant amidase[END_REF] a-amylase [START_REF] Schindler | Simultaneous determination of α-amylase and amylogucosidase activities using flow injection analysis with Fourier transform infrared spectroscopic detection and partial least-squares data treatment[END_REF][START_REF] Krieg | Determination of alpha-amylase activity using Fourier transform infrared spectroscopy[END_REF], amyloglucosidase [START_REF] Schindler | Simultaneous determination of enzyme activities by FTIRspectroscopy in a one-step assay[END_REF], Ca 2+ ATPase [START_REF] Barth | Infrared spectroscopic signals arising from ligand binding and conformational changes in the catalytic cycle of sarcoplasmic reticulum calcium ATPase[END_REF][START_REF] Barth | Molecular changes in the sarcoplasmic reticulum calcium ATPase during catalytic activity. A Fourier transform infrared (FTIR) study using photolysis of caged ATP to trigger the reaction cycle[END_REF][START_REF] Thoenges | Direct measurement of enzyme activity with infrared spectroscopy[END_REF], creatine kinase [START_REF] Raimbault | ADP-binding and ATP-binding sites in native and proteinase-K-digested creatine kinase, probed by reaction-induced difference infrared spectroscopy[END_REF], diisopropylfluorophosphatase [START_REF] Gäb | Quantification of hydrolysis of toxic organophosphates and organophosphonates by diisopropyl fluorophosphatase from Loligo vulgaris by in situ Fourier transform infrared spectroscopy[END_REF], bfructofuranosidase [START_REF] Cadet | Enzyme kinetics by mid-infrared spectroscopy: beta-fructosidase study by a one-step assay[END_REF][START_REF] Schindler | Determination of enzyme kinetics and chemometric evaluation of reaction products by FTIR spectroscopy on the example of ßfructofuranosidase[END_REF], fructose 1,6 biphosphatase [START_REF] López-Sánchez | Determination of enzyme activity inhibition by FTIR spectroscopy on the example of fructose bisphosphatase[END_REF], glucose oxidase [START_REF] Karmali | Assay for glucose oxidase from Aspergillus niger and Penicillium amagasakiense by Fourier transform infrared spectroscopy[END_REF], glutamic pyruvic transaminase [START_REF] Wright | Use of IR absorption of the carboxyl group of amino acids and their metabolites to determine pKs, to study proteins, and to monitor enzymatic activity[END_REF], blactamase [START_REF] Fisher | beta-Lactamase proceeds via an acylenzyme intermediate. Interaction of the Escherichia coli RTEM enzyme with cefoxitin[END_REF], oxalate decarboxylase [START_REF] Muthusamy | Real-time monitoring of the oxalate decarboxylase reaction and probing hydron exchange in the product, formate, using fourier transform infrared spectroscopy[END_REF], urease [START_REF] Karmali | The use of Fourier transform infrared spectroscopy to assay for urease from Pseudomonas aeruginosa and Canavalia ensiformis[END_REF] and phopholipases [START_REF] Estrela-Lopis | Dipalmitoyl-phosphatidylcholine phospholipase D interactions investigated with polarization-modulated infrared reflection absorption spectroscopy[END_REF][START_REF] Yamamoto | Regulation of phospholipase D activity by neutral lipids in egg-yolk phosphatidylcholine small unilamellar vesicles and by calcium ion in aqueous medium[END_REF][START_REF] Do Ld | Direct determination of phospholipase D activity by infrared spectroscopy[END_REF]. IR can identify specific finger-like signatures in microbial cells [START_REF] Bosch | Type-IV pili spectroscopic markers: applications in the quantification of piliation levels in Moraxella bovis cells by a FT-IR ANN-based model[END_REF][START_REF] Naumann | Microbiological characterizations by FT-IR spectroscopy[END_REF][START_REF] Helm | Classification and identification of bacteria by Fourier-transform infrared spectroscopy[END_REF][START_REF] Rebuffo | Reliable and rapid identification of Listeria monocytogenes and Listeria species by artificial neural networkbased Fourier transform infrared spectroscopy[END_REF][START_REF] Bosch | Fourier transform infrared spectroscopy for rapid identification of nonfermenting gram-negative bacteria isolated from sputum samples from cystic fibrosis patients[END_REF] tumor cells [START_REF] Harvey | Discrimination of prostate cancer cells by reflection mode FTIR photoacoustic spectroscopy[END_REF][START_REF] Holton | Subcellular localization of early biochemical transformations in cancer-activated fibroblasts using infrared spectroscopic imaging[END_REF][START_REF] Gaigneaux | The infrared spectrum of human glioma cells is related to their in vitro and in vivo behavior[END_REF] or in tissues [START_REF] Harvey | Discrimination of prostate cancer cells by reflection mode FTIR photoacoustic spectroscopy[END_REF] giving information on biochemical cell composition or changes induced by anti-tumor drugs in cells [START_REF] Diem | A decade of vibrational micro-spectroscopy of human cells and tissue[END_REF][START_REF] Krishna | Combined Fourier transform infrared and Raman spectroscopic identification approach for identification of multidrug resistance phenotype in cancer cell lines[END_REF][START_REF] Bhargava | Towards a practical Fourier transform infrared chemical imaging protocol for cancer histopathology[END_REF][START_REF] Matthäus | Infrared and Raman Microscopy in Cell Biology[END_REF][START_REF] Zwielly | Discrimination between drug-resistant and non-resistant human melanoma cell lines by FTIR spectroscopy[END_REF][START_REF] Gasper | IR spectroscopy as a new tool for evidencing antitumor drug signatures[END_REF][START_REF] Draux | IR spectroscopy reveals effect of non-cytotoxic doses of anti-tumour drug on cancer cells[END_REF][START_REF] Diem | The infrared spectral signatures of disease: extracting the distinguishing spectral features between normal and diseased states[END_REF][START_REF] Vileno | Evidence of lipid proxydation and protein phosphorylation in cells upon oxydative stress photo-generated by fullerols[END_REF][START_REF] Gasper | FTIR spectral signature of the effect of cardiotonic steroids with antitumoral properties on a prostate cancer cell line[END_REF][START_REF] Flower | Synchotron FTIR analysis of drug treated ovarian A2780 cells: an ability to differenciate cell response to different drugs?[END_REF][122]. Combination of metabolic fingerprinting spectra obtained by infrared spectroscopy and multivariate statistical analyses [START_REF] Diem | Infrared spectroscopy of cells and tissues: shining light onto a novel subject[END_REF][START_REF] Ellis | Metabolic fingerprinting in disease diagnosis: biomedical applications of infrared and Raman spectroscopy[END_REF][START_REF] Ellis | Metabolomics-assisted synthetic biology[END_REF] as well as infrared micro-spectroscopy to monitor cell biological processes of live or fixed cells have been employed successfully [START_REF] Romeo | Infrared micro-spectroscopic studies of epithelial cells[END_REF][START_REF] Bellilosa | Tracking Infra-Red signatures of drugs in cancer cells by Fourier Transform microspectroscopy[END_REF][128]. Activities of alkaline phosphatase in aqueous buffer [START_REF] Wong | FTIR spectroscopic kinetic analysis of alkaline phosphatase under hyperbaric manipulation[END_REF] or in sera [START_REF] Thouverey | Inorganic pyrophosphate as a regulator of hydroxyapatite of calcium pyrophosphate dihydrate mineral deposition by matrix vesicles Osteoarthritis and cartilage[END_REF] have been determined by IR, suggesting the possibility of using this method for quantitative determination of a specific enzymatic activity in whole cells by IR in one single assay.

CHAPTER II

AIMS

Context of the Ph D thesis

The The first one is centered on the effects of azadipeptide nitrile cathepsin K inhibitors (CKIs) on bone formation mediated by osteoblasts and on bone resorption controlled by osteoclasts. I synthetized the cathepsin K inhibitors as it has been reported in the experimental part. However the organic synthesis has been published [42,43], therefore I did not include the organic synthesis of CKIs in the Result section.

The second part of my Ph D thesis is focused on the development of an alkaline phosphatase activity assay in matrix vesicles by infrared spectroscopy.

The third part is the extension of the infrared assay of the kinetics of phosphate-release from natural substrates in living cells. This allowed us to determine in a continuously manner the alkaline phosphatase activity by infrared spectroscopy under physiological condition and in cellular medium. This method could be used for screening inhibitors of alkaline phosphatase, a biological marker of mineral formation. Below are the detailed three specific aims which formed the basis of my thesis.

Effects of Cat K inhibitors on mineralization and bone resorption

In order to provide a proof of concept of the potential use of our CKIs [42,43] in drug treatments for osteoporosis, as well as to consolidate the efficiency/biological evaluation of these compounds, prior to preclinical trials, we compared the effects of two inhibitors, CKI-8

and CKI-13 (scheme 1) with those of the commercial inhibitor E64 on cell viability, osteoblast-induced mineralization and osteoclast-induced bone resorption.

The objective is to determine whether CKIs (CKI-8 and CKI-13) can affect mineralization using osteoblast cells and to evaluate their bone resorption ability on osteoclasts. Cell viability assay is performed on osteoblasts and on osteoclasts to check the toxicity of CKIs before mineralization and resorption treatment. The effect of CKIs on mineralization is evaluated on osteoblasts (human osteoblast-like Saos-2 cells and primary osteoblast from murine calvaria) which underwent the entire osteoblastic differentiation program from proliferation to mineralization. Mineralization is monitored by detecting calcium nodules with Alizarin Red-S and by measuring TNAP activity. Inhibition of Cat K in osteoclast-derived intracellular medium is evaluated by a gelatin zymography method, which could be used as a screening test of inhibitors before checking their effects on bone resorption. Bone resorption assay was assayed on the surface of an inorganic crystal coating surface (Corning Bone assay surface) and on bovine bone slices. Resorption pit area per osteoclast was measured to determine the effects of Cat K inhibitors. The overall findings of the regulating effect of cat K inhibitors were presented in Chapter IV (part I).

Development of a new Infrared assay to monitor pyrophosphate hydrolysis by Matrix

Vesicles

Tissue non specific alkaline phosphatase (TNAP) hydrolyses pyrophosphate (PP i ) and is an inhibitor of hydroxyapatite (HA) formation. Inorganic phosphate (P i )/PP i ratio is a key factor determining the mineralization efficiency. A P i /PP i ratio greater than 140 leads to the formation of HA, while a ratio less than 70 inhibited completely HA formation. A P i /PP i ratio below 6 leads to the formation of calcium pyrophosphate dehydrate (CPPD), characteristic for CPPD pathological mineralization in osteoarthritis cartilage [START_REF] Thouverey | Inorganic pyrophosphate as a regulator of hydroxyapatite of calcium pyrophosphate dihydrate mineral deposition by matrix vesicles Osteoarthritis and cartilage[END_REF] . Therefore, a decrease in PP i concentration, due to high TNAP activity, will induce HA mineralization.

Since TNAP as well as other enzymes were enrichied in MVs implicated in the mineralization, the MVs will be used as a primary source of TNAP to test its activity.

An infrared assay of the kinetics of phosphate-release from physiological substrates in living cells

MVs are released from chondrocyte as well as from osteoblasts. Having demonstrating that the infrared assay can monitor phosphatase activity in MVs. Here, we developed a continuous infrared (IR) assay to determine phosphatase activity in Saos-2 cells and in primary osteoblasts from exogenous substrates such as PP i , AMP, ADP, ATP, UTP, βglycerophosphate or α-D-glucose-1-phosphate and para-nitrophenylphosphate (pNPP). The IR assay can be employed as a cost-effective label-free approach to determine the phosphatase activity from extracellular physiological substrates in whole cells. It may serve to screen the phosphatase inhibitors in TNAP-enriched cells. An overview of the faisability to determine the phosphatase activity in the different cellular models of TNAP-enriched is presented and discussed in in Chapter IV (part III).

CHAPTER III MATERIALS AND METHODS

Organic synthesis:

The compounds CKI-8 and CKI-13 have been previously synthesized [42,43,[START_REF] Frizler | Structural optimization of azadipeptide nitriles strongly increases association rates and allows the development of selective cathepsin inhibitors[END_REF]. Here we present a short overview.

Scheme 2: Synthesis route for compounds A and B

4-bromobenzoyl chloride:

2.01 g 4-bromobenzoic acid (10 mmol) was added to a 50 mL round-bottomed flask equipped with a magnetic stir bar, 20 mL thionyl was then added. The mixture was stirred for 30 min, then it was heated at reflux for 60 min. After evaporation of the excessive thionyl, the solid residue was suspended in 30 mL cyclohexane for recrystallization. The mixture was heated at reflux for 30 min, then cooled slowly to room temperature and next to 0 °C in an ice bath. It was filtered through a fritted-glass funnel, the solid was washed with 15 mL cold cyclohexane and then dried under vacuum to afford 4-bromobenzoyl chloride as a white solid.

L-leucine methyl ester hydrochloride:

h 2
In an ice-bath, 5 mL thionyl was added drop wise under stirring to a 50 mL round-bottomed flask containing 30 mL anhydrous methanol. 10 min later 5 g L-leucine was added. After the mixture was stirred for 15 min in ice-bath, it was allowed to warm to room temperature and stirred for additional 30 min and then the mixture was heated at reflux for 60 min. The heated mixture was concentrated in a rotary evaporator to leave an oily residue, which was then suspended in 40 mL anhydrous ether. A white solid precipitated out and then was cooled to -20 °C for 2 h, the mixture was filtered through a fritted-glass funnel and the solid was washed with 15 mL cold anhydrous ether and finally dried under vacuum to afford L-leucine methyl ester hydrochloride as a white solid.

Methyl 2-(4-bromobenzamido)-4-methylpentanoate:

0.5 g sodium bicarbonate was dissolved in 20 mL water, and 1.82 g L-leucine methyl ester hydrochloride (10 mmol) was added under stirring. After 15 min, the solution was extracted with dichloromethane for four times (15 mL×4). The combined organic layers were washed with H 2 O (2×15 mL), dried with Na 2 SO 4 and then concentrated in a rotary evaporator. 2.19 g 4-bromobenzoyl chloride (10 mmol) was added to the solution under ice-cooling and 1.40 mL anhydrous triethylamine was added drop wise. The mixture was stirred at 0 °C for 30 min, then it was allowed to warm to room temperature and stirred for additional 3 h. The mixture was washed with HCl (1×15 mL), saturated NaHCO3 (1×15 mL), H2O (1×15 mL) and brine (1×15 mL) in turn. The solvent was dried by Na 2 SO 4 and then evaporated to obtain a white solid, then it was purified by column chromatography on silica gel with petroleum ether/ethyl acetate (1:1) as eluent to obtain methyl 2-(4-bromobenzamido)-4-methylpentanoate as white solid.

3-(4-bromobenzamido)-5-methyl-2-oxohexanoic acid:

3.42 g methyl 2-(4-bromobenzamido)-4-methylpentanoate (10 mmol) was dissolved in 15 mL THF, then 20 mL 1M NaOH was added under ice-cooling and then stirring was last for 3h.

THF was removed in a rotary evaporator. The pH of the mixture was adjusted to 1-2, inducing a white solid precipitates. Then ethyl acetate was added to dissolve the precipitates, the organic layer was separated and the aqueous layer was extracted with ethyl acetate (15 mL×3). The combined organic phases were washed with brine twice (2×15 mL), then dried with Na 2 SO 4 and evaporated to obtain 3-(4-bromobenzamido)-5-methyl-2-oxohexanoic acid as a white solid.

4-bromo-N-(1-(1, 2-dimethylhydrazinyl)-4-methyl

3-bromo-N-(1-(2-cyano-1,2-dimethylhydrazinyl)-4-methyl-1-oxopentan-2-yl)benzamide (2)

This compound was produced by using the similar procedures as for compound 1 but staring with 3-bromobenzoic acid instead of 4-bromobenzoic acid.

N-(1-(2-cyano-1,2-dimethylhydrazinyl)-4-methyl-1-oxopentan-2-yl)-4'-phenyl-biphenyl-

4-carboxamide (CKI-8)

This compound was produced by using SUZUKI reaction in staring with compound 1 as maternal compound using biphenyl-4-ylboronic acid. Hz, 2H), 1.10 (dd, J = 28.6, 6.5 Hz, 6H). 13

N-(1-(2-cyano-1,2-dimethylhydrazinyl)-4-methyl-1-oxopentan-2-yl)-4'-phenyl-biphenyl-

3-carboxamide (CKI-13)

This compound was produced by using the same procedure as for CKI-8 in staring with compound 2 as maternal compound using biphenyl-4-ylboronic acid. The preparation of primary cells were performed at INSA of Lyon, IMBL Building, 69100

Villeurbanne, France.

1. Primary osteoblast cells were enzymatically isolated from calvaria (frontal and parietal bones) of 5-6 day old mice (C57BL/6J strain). Calvaria were dissected aseptically and cells were isolated using sequential digestion at 37 °C with 0.05 % trypsin/ethylenediaminetetraacetic acid (EDTA) for 20 min and then with 0.8 U mL -1 liberase for 20 min. The first two digestions were discarded, and the cells obtained after two digestions (each time incubated with 0.8 U mL -1 liberase for 45 min) were collected, pooled and then filtered through a 100 μm-diameter-pore cell strainer. The cells were plated at a density of 100,000 cells/well in 12-well plates (Corning Inc) in Dulbecco modified Eagles medium (DMEM) containing 15% (mL/mL) fetal bovine serum (FBS), 100 U ml -1 penicillin and 100 μg ml -1 streptomycin, and 24 h later switched to DMEM containing 10 % FBS (mL/mL) supplemented with 50 g mL -1 L-ascorbic acid (AA) for six days. They were then transferred to DMEM containing 10% FBS (mL/mL) supplemented with 50 g mL -1 L-ascorbic acid (AA) [START_REF] Gillette | The role of annexin 2 in osteoblastic mineralization[END_REF][START_REF] Vaingankar | Subcellular targeting and function of osteoblast nucleotide pyrophosphatase/phosphodiesterase 1[END_REF] with 0.1 % DMSO (mL/mL) and 7.5 mM β-glycerophosphate (β-GP) without (control) or with CKI at the indicated concentrations for one more week. AA and β-GP are two osteogenic factors commonly used to stimulate osteoblast differentiation and mineralization [START_REF] Gillette | The role of annexin 2 in osteoblastic mineralization[END_REF][START_REF] Vaingankar | Subcellular targeting and function of osteoblast nucleotide pyrophosphatase/phosphodiesterase 1[END_REF]. Cultures were maintained in a humidified atmosphere consisting of 95 % air and 5% CO 2 at 37 °C. Primary cell cultures were used without passage. Primary osteoblasts as prepared were used for the mineralization (calcium nodule detection) and TNAP activity assays.

2.

Human osteosarcoma Saos-2 cells (ATCC HTB-85) were cultured in so-called growth medium consisting of Dulbecco modified Eagles medium DMEM (ATCC) supplemented with 100 U mL -1 penicillin, 100 g mL -1 streptomycin (both from Sigma) and 10 % FBS (mL/mL); Gibco), at 37 °C, in an atmosphere of 5 % CO 2 and 95% air. For the MTT test (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide), Saos-2 cells were plated in 96-

well culture plates at a cell density of 10,000 cells/well in growth medium supplemented with 0.1 % DMSO (mL/mL) , 50 µg mL -1 AA and 7.5 mM β-GP for three days without (control) or with CKI at the indicated concentrations. For mineralization (calcium nodule detection) and TNAP assays, Saos-2 cells were plated in 12-well culture plates at a cell density of 100,000 cells/well in growth medium supplemented with 0.1 % DMSO (mL/mL) , 50 µg mL -1 AA and 7.5 mM β-GP for eight days without (control) or with CKI at the indicated concentrations.

3.

The transformed murine monocytic cell line RAW 264.7 (ATCC, Manassas, VA, USA) was cultured at 37 °C in 5 % CO 2 atmosphere in DMEM supplemented with 10 %FBS (mL/mL) and antibiotics (100 U mL -1 penicillin and 100 µg mL -1 streptomycin), so-called growth medium. For MTT tests, cells were plated in a 96-well plate at a density of 10,000 cells/well in growth medium containing 0.1 % DMSO (mL/mL), 20ng mL-1 recombinant mouse receptor-activator of NF-κB ligand (mRANK-L), without (control) or with CKI at the indicated concentrations for three days. mRANK-L was produced in our facility (PAP; SFR Biosciences). For the conditioned medium used for zymography, cells were plated in a 6well plate at a density of 350,000 cells/well in growth medium containing 20 ng mL -1 mRANK-L for five days.

4.

Primary osteoclasts from bone marrow cultures from posterior limbs were collected from 7-9 week-old mice (C57BL/6J strain). Cell suspensions were prepared by flushing bone marrow cells using complete medium (α-MEM) (Invitrogen, Cergy Pontoise, France) supplemented with 10% FBS (mL/mL), 100 U mL -1 penicillin, 100 µg mL -1 streptomycin and 2 mM L-glutamine (Invitrogen)). Mononuclear cells isolated using lymphocyte separation medium (EuroBio, Courtaboeuf, France) were seeded at 37° C and 5 % CO 2 in complete α-MEM medium supplemented with 30 ng mL -1 mRANKL and 25 ng mL -1 recombinant mouse macrophage-colony stimulating factor produced in our facility (PAP; SFR Biosciences), so-called differentiation medium. Bone marrow mononuclear cells from mice, were seeded at 350,000 cells/well, in 6-well plates, with a change of medium every second day, over five days. Once the cells were mature, they were ready for the MTT and bone resorption assays. For the MTT assay, mature cells were detached from the plastic plates using PBS with 25 mM EDTA. They were plated at a density of 10,000 cells/well in 96-well plates in differentiation medium containing 0.1 % DMSO (mL/mL) without (control) and with CKI at the indicated concentrations for three days.

Cell viability assay

The viability of cultured cells (Saos-2 or RAW 264.7 cell lines or osteoclast primary cells) was measured using the MTT colorimetric assay (Roche Diagnostics, Meylan, France) as described previously [START_REF] Mosmann | Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays[END_REF]. Briefly, cells were prepared as described above. Then the MTT labeling reagent (0.5 mg mL -1 final concentration) was added to each well. The cells were further incubated for 4 h. 100 µl of solubilization solution (10 % SDS g:mL in 0.01M HCl)

were then added and plates were allowed to stand overnight at 37 °C in a humidified atmosphere. The absorbance was measured at 550 and 690 nm wavelengths using a Tecan Infinite M200 (Salzburg, Austria) micro-titer plate reader. Cell viability was directly related to the difference in absorbance measured at 550 and 690 nm. Results were normalized relative to their respective controls (without inhibitors) taken as 100. For each inhibitor/cell combination, three distinct sample pools were analyzed in a triplicate manner (n = 9).

Calcium nodule detection

Cell layers (Saos-2 cells or primary osteoblasts isolated from mouse calvaria) were washed with phosphate-buffered saline (PBS) (PAA) and stained with 0.5 % (g/mL) Alizarin

Red-S (AR-S) (Sigma) in PBS (pH 5.0) for 30 min at room temperature. After washing four times with PBS to remove free calcium ions, cell cultures were destained with 3.6 % (g/mL) cetylpyridinium chloride (Sigma) in PBS pH 7.0 for 60 min at room temperature. The AR-S concentration was determined by measuring the absorbance at 562 nm [START_REF] Stanford | Rapidly forming apatitic mineral in an osteoblastic cell line[END_REF].

Tissue-nonspecific alkaline phosphatase activity assay

To determine tissue-nonspecific alkaline phosphatase (TNAP) activity, cells (Saos-2 cells or primary osteoblasts isolated from mouse calvaria) cultured in 6-well plates were harvested in 0.2 % (g/mL) Nonidet P-40 and disrupted by sonication. The homogenate was centrifuged at 1,500 g for 5 min, and TNAP activity in the supernatant was determined by measuring the absorbance at 405 nm in buffer (pH 10.4) containing A-M-P [START_REF] Cyboron | Purification and initial characterization of intrinsic membrane-bound alkaline phosphatase from chicken epiphyseal cartilage[END_REF]. In the same lysates, the protein content was determined using a bicinchoninic acid (BCA) assay (Pierce)

or Bradford (see below). Results are shown as µmol para-nitrophenolphosphate (pNPP) min - 1 mg protein -1 .

Zymography of cathepsin K in cell lysates

Zymography was performed as reported [START_REF] Li | Detection of femtomole quantities of mature cathepsin K with zymography[END_REF]. (mL/mL) glycerol for 10 min. The washing was repeated twice for 10 min each time. Gels were then incubated for 30 min at room temperature in activity buffer containing 100 mM sodium phosphate buffer pH 6.0, 1 mM EDTA and 2 mM dithiothreitol (DTT). Then this activity buffer was exchanged for fresh activity buffer containing 0.1% DMSO (mL/mL) without (control) or with CKI at the indicated concentrations for 24 h incubation at 37 °C.

The gels were rinsed twice with deionized water and were incubated for 1 h in Coomassie stain (10 % mL/mL acetic acid, 25 % mL/mL isopropanol, and 4.5 % mL/mL Coomassie blue) labeled the gelatin. This was followed by destaining (10 % mL/mL isopropanol and 10 % mL/mL acetic acid). Gels were scanned using a Canon scanner. Densitometry was performed using Image J software (developed by the National Institutes of Health), and the IC 50 values for CKI-8 and CKI-13 inhibition on human catK were calculated based on the results of gelatin zymography.

TRAP staining of RAW 264.7 cells and osteoclast primary cells

To confirm the generation of multinucleated osteoclast cells, the cultured monocytic cell line RAW 264.7 cells and also osteoclast primary cells were stained for TRAP using the TRAP-staining kit (Sigma-Aldrich), according to the manufacturer's instructions. TRAPpositive multinucleated (3 or more nuclei) osteoclasts were visualized by light microscopy and photographed. Each osteoclast formation assay was performed independently at least 3 times.

Resorption pit assay

Assessment of bone resorption per single osteoclast (resorption index) was performed as described [START_REF] Georgess | Comparative transcriptomics reveals RhoE as a novel regulator of actin dynamics in bone-resorbing osteoclasts[END_REF]. Briefly, osteoclasts (RAW 264.7 cells and primary mouse osteoclasts), after 5 days of differentiation (see above for details), were detached from plastic plates using PBS 25 mM EDTA and replated on bovine bone slices at a density of 50,000 cells/well in 96-well plates or on a 96-well Corning Osteo Assay Surface plate at a density of 30,000 cells/well.

They were treated during the entire experiment with 30 ng mL -1 mRANKL and M-CSF at 25 ng mL -1 , in the presence or absence of CKI-8 and CKI-13. After 48 h, mature cell numbers were first determined by staining cells for TRAP using a leukocyte acid phosphatase kit. The cells were removed with water from the Osteo Corning plates and from the bone surface, then stained with 5 % (g/mL) silver nitrate solution to determine bone resorption. In the case of the bovine bone slices, osteoclasts were scraped from the slices using cotton buds, and slices were stained for 3 min with a 1 % (g/mL) toluidine solution. Images were acquired using a widefield microscope with side illumination, followed by quantitative analysis of the resorption area using Image J software 1.44p (NIH, USA). The results for each bone slice were expressed as the ratio of the area of resorption to the number of mature osteoclasts, and were normalized relative to their respective controls (without inhibitors), taken as 100 (corresponding to 3914 µm 2 / osteoclast). For each inhibitor in the cells three distinct sample pools were analyzed in a duplicate manner (n = 6).

Preparation of Matrix Vesicles

Matrix vesicles (MVs) were prepared according to Wuthier's methods [START_REF] Wu | Characterization of the nucleational core complex responsible for mineral induction by growth plate cartilage matrix vesicles[END_REF]. Femurs from twenty 17-day-chicken embryos were taken. Slices (

SDS-PAGE Analysis

Proteins of each fraction were separated on 12 % (w:v) SDS-polyacrylamide gels [START_REF] Laemmli | Cleavage of structural proteins during the assembly of the head of bacteriophage T4[END_REF].

Composition of the SDS-polyacrylamide gels are indicated in Table 2. Mineralization is followed by measuring the turbidity at 340 nm at 37 ° C overnight by UV spectrophotometer.

Determination of protein concentration

The protein content was determined using a Bradford assay [START_REF] Bradford | A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF]. Enzyme units are µmol of p-nitrophenolate released per min per mg of proteins. The amount of protein in the different fractions was determined by the Bradford method . This is a colorimetric method based on the adsorption of Coomassie blue R250 to the side chains of basic amino acid residues and hydrophobic proteins, forming a complex having a color absorption maximum at 595 nm. The absorbance change is proportional to the amount of dye fixed and therefore the amount of protein in the sample. To determine the amounts of protein in the different fractions, a reference range is made using increasing amounts of protein of known concentration , bovine serum albumin BSA 5μg/μL ( 0 , 2 , 4, 6 , 8, 10, 15 mg) . In parallel, 5 μL of each fraction were prepared . Bradford reagent ( 200 uL) is added last at the same time in all tubes containing equal volumes of 800 mL after adding water.

Infrared Spectra

IR data were acquired with a Thermo Scientific Nicolet iS10 spectrometer equipped with a DTGS detector. The IR spectra were recorded with a thermostated cell kept at 37°C with 256 interferograms at 4 cm -1 resolution each and Fourier transformed. During data acquisition, the spectrometer was continuously purged with dry filtered air (Balston regenerating desiccant dryer, model 75-45 12 VDC). At least three independent measurements have been performed to determine the nature of minerals and to obtain kinetics parameters.

Identification of Mineral Complexes by Infrared Spectroscopy

From the mineralization assay, the formed minerals were centrifuged and washed two times with water, then dried under N 2 . Dry material (0.5 mg) was put on a diamond ATR support. The infrared spectra were measured by the means of Thermo Scientific Nicolet iS10 spectrometer as stated above.

Protein determination by infrared spectroscopy

To determine protein concentration, we measured the intensity of the amide I and II bands in the infrared spectra (Fig. 3) [START_REF] Jackson | The use and misuse of FTIR spectroscopy in the determination of protein structure[END_REF]. The IR spectra were recorded with a thermostated IR cell kept at 37 °C with 128 interferograms at 4 cm -1 resolution each and then Fourier transformed. All measurements were performed by taking 3 μL of sample solution, deposited between two BaF 2 windows of a demountable thermostated cell (model Harrick) separated with a 6 µm. A series of five concentrations of BSA from 5 mg mL -1 to 20 mg mL -1 served to as a calibration curve for protein determination. Final protein concentrations in cells were determined directly by measuring the intensity of the amide-II band at 1550 cm -1 using ε amide II = 3.6 mg -1 ml cm -1 .

Phosphatase activity determination of matrix vesicles by infrared spectroscopy

To start the enzymatic reaction, an aliquot of freshly prepared 0.2 to 8 mg MV mL -1 was taken and mixed with the reaction mixture containing stock solution of 100 mM PPi in aqueous buffer 100 mM Tris-HCl, pH 8.0, 5 µM de ZnCl 2 and 5 mM MgCl 2 so that final PP i concentration was 40-60 mM. Final protein concentration in MVs was 0.250 to 4 mg MV protein mL -1 . Three µL of reaction mixture containing MVs was taken and deposited between two ZnSe windows of a demountable thermostated cell (model Harrick) separated with a 12 µm (for activity measurements) or with a 6 µm spacer (for protein determination and activity measurements). Protein concentration was measured with the amide-II band using ε amideII = 3.6 mg -1 ml cm -1 . PP i band located at 1107 cm -1 (Molar absportion coefficient = 2158 ± 211 M -1 .cm -1 ) and P i bands located at 1076 cm -1 (Molar absportion coefficient = 1346 ± 116 M - 1 .cm -1 ) and at 990 cm -1 (Molar absportion coefficient = 493 ± 49 M -1 .cm -1 ) served to measure the substrate and the product concentrations. IR data were acquired with a Thermo Scientific Nicolet iS10 spectrometer equipped with a DTGS detector as specified above.

Phosphatase activity determination of osteoblasts by infrared spectroscop

To start the enzymatic reaction, an aliquot of either Saos-2 cells (6 to 28 mg protein mL -1 ) or primary osteoblast (20 to 45 mg protein mL -1 ) was taken and mixed with the reaction medium containing 40-80 mM final PP i or one of other substrates (AMP, ADP, ATP, UTP, G-1-P, β-GP and p-NPP), 5 mM MgCl 2 and 5 µM ZnCl 2 in either DMEM (10% FBS) and 100 mM Tris-HCl buffer pH= 7.8. Three µL of the reaction mixture containing either Saos-2 cells or primary osteoblast cells were taken and deposited between two BaF 2 windows of a demountable thermostated cell (model Harrick) separated with a 12 µm (for IR spectra of substrates) or 6 µm (for IR spectra of cells with substrates) Teflon spacer. Tissue non-specific alkaline phosphatase activity was assayed at 37 °C by recording absorbance from 1300 to 900 cm -1 wavenumbers. Protein concentration was measured with the amide-II band using ε amide II = 3.6 mg -1 ml cm -1 . PP i band located at 1107 cm -1 (Molar absorption coefficient = 2158 ± 211 M -1 .cm -1 ) and P i bands located at 1076 cm -1 (Molar absorption coefficient = 1346 ± 116 M - the substrate and the product concentrations. IR data were acquired with a Thermo Scientific Nicolet iS10 spectrometer equipped with a DTGS detector as specified above.

Statistics

All data are presented as mean values ± SEM (standard error of the mean). Differences between groups were assessed using the Mann-Whitney test, depending on their normal distribution. Results were considered statistically significant at p-values <0.05 indicated by (*) while p<0.01 is indicated by (**) in all figures.

CHAPTER IV

RESULTS and DISCUSSION 1 Effects of Azanitrile Cathepsin K Inhibitors on Mineralization and Bone

Resorption

Cytotoxicity of CKI-8 and CKI-13 on cells (colorimetric MTT assay for cell viability)

The cytotoxicity of CKI-8 and CKI-13 (Scheme 1) were tested on three distinct types of cells: human osteoblast-like Saos-2, the mouse monocyte cell line RAW 264.7 and the mature mouse primary osteoclast cells, using a MTT assay [START_REF] Mosmann | Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays[END_REF]. These tests were performed after the cells had been incubated without (control) or with the compounds for 3 days. CKI-8 and CKI-13 were not toxic in the concentration range of 0.1 to 1000 nM on osteoblast-like Saos-2 cells (Fig. 4a) and on RAW 264.7 cells (Fig. 4b). On mature mouse primary osteoclasts, there was no toxicity up to 100nM, while at 1000 nM, less than 40-60 % cells were viable (Fig. 4c). On the other hand, the commercial inhibitor E64

was not toxic throughout this concentration range (Fig. 4c). The addition of 0.1 % (mL/mL) DMSO used to solubilize the inhibitors did not affect cell toxicity.

Differentiation and mineralization of osteoblast-like Saos-2 cells and primary mouse calvarial osteoblasts

To check whether CKI-8 and CKI-13 could inhibit the mineralization process, human Saos-2 cells and mouse primary osteoblasts were selected as cell models to monitor mineralization. To stimulate mineralization, AA and β-GP, as osteogenic factors, were added [START_REF] Gillette | The role of annexin 2 in osteoblastic mineralization[END_REF][START_REF] Vaingankar | Subcellular targeting and function of osteoblast nucleotide pyrophosphatase/phosphodiesterase 1[END_REF][START_REF] Stanford | Rapidly forming apatitic mineral in an osteoblastic cell line[END_REF]. The extent of AR-S staining was measured to quantify calcium nodules (Fig. 5a andb). As reported earlier [START_REF] Mosmann | Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays[END_REF], mineralization induced by Saos-2 cells was strongly enhanced by the combined addition of AA and β-GP in the cell cultures (Fig. 5b). We determined the effects of the inhibitors CKI-8 and CK-13 on the mineralization process induced by Saos-2 cells after stimulation (Fig 5c andd) and on primary osteoblasts (Fig 5e andf). As shown by AR-S staining, CKI-13 significantly inhibited mineralization with Saos-2 cells at 10 and 100 nM concentrations to around 60 % of control, while CKI-8 did not inhibit significantly, although there were slight variations (Fig. 5c). We then measured the effect of CKI-8 and CKI-13 on the activity of tissue-non-specific alkaline phosphatase (TNAP), a mineralization marker [START_REF] Cyboron | Purification and initial characterization of intrinsic membrane-bound alkaline phosphatase from chicken epiphyseal cartilage[END_REF], in cells. CKI-13 reduced TNAP activity significantly on Saos-2 cells at 1000 nM to 80 % of control (Fig. 5d), suggesting that CKI-13 can slow down TNAP activity and mineralization. We determined the effects of both CKI-8 and CK-13 at concentrations ranging from 100 to 1000 nM in primary osteoblasts. AR-S staining did not indicate any significant effect of either CKI-8 or CK-13 on mineralization induced by primary cells (Fig. 5e). However, TNAP activity decreased from 100% (control) to 58 % of control using 100 nM CK-13 (Fig. 5f). In contrast, CKI-8 did not inhibit TNAP significantly, consistent with the AR-S staining results. In all cases, the addition of 0.1 % (v/v) DMSO to solubilize the inhibitors did not affect calcium nodule formation and TNAP activity. CKI-13 could inhibit TNAP activity slightly in murine osteoblasts, as well as in human Saos-2 cells accompanied by a decrease in mineralization in murine osteoblasts but not in human Saos-2 cells.

Gelatin zymography assay on cathepsin K activity in RAW 264.7 cells

As CKI-8 and CKI-13 inhibited human recombinant CatK in vitro [43], we wanted to verify whether they could inhibit mouse CatK from RAW 264.7 and from primary murine mature osteoclasts since we are using these cells. Recombinant human CatK was used as a control (600 ng) and as a protein marker in zymography. Cellular proteins (300 ng) including CatK were extracted from RAW 264.7 cells or primary murine mature osteoclasts.

Recombinant CatK and extracted cell proteins were then loaded onto 12.5% SDSpolyacrylamide gels containing 0.2 % (g/mL) gelatin at 4 °C for zymography. Human CatK (Fig. 6a, left lanes) migrated at the same position as CatK in RAW 264.7 cell extracts on the zymography gel (Fig. 6a, right lanes). Gels were then pre-incubated with CKI-8 (Fig. 6a, top lanes) and CKI-13 (Fig. 6a, bottom lanes), respectively, at concentration ranging from 5 to 1000 nM. At 1000 nM, both inhibitors were able to completely block the gelatinolytic activity of human CatK (Fig. 6a, left lanes) or mouse CatK (Fig. 6a, right lanes). We also observed a slight inhibition of gelatinolytic activity of CatK in extracts of primary osteoclasts (Fig. 6b), albeit less pronounced than observed for RAW 264.7 cells or human CatK (Fig. 6a). From the results of gelatin zymography, it is possible to calculate the apparent IC 50 of inhibition of CKI-8 and CKI-13 by human CatK (Fig. 6c andd). A value of IC 50 = 29 ± 11 nM was obtained for CKI-13, and a value of IC 50 = 51 ± 20 nM was obtained for CKI-8.

The addition of 0.1 % (mL/mL) DMSO used to solubilize the inhibitors did not affect the inhibition. These IC 50 values were higher than those reported from in vitro enzymatic assays based on a synthetic dipepdide containg fluorescent labeling [43]. On the other hand, the IC 50 of CKI-13 is lower than that of CKI-8, being consistent with a previous in vitro report [42].

Assessment of bone resorption index on bone model matrix 1. Osteo corning plate assay

Osteoclastogenesis was induced using osteoclasts from murine bone marrow on Osteo Assay plates mimicking the bone surface, as well as on bovine bone slices. Typical mineralized matrix resorption by primary osteoclasts on Osteo Assay plates are shown, incubated in the absence (Fig. 7a) or presence of 100 nM CKI-13 (Fig. 7b) for 48 hours.

Incubation with CKI-13 at 100 nM significantly decreased resorption of this mineralized surface. The effects of CKI on resorption on Osteo Assay plates were quantified by comparing the resorption index in the control sample with 0.1 % DMSO (mL/mL) with that obtained by osteoclasts in the presence of CKI-8 and CKI-13 in 0.1 % DMSO (mL/mL). The presence or absence of DMSO at concentrations of up to 0.1 % (mL/mL) did not affect the resorption index on Osteo Assay plates by primary osteoclasts. CKI-13 at 100 nM decreased significantly the resorption index of primary osteoclasts (Fig. 7c), while the commercial inhibitor E64 and CKI-8 did not (Fig. 7c). CKI-8, CKI-13 and E64 at 10 nM tend to increase the resorption. This is probably due to an acidic secretion by osteoclast, dissolving hydroxyapatite crystals. The Corning Osteo Assay surface does not contain any protein and consists of an inorganic crystalline calcium phosphate, mostly hydroxyapatite as probed by infrared spectroscopy (Fig. 7d) coated on a polystyrene plate [START_REF] Pathi | A novel 3-D mineralized tumor model to study breast cancer bone metastasis[END_REF]. respectively. Results are expressed as percentage ofcontrol containing DMSO 0.1% (mL/mL) without inhibitor. DMSO 0.1 % (mL/mL) has no effect on the resorption activity. The experiments were repeated in triplicate for each inhibitor (n=6). d) The composition of osteoassay plate was identified by infrared spectroscopy. The infrared spectrum exhibited four peaks at 1030 cm -1 , 960 cm -1 , 600 cm -1 and 560 cm -1 , corresponding to the peaks of HA.

Bovine bone slice assay

Since E64, CKI-8 and CKI-13 are inhibitors of CatK acting on collagen, we needed to confirm their anti-resorption properties on bone slices. Furthermore, there are differences in the way that osteoclasts act on the hydrated mineral interspaced with extracellular matrix proteins, as in bovine bone slices, compared with dissolving a continuous microcrystalline surface, as in Corning Osteo Assay plate. Therefore we evaluated the effects of CKI-8 and CKI-13 on osteoclast resorbing bovine bone slices. The results showed that CKI-13 at 10 and 100 nM, while CKI-8 and E64 both at 100nM decreased resorption significantly by primary osteoclasts on bovine bone slices (Fig. 8a). Moreover, the resorption lacunae on bovine bone slices in the presence of CKI-8 (Fig. 8d) and CKI-13 (Fig. 8e) were clearly smaller than the control (Fig. 8b) or the E64-treated bone slices (Fig. 8c). Significant difference in the mean area of resorption pits was observed between CKI-8, CKI-13 and E64 at 100nM (Fig. 8f).

These differences in pit morphology could be due to CKI-8 and CKI-13 slowing down osteoclast spreading or motility.

DISCUSSION

We report the properties of a series of cathepsin K, B, L and S inhibitors, among these CKI-13 having an IC 50 = 0.006 nM and being highly selective for CatK over B and S (~1000fold) [43]. As these inhibitors were selective under in vitro conditions using human recombinant CatK as a target enzyme, zymography was used to ascertain their inhibition properties on mouse CatK. Both CKI-8 and CKI-13 inhibited CatK in extracts of mouse monocyte-derived macrophage RAW 264.7 cells and from primary osteoclasts from mouse calvaria at higher concentrations than expected from in vitro values [43]. The IC 50 values determined from zymography of CKI-8 (51 ± 20 nM) and of CKI-13 (29 ± 11 nM) were higher than those of CKI-8 (0.263 nM) and CKI-13 (0.006 nM) acting on human CatK determined using the colorimetric method on dipeptides containing fluorophore [43]. This discrepancy is probably due to the nature of the substrate: dipeptides and gelatin are different in size and structure, affecting their accessibility to the active site of catK, as well as the inhibitor's binding site. Alternatively gel zymography can over estimate the apparent IC 50 due to the long exposure of inhibitors in the gel since the inhibitors may not stop completely the gelatinolytic activity.

To validate the feasibility of the possible therapeutic use of the candidate inhibitors prior to preclinical trials, we tested their toxicities, their effects on the mineralization process induced by osteoblasts and their influence on resorption mediated by osteoclasts. CKI-8 and CKI-13 showed no toxicity up to 1000 nM on osteoblast-like Saos-2 cells and on osteoclast RAW 264.7 cells. However, they were toxic when their concentrations exceeded 100 nM in primary osteoclasts. This suggested that the maximum concentration in cells without producing any cytoxicity was around 100 nM for the candidate molecules. CKIs cell treatments were further verified for a possible side effect on osteoblast-induced mineralization. Neither CKI-8 nor CKI-13 affected the mineralization process induced by primary osteoblasts from mouse calvaria although a slight inhibition of TNAP activity by CKI-13 was observed. CKI-13 slowed down significantly the mineralization process induced by the Saos-2 cell line, as well as inhibiting slightly the TNAP activity. The different effects on mineralization of CKI-13 on both types of cells are probably due to their distinct maturation times, as well as their mouse vs human origin.

Direct determination of pyrophosphate hydrolysis in Matrix Vesicles by

Infrared Spectroscopy

Characterization of matrix vesicles

MVs were isolated from femurs of 17-day-old chicken embryos. To check the purity of matrix vesicles, we analyzed the SDS-PAGE of the distinct fractions during differential centrifugation (Fig. 2) After a first centrifugation at 600 x g to discard cellular debris, pellet 1 (P1) was extracted, then the supernatant 1 (SN1) was subjected to a second centrifugation at 20 000 x g where pellet 2 (P2) was extracted, supernatant 2 (SN2) was subjected to a third centrifugation at 80 000 x g to obtain supernatant 3 (SN3) and MVs in pellet 3 (P3). P1, P2, SN1, SN3 and P3 were analyzed by SDS-PAGE (Fig. 9). MVs are enriched in three bands with apparent molecular masses of 45, 37, and 31 kDa (Fig. 9), consistent with the finding of Wu et al. [START_REF] Wu | Characterization of the nucleational core complex responsible for mineral induction by growth plate cartilage matrix vesicles[END_REF]. Since TNAP is a marker of mineralization process, we determined its activity in different fractions P1, P2, SN1, SN3 and P3 during differential centrifugation (Fig. 10). The specific activity of pellet 3 in the TNAP is 19.61 U/mg at pH 10.4 (Table 3), which is more than two times higher in other chondrocyte membranes. The results indicated an enriched amount of TNAP characteristic of MVs.

Fractions

Protein

Concentration

(µg/µL) The ability of isolated MVs to induce mineral formation was monitored by light scattering at 340 nm. MVs in the SCL mineralization medium containing 2mM Ca 2+ (total concentration) and 3.42mM P i (total concentration) induced mineralization after a lag period of about 6 h when P i and Ca 2+ accumulated within MVs. The absorbance at 340 nm increased rapidly after Minerals formed in the presence of MVs were analyzed by IR spectroscopy and compared with IR spectra of HA. The mineral formed under the same conditions for the turbidity measurements were determined by IR assay (Fig. 12). The positions of bands in the infrared spectrum at 1090 cm -1 , 1020 cm -1 , 962 cm -1 , 633 cm -1 , and 602 cm -1 (Figure 12. HA), corresponding to mineral formed in the MVs, matched almost exactly the positions of bands in the infrared spectrum of HA (Fig. 12. MVs), indicating the ability of MVs to produce HA.

Minor variations around 1020 cm -1 are associated to the presence of MVs membrane phospholipids. These results confirmed that MVs are functional and able to produce HA. Having demonstrated that P3 contains enriched amount of MVs as revealed by SDS-PAGE (Fig. 9) and by TNAP activity (Fig. 10) in addition to their functional property as indicated by turbidity measurements (Fig. 11) and their ability to form HA as shown by infrared spectroscopy (Fig. 12), P3 sample served as a source of MVs.

Development of an IR assay to determine TNAP enzymatic activity in MVs

Up to now, TNAP activity is measured at non-physiological alkaline pH (10.4) using a non-natural substrate para-nitrophenyl phosphate (p-NPP). To screen inhibitors, it is necessary to develop an assay using natural substrates in turbid medium at physiological pH. Another problem often encountered with our types of inhibitors is that they contain aromatic groups which can interfere with the band absorption of nitrophenolate rending the determination of TNAP activity difficult by using colorimetric method. Therefore we developed an infrared (IR) assay that may solve the problems encountered with the hydrolysis of p-NPP. IR spectroscopy will allow the use of a natural substrate pyrophosphate (PP i ) at physiological pH 8. In addition the activity measurement will not be affected by colored or turbid medium. Hydrolysis of PP i by TNAP released inorganic phosphate (P i ) [44]. The IR spectrum of HPO 3 2-anion in aqueous buffer containing 50 mM Tris-HCl, pH 8.0, 5 µM ZnCl 2 and 5 mM MgCl 2 indicates two bands at around 1076 and 990 cm -1 (Fig. 13) that are assigned respectively to asymmetric stretching and symmetric stretching vibrations [START_REF] Tsuboi | Vibrational spectra of phosphite and hypophosphite anions and the 24 characteristic frequencies of PO3--and PO2--groups[END_REF]. The position of the phosphate bands is sensitive to their ionic environment as in the case of nucleotides [START_REF] Wang | Raman difference studies of GDP and GTP binding to c-Harvey ras[END_REF] or phospholipids [START_REF] Yamamoto | Regulation of phospholipase D activity by neutral lipids in egg-yolk phosphatidylcholine small unilamellar vesicles and by calcium ion in aqueous medium[END_REF]. The IR spectrum of 50 mM PP i shows one band located at around 1107 cm -1 (Fig. 13). The IR spectra of P i and of PP i present sufficient differences in band shapes and positions (Fig. 13) to be used for analytical application. To illustrate the potential of the enzymatic assay, we measured the hydrolytic activity of MVs towards PP i in reaction medium containing 5 µM ZnCl 2 and 5 mM MgCl 2 , 100 mM Tris-HCL, pH 8.0. IR spectra of MVs in reaction medium after 5 min and after 60 min (Fig. 14a) incubation time at 37°C have been recorded. As a control IR spectra of MVs in reaction medium without PPi have been measured after 5 min and after 60 min indicating no changes (data not shown). The 1300 -900 cm -1 wavenumber ranges was selected to reveal the PP i hydrolysis (Fig. 14 a). Difference IR spectra of MVs with PPi (spectrum recorded at the indicated time minus that recorded immediately). Significant variation in the intensity of absorption bands of PP i located at 1120-1100 cm -1 as well as other variations attributed to P i at 1075-1080 cm -1 and 990 cm -1 were observed. (c) Expected IR difference spectrum of product and substrate (2 x P i minus PP i ) (dashed line). IR difference spectrum of the hydrolysis of 50 mM PP i by 2.5 mg MV protein mL -1 in buffer A (spectrum recorded at 60 min minus spectrum recorded immediately) (Full line) (d) Sensitivity of the infrared assay to determine the hydrolysis of 50 mM PP i by 2.5 mg MV protein mL -1 (full line) and by 0.2 mg MV protein mL -1 (dashed line) as shown by their difference spectrum respectively (spectrum of MVs with 50 mM PP i recorded at 30 min minus spectrum recorded immediately).

The IR spectra of MVs in the reactive medium did not indicate any changes in the 4000-1400 cm -1 region of the IR spectrum. In the 1400-900-cm -1 range, the IR spectral changes are dominated by the hydrolysis of PP i by MVs due to the relative large amount of PP i (Fig. 14a).

The absorbance decrease of PP i band at 1100-1107 cm -1 (Fig. 14a) is associated to a decrease of PP i , while the increase of the 1076-1080-cm -1 and 990-cm -1 bands signaled the apparition of P i . This is better seen on the difference spectra of MVs with PP i (Fig. 14b) (spectrum recorded at the indicated time minus that recorded immediately). The negative band at 1100-1120 cm -1 and the two positive bands located at 1076-1080cm -1 and 990-cm -1 bands indicated the hydrolysis of PP i . This was confirmed by calculating the difference spectra corresponded to the hydrolysis (spectrum of two P i minus spectrum PP i since one molecule PP i gives to two molecules of P i (Fig. 14c, dashed line). The IR difference spectrum of MVs in reactive medium containing PP i (Spectrum of sample incubated for 60 min minus spectrum of sample incubated for 5 min) (Fig. 14c, full line) is identical to that of the calculated difference (Fig. 14c, dashed line). This confirms that the IR changes are solely related to the hydrolysis of PP i by MVs. The limit to detect significant infrared changes of the hydrolysis of PP i was estimated to be around 0.2 mg MV-proteins mL -1 (Fig. 14d, dashed line). For comparison, the hydrolysis of PP i by 2.5 mg MV-proteins mL -1 induced about ten times larger infrared changes (Fig. 14d, full line). At 0.2 mg mL -1 MV protein, only faint bands not much more intensive than the noise signal are detected (Figure 14. d, dashed line), while at 2.5 mg mL -1

MVs protein, the IR bands indicating disappearance of PPi and appearance of Pi are well resolved (Figure 14. d, full line).

We selected the PP i band (located at 1100-1120 cm -1 ) and the two P i bands (located at 1076-1080-cm -1 and 990-cm -1 ) to determine the hydrolytic activity of PP i . by MVs (Fig. 15). We can determine the concentration changes during PP i hydrolysis by MVs and determine activity of PP i hydrolysis by MVs. Using the 1107 cm -1 bands we calculated an activity of around 1.367-1.83 μmole mg -1 min -1 for MV-protein concentration range of 1 to 2.5 mg mL -1 (Table 4). For comparison, the TNAP activity of MVs determined at pH 10.4 using the p-NPP as a substrate, amounted to 30 ± 10 μmole mg -1 min -1 . This is consistent with the reported values for alkaline phosphatase extracted from HeLa Ch cells, having a PP i hydrolytic activity at pH 8.5 about 26 to 30 times smaller than the pNPP hydrolytic activity at pH 10.4 [START_REF] Cox | Alkaline Inorganic Pyrophosphatase Activity of Mammalian-Cell Alkaline Phosphatase[END_REF].

To test if the hydrolysis of PP i is specifically associated to TNAP, we incubated the reactive medium containing MVs and 50 mM PP i with 5 mM levamisole, a TNAP specific inhibitor. Addition of levamisole induced a smaller slope in the absorbance changes at 1107 cm -1 (Figure 16.a red symbols), at 990 cm -1 (Figure 16. b red symbols) and at 1076 cm -1 (Figure 16. c, red symbols) as compared with the control sample without levamisole (Figure 16. blue symbols). We observed a significant inhibition of PP i hydrolysis in the presence of 5 mM levamisole from 1132 ± 370 nmol min -1 mg -1 (without levamisole) to 343 ± 112 nmol min -1 mg -1 (with 5 mM Levamisole) confirming that TNAP is to a large extent responsible for PP i hydrolysis. Absorption changes of Pi band at 1076 cm -1 during PPi hydrolysis by 2 mg mL -1 MVs in buffer A at 37 °C in the absence (blue symbols) or presence (red symbols) of 5 mM levamisole.

DISCUSSION

It has been recognized that IR spectroscopy in combination with the appropriate multivariate analysis has considerable potential as a metabolic fingerprinting tool for the diagnosis of disease or dysfunction [START_REF] Ellis | Metabolic fingerprinting in disease diagnosis: biomedical applications of infrared and Raman spectroscopy[END_REF][START_REF] Ellis | Metabolomics-assisted synthetic biology[END_REF] and can assess biochemical changes of cells exposed to various inhibitors such as cardiotoxic glycoside inhibitor ouabain [START_REF] Gasper | FTIR spectral signature of the effect of cardiotonic steroids with antitumoral properties on a prostate cancer cell line[END_REF]122], tyrosine inhibitor imatinib [START_REF] Baran | The roles of macromolecules in imatinib resistance of chronic myeloid leukemia cells by Fourier transform infrared spectroscopy[END_REF] or AKT kinase inhibitor [START_REF] Travo | Basis of a FTIR spectroscopy methodology for automated evaluation of Akt kinase inhibitor on leukemic cell lines used as model[END_REF]. It is quite rare to find an infrared assay to monitor directly one specific activity in the extracellular vesicles, cells or tissues which can be used to screen inhibitors. In this work, we purified matrix vesicles derived from chicken embryo chondrocytes and we verified their characteristics. They are able to initiate mineralization and they have a high activity in TNAP. Matrix vesicles were selected as model of extracellular vesicles due to their high alkaline phosphatase activity. In addition, inhibitors of TNAP may serve as a drug therapy to cure vascular calcification. The TNAP highly expressed in mineralized tissues , and essential for the mineralization of the bone, is an important therapeutic target for the treatment of pathological calcifications.

Diseases associated with calcifications belong to a large family of diseases that can be caused not only in skeletal tissues but also in non-skeletal tissues such as vascular calcification tissues.

The aim of our work was to develop an assay of the enzymatic activity in the hydrolysis TNAP as PP i a natural substrate for MVs activity assay. We demonstrate the feasibility of the infrared assay by measuring the TNAP activity in MVs that was around 1.4 μmol min -1 mg -1 , while we observed a significant inhibition of PP i hydrolysis in the presence of 5 mM levamisole (a TNAP inhibitor) from 343 ± 112 nmol min -1 mg -1 confirming that the pyrophosphatase activity originated mostly from TNAP. The main advantages of the IR assay lie in the possibility to determine directly in situ hydrolytic activity of TNAP and to obtain IC 50 of inhibitors using natural substrates in an almost continuous manner at physiological pH.

In this respect, IR is insensitive to light scattering effects caused by the turbidity of the samples and relatively large concentrations of protein can be employed. However, for the IR assay, this technique presents some limitations related to the accuracy of determination of the base line but also in the thickness of the spacer. We estimated that the sensitivity based on a 30-min assay is around 0.2 mg cell proteins mL -1 in a 5-10 μL sample containing 50 mM PP i .

Lower protein concentration could be used with longer incubation times. The other limitation concerns the minimal amount of P i or PP i concentrations which should be at least around 1 mM to be reliabily detected.

An infrared assay of the kinetics of phosphate-release from physiological substrates in living cells

Infrared spectra of different substrates

Having demonstrated that the hydrolysis of pyrophosphate in matrix vesicles can be measured by infrared spectroscopy, we wanted to explore if other types of substrates could serve to To determine the molar absorption coefficient, we used the Beer-Lambert law : A=ε•l•C.

Where A is the absorbance, ε is the molar absorption coefficient (M -1 cm -1 ), l is the path length (cm), and C is the protein concentration (M). Table 5 contained the values of molar absorption coefficients for P i (ε Pi = 1346 ± 116 M -1 .cm -1 for the 1076-cm -1 band and ε Pi = 493 ± 49 M -1 .cm -1 for the 990 cm -1 band) and for other substrates: AMP, ADP, ATP, UTP, glucose-1-phosphate (G-1-P), β-glycerophosphate (β-GP), p-nitrophenylphosphate (p-NPP), PP i .

Table 5. Molar absorbance of different physiological substrates. ε value= mean ± mean deviation and the results were based on three independent measurements.

Substrates

Band position

cm -1 Molar Absorbance L•mol -1 •cm -1 Substrates Band position cm -1 Molar Absorbance L•mol -1 •cm

Determination of protein concentration

The determination of protein concentration by infrared spectra was performed using NISTcertified BSA solution in buffer A. A series of five dilutions (in triplicates) spanning the range from 5 mg/mL to 20 mg/mL served to obtain an accurate calibration curve (Fig. 18). We obtained a mean molar absorption coefficient of the amide-II band: ε amide II =3.6 mg -1 ml cm -1 . The amide-I band intensity is slightly affected by the intensity of water band (especially at low protein concentration) due to the subtraction factor for the water band (Fig. 18). Therefore the amide-II band served to determine the protein concentration since this band is outside the absorption of water band.

IR assay to determine TNAP enzymatic activity in Saos-2 cells

To illustrate the potential of the IR assay in assessing phosphatase activity in cells, we selected human osteoblast-like Saos-2 cells which are known for their high TNAP activity.

These cells express the entire osteoblastic differentiation program from proliferation to mineralization [START_REF] Hausser | Low doses and high doses of heparin have different effects on osteoblast-like Saos-2 cells in vitro[END_REF], produce a collagenous extracellular matrix [START_REF] Mcquillan | Matrix deposition by a calcifying human osteogenic sarcoma cell line (Saos-2)[END_REF] and spontaneously release mineralization-competent MVs [START_REF] Fedde | Human osteosarcoma cells spontaneously release matrix-vesicle-like structures with the capacity to mineralize[END_REF]. We measured IR spectrum immediately ( (positive 1088-1080 cm -1 and 990-cm -1 bands) and AMP (positive 977 cm -1 band), however it was not possible to ascertain the formation of PP i since there were no significant positive 1107cm -1 PP i band. We also observed hydrolysis of G-1P, β-GP and pNPP and PP i in Saos-2 cells indicating a broad phosphatase activity.

The feasibility to monitor a phosphatase activity by Saos-2 cells in buffer A was verified. Now, we want to demonstrate the possibility to determine protein concentration using the amide-II band so that we could determine a specific phosphatase activity in one single assay. That is to determine protein concentration in cells and the phosphatase activity simultaneously. Typical IR spectra of 50 mM PP i with Saos-2 cells in buffer A (Fig. 21) immediately and after mixing 30 min incubation (full trace) indicated the decrease of the 1100 cm -1 band of PP i and the increase of the 1080 cm -1 band of P i , confirming the presence of pyrophosphatase activity. Furthermore, the Table 6. Specific of phosphatase activity (nmol mg -1 min -1 ) of different substrates in Saos-2 cells as determined by using phosphate bands (990 and 1080-1070 cm -1 ) and other substrates bands. Buffer A has the disadvantage of inducing cell aggregation after several hours incubation but its absorption in the 1200-1000 cm -1 (Fig. 22) region is lower than that of DMEM (cellular medium) and its composition can be controlled. measured immediately after mixing PP i with cells) recorded for Saos-2 cells in buffer A (Fig. 24A) and in DMEM medium (Fig. 24B) which were higher than that those of primary osteoblasts in buffer A (Fig. 24C) and in DMEM (Fig. 24D) respectively. Infrared spectra indicated both of the disappearance of the 1100-1120-cm -1 band of PP i and the appearance of the 1076-1080-cm -1 and 990-cm -1 bands of P i , confirming the presence of a pyrophosphatase activity.

Substrates

The kinetic parameters are easily obtained from IR spectra. The absorbance changes of the 1120-1100-cm -1 band of PP i (Fig. 25a) and the 990 cm -1 band of P i (Fig. 25b) served to determine the pyrophosphatase activity and the amide-II band allowed us to determine protein concentration. As expected, increasing the amount of Saos-2 cells, from 7.5 mg mL -1 , 12 mg mL -1 to 18 mg mL -1 as determined by the amide-II protein band, increased the pyrophosphatase activity (expressed as nmol min -1 ) but the specific activity remained at the level of 116 ± 13 nmol min -1 mg -1 as expected (Fig. 25c ). To check if the pyrophosphatase activity originates from TNAPenriched cells, levamisole (a TNAP inhibitor) was added to the medium. At the concentrations of 5 mM (Fig. 25d), levamisole almost completely inhibited the PP i hydrolysis by Saos-2 cells (IC 50 = 1.16 ± 0.03 mM), suggesting TNAP contributed mostly to the pyrophosphatase activity of Saos-2 cells. The IC 50 value of 1.16 ± 0.03 mM is comparable with the reported concentration of 1 mM levamisole that blocked calcification in cultures of aortas from uremic rats [START_REF] Lomashvili | Upregulation of alkaline phosphatase and pyrophosphate hydrolysis: potential mechanism for uremic vascular calcification[END_REF].

DISCUSSION

Enzymes, which release phosphate (P i ), are among one of the most diverse sets of proteins with respect to the reactions they catalyze [START_REF] Pegan | A Universal, Fully Automated High Throughput Screening Assay for Pyrophosphate and Phosphate Release from Enzymatic Reactions[END_REF]. Among the enzymes having a phosphatase activity and releasing P i , tissue non-specific alkaline phosphatase (TNAP) performs indispensable, multisystemic functions in humans [48,[START_REF] Buchet | Multisystemic functions of alkaline phosphatases[END_REF].

TNAP is thus today recognized as an attractive drug target for the treatment of vascular calcification. [START_REF] Mebarek | Ankylosing spondylitis, late osteoarthritis, vascular calcification, chondrocalcinosis and pseudo gout: toward a possible drug therapy[END_REF][START_REF] Dahl | Discovery and validation of a series of aryl sulfonamides as selective inhibitors of tissuenonspecific alkaline phosphatase (TNAP)[END_REF]. The screening of TNAP inhibitors has relied on the determination of the recombinant TNAP activity at alkaline pH using para-nitrophenylphosphate (p-NPP). A 1,000fold more sensitive and 10-fold faster than the p-NPP assay has been developed with p-NPP dioxetane-based substrate as TNAP substrate and with recombinant TNAP [START_REF] Sergienko | High-throughput screening of tissue-nonspecific alkaline phosphatase for identification of effectors with diverse modes of action[END_REF]. However, TNAP inhibitors selected under such conditions may act differently on the living cells.

Therefore, there is a strong demand for flexible, fast and cost-effective strategies to select inhibitors with improved prospects for clinical success [START_REF] Bleicher | Hit and lead generation: beyond highthroughput screening[END_REF].

Here we demonstrated the ability of IR spectroscopy to directly determine in situ a phosphatase activity in osteoblasts using natural substrates without any labeling. We showed the ability of IR spectroscopy to directly determine simultaneously protein concentration and the phosphatase activity in in matrix vesicles as well in cultured osteoblasts using physiological substrates such as AMP, ADP, ATP, UTP, and PP i . The method has been validated for osteoblasts (Saos-2 cells and primary osteoblasts) and chondrocyte-derived matrix vesicles which are all TNAP enriched. This approach could be extended to determine any type of phosphatase activity in other cells. It may serve as a metabolomics tool to characterize any types of phosphatase activities and to screen the phosphatase inhibitors in cells.

Effects of Cathepsin K inhibitors on bone resorption and mineralization

In China we developed a series of Cathepsin K, B, L and S inhibitors [42,43]. The best is CKI-13 having an IC 50 = 0.006 nM and is highly selective over Cathepsin B and S (~1000fold). I synthetized several inhibitors including CKI-8 and CKI-13 in China. In France, I examined the effects of these inhibitors on mineralization using human Saos-2 osteoblast-like cells and murine primary calvaria cells. Survival MTT tests on Saos-2 osteoblast-like, RAW264.7 cell and murine primary osteoclast have been performed in the presence of the inhibitors. The alizarin red staining and TNAP activity assay were performed to check the possible effects of inhibitors on mineralization. In addition, a gelatin zymography method was optimized to observe the effects of inhibitors on cathepsin K activity in osteoclastsderived extracellular medium, which would be served as a screening test of inhibitors before checking their effects on bone resorption. Bone resorption by RAW 264.7 cells and osteoclast primary cells were performed on the surface of an inorganic crystal coating surface (Corning Bone assay surface) and on bovine bone slices. Resorption pit area per osteoclast was measured to determine the effects of Cat K inhibitors. Taken together, the overall findings indicate that both CKI-8 and CKI-13 can inhibit cathepsin K in cellular medium at the notoxicity concentration ranges and can inhibit bone resorption, validating the proof of concept of the possible therapeutic use of the small molecules as cathepsin K inhibitors.

At this stage, several points need to be considered. 1) CKI-13 slowed down significantly the mineralization process induced by the Saos-2 cell line, as well as inhibiting slightly the TNAP activity, while it did not affect the mineralization process in primary cell lines. This indicates that further work is needed to optimize chemical structures to obtain the best candidate molecules having strong inhibition of bone resorption and having no effects on the mineralization. 2) We observed that our CKI-8 and CKI-13 treatments produced smaller pits when compared with the larger pits induced by E64 treatment. This is an important observation since this suggests that CKI-8 and CKI-13 can slow down the mobility of osteoclasts. Actually we do not know how these inhibitors can slow down the mobility of osteoclasts. 3) So far, all of our biological assays were performed on in vitro cellular models.

Preclinical trials on animal models are a necessity to validate the proof of concept of the use of cathepsin-K inhibitors as a drug treatment to cure osteoporosis.

Development of an IR assay to detect TNAP activity in matrix vesicles and living cells

One of the main problems for drug screening is that the small molecules selected during the drug screening tests fail often to pass the preclinical and clinical trials. This is not surprising considering the fact that most of the drug screening process use labeled substrates, which often are not natural substrates, sometimes under non physiological conditions and the tests are performed on recombinant enzymes and not on cells or tissues.

Therefore there is a necessity to develop cost-effective drug screening tests, which can mimic as well as possible the drug target in a biological system.

The main advantages of the IR assay lie in the possibility to determine directly in situ hydrolytic activity of phosphatase and to obtain IC 50 of inhibitors using natural substrates in an almost continuous manner at physiological pH and in a single assay (determination of protein concentration and enzymatic activity in one single measurement). In addition, we demonstrated that the IR assay could monitor a phosphatase activity in matrix vesicles and in the whole cells in cellular medium close to physiological conditions.

The main disadvantages of the IR assay lie in its low sensitivity, experimental errors can be as large than 20 % due to a difficulty to control the thickness of the cell, although ATR could solve partly this problem. There is a limit in detection, which is around 1 mM of substrate (phosphate compounds). It is not possible to determine reliably the concentration of substrate or product below this concentration. However, the IR assay is well adapted to determine Vmax as a kinetic parameter due to saturate concentrations of substrate or IC 50 . Another problem is the sample-to-sample reproducibility; especially when the cells have variable activities and maturation degrees. This necessitates to prepare the same types of cells under the same conditions for screening the small molecules, which limitates the numbers of molecules to be tested.

Nevertheless, despite these difficulties, the IR assay can serve as a metabolic tool to evaluate the phosphatase activity in the cells. Plusieurs de ces traitements ont des inconvénients soit au niveau de leur efficacité générale, de leur mode d'application ou de leurs effets secondaires. C'est pourquoi, il y a un besoin urgent de développer de traitements plus efficaces et la demande en médicaments sûrs pour le traitement de l'ostéoporose demeure élevée [7].

Un des traitements de l'ostéoporose les plus prometteurs est un traitement par voie orale d'inhibiteur de cathepsine K (CKI) permettant de ralentir la résorption osseuse [8,9]. Des efforts considérables ont été effectués dans le développement des inhibiteurs hautement sélectifs [10]. Cinq CKIs, Balicatib, Relacatib, Odanacatib (ODN) et MIV-711 ont été évalués comme traitements thérapeutiques de l'ostéoporose [11,12]. MIV-711 a déjà passé avec succès la phase I en recherches cliniques pour le traitement de l'arthrose ainsi que pour ceux d'autres maladies osseuses [13]. Seul l'ODN a passé avec succès la phase clinique III [14,15]. Bien que ces molécules sont des agents anti-résorption, certains des composés ont des effets secondaires [16,13]. C'est pourquoi le développement d'autres CKIs se poursuit encore, une attention particulière est portée sur les azanitriles [37][38][39][40]. Tout récemment, nous avons développé des CKIs dont le CKI-13, IC 50 = 0.006 nM (schéma 1) ayant une très bonne sélectivité et un Ki de l'ordre du pico molaire [17,18]. La minéralisation est fortement stimulée en ajoutant les deux facteurs ostéogéniques l'acide ascorbique (AA) et le béta-glycéro-phosphate (Fig. 2b) comme attendu [22]. CKI-8 n'affecte pas l'activité de phosphatase alcaline (Fig. 2d) ainsi que la minéralisation induite par les Saos-2 (Fig. 2c) et par les ostéoblastes primaires (Fig. 2f). CKI-13 diminue de 35 % la minéralisation induite par les Saos-2 (Fig. 2c) tandis qu'il n'affecte pas la minéralisation induite par les osteoblastes primaires (Fig. 2f). La résorption osseuse sur des tranches d'os d'origine bovine est diminuée avec 10 nM de CKI-13, 100 nM de CKI-8 et l'inhibiteur commercial E64 (Fig. 3). CKI-8 et CKI-13 diminuent la mobilité des ostéoclastes. 1. Développement de mesure de l'activité de la phosphatase alcaline dans les vésicules matricielles et dans les ostéoblastes au moyen de la spectroscopie IR La phosphatase alcaline de tissue non-spécifique (TNAP) est exprimée fortement dans l'os, le foie et les reins [23]. Il y a trois enzymes clefs dans la régulation du pyrophosphate extracellulaire (ePP i ), qui est le substrat naturel de la TNAP dans le tissu osseux [24,25]. La première: l'enzyme ankylosante progressive (ANK) est un transporteur de PP i et la seconde est l'enzyme nucléotide extracellaire pyrophosphatase/phosphodiesterase-1 (NPP1) hydrolyse les nucléotides triphosphates en formant du PPi. ANK et NPP1 [26,27] contribuent à produire du ePP i tandis que la troisième enzyme TNAP hydrolyse l'ePP i [28]. L'ePP i est un inhibiteur de la formation d'hydroxyapatite (HA). Selon les conditions physiologiques, HA est formé dans l'os. Par contre de l'HA peut être accumulé dans les tissus non squelettiques selon les conditions pathologiques. La TNAP qui hydrolyse ePP i est considérée comme une enzyme antagoniste à ANK et NPP1. Le rôle de la phosphatase alcaline a été démontré dans le cas de l'hypophosphatasie chez les patients humains. Cette maladie est causée par une mutation de la TNAP qui conduit à une activité déficiente en TNAP [29,30]. Une forte concentration d' ePP i , inhibe la formation d'HA donnant lieu à une ostéomalacie ou à une mauvaise formation de l'os [30,31]. Dans le cas contraire, une forte activité de TNAP peut causer des calcifications pathologiques comme dans le cas des calcifications vasculaires. C'est pourquoi la TNAP, qui est considérée comme un marqueur de la formation osseuse, est une cible thérapeutique attractive pour le traitement des maladies associées aux calcifications vasculaires [32].

Actuellement la sélection des inhibiteurs de la TNAP s'effectue au moyen d'un substrat coloré : le para-nitrophenylphosphate (p-NPP) qui n'est pas un substrat naturel [33]. Les petites molécules sélectionnées de cette façon peuvent agir différemment dans les cellules osseuses. Il y a une forte nécessité de développer des stratégies de criblage des inhibiteurs qui se comportent le plus efficacement possible in vivo.

La plupart des mesures de dosage du ePP i sont basées sur les essais enzymatiques couplés dosant le NADPH [34], le NADH [35,36] ou par l'intermédiaire de la luciférase [37][38][39][40][41][42][43] ou en utilisant le 2-amino-6-mercapto-7-methylpurine [44,45] ou le luminol [46]. D'autres méthodes colorimétriques ont été développées avec du malachite green [47] des complexes avec des dérivés de coumarin contenant du Zn (II) et Cd (II) [48]. Aucune de ces méthodes ne permettent de doser simultanément la concentration de protéine, celle du substrat et celle du produit de la réaction, simultanément.

La spectroscopie IR est la seule technique permettant de suivre l'hydrolyse des substrats naturels de la TNAP et de mesurer la concentration de protéine en même temps. Les Nous avons développé un dosage d'hydrolyse de PP i par la phosphatase alcaline par IR, ayant l'avantage de fonctionner sur des vésicules matricielles et des cellules ostéoblastes avec des substrats naturels à un pH physiologique. Par exemple, la bande de PP i localisée à 1107 cm -1 (ε= 2158 ± 211 M -1 .cm -1 ) et celles de P i localisées à 1076-1080 cm -1 (ε= 1346 ± 116 M - 1 .cm -1 ) et à 990-991 cm -1 (ε= 493 ± 49 M -1 .cm -1 ) ont servis à mesurer les concentrations du substrat et du produit (Fig. 5). Parfois, il est difficile d'observer finement les changements produits au cours de l'hydrolyse des substrats sur les spectres IR. C'est pourquoi nous avons effectué des spectres de différence: soit spectre mesuré après un temps d'incubation moins spectre mesuré à temps =0 (Fig. 6).

Faculté

  Abbreviations

Figure 1 .

 1 Figure 1. Actions of osteoblasts and osteoclasts during the resorption and formation of bone

- 1 - 1 .

 11 oxopentan-2-yl)benzamide:3.56 g 3-(4-bromobenzamido)-5-methyl-2-oxohexanoic acid (10 mmol) was dissolved in THF(15 mL) and cooled to -25 °C. To the stirred solution, 1.11 mL N-methylmorpholine (10 mmol) and 1.32 mL isobutylchloroformate (10 mmol) were added consecutively. 2.66 g 1, 2dimethylhydrazine dihydrochloride (20 mmol) was dissolved in 5 mL H 2 O, and 5 M NaOH (8 mL) was added under ice-cooling. When the precipitation of N-methylmorpholine hydrochloride occurred this solution was added to the reaction mixture. It was allowed to warm to room temperature within 30 min and stirred for additional 90 min. After evaporation of the solvent, the resulting aqueous residue was extracted with ethyl acetate (1×40 mL, 3×10 mL). The combined organic layers were washed with H 2 O (1×15 mL), saturated NaHCO 3 twice (2×15 mL), H 2 O (1×15 mL) and brine (1×15 mL) in turn. The solvent was dried with Na 2 SO 4 and evaporated to obtain colorless oil. It was purified by column chromatography on silica gel with petroleum ether/ethyl acetate (1:1) as eluent to obtain 4-bromo-N-(1-(1, 2-dimethylhydrazinyl)-4-methyl-1-oxopentan-2-yl)benzamide as a colorless oil, which was then slowly solidified at room temperature. 23 g sodium acetate (15 mmol) and 1.58 g cyanogen bromide (15 mmol) were added to a MeOH solution (20 mL) containing 1.78g (5 mmol) 4-bromo-N-(1-(1,2-dimethylhydrazinyl)-4-methyl-1-oxopentan-2-yl)benzamide. The mixture was stirred at room temperature for 5 h and then the solvent was removed under reduced pressure. The residue was suspended in 10 mL H 2 O, a pH of 1-2 was adjusted by using 10% KHSO 4 , and then it was extracted with ethyl acetate for three times (3×20 mL). The combined organic layers were washed with H 2 O (1×10 mL), sat. NaHCO 3 (2×10 mL) and brine (1×10 mL) in turn. The solvent was dried by Na 2 SO 4 and removed in vacuo. The oily residue was purified by column chromatography on silica gel using petroleum ether / ethyl acetate (2:1) as eluent. The obtained oil was dried in a desiccator at room temperature for several days to obtain 4-bromo-N-(1-(2-cyano-1,2dimethylhydrazinyl)-4-methyl-1-oxopentan-2-yl)benzamide as a colorless solid.

1 H

 1 NMR (500 MHz, CDCl 3 ): δ 7.91 (d, J = 8.1 Hz, 2H), 7.76 -7.71 (m, 6H), 7.67 (d, J = 7.5 Hz, 2H), 7.50 (t, J = 7.5 Hz, 2H), 7.41 (t, J = 7.3 Hz, 1H), 6.61 (d, J = 8.4 Hz, 1H), 5.38 (td, J = 10.0, 3.8 Hz, 1H), 3.39 (s, 3H), 3.27 (s, 3H), 1.87 (s, 1H), 1.73 (ddd, J = 23.4, 21.8, 12.1

  were discarded and the supernatant was centrifuged at 20 000 g for 20 min at 4 °C. A second centrifugation at 80 000g for 60 min at 4°C was then performed. The supernatant was discarded and the pellet was gently washed with 1 mL Tris HCL (100 mM, pH 8.0) containing 5 µM ZnCl 2 and 5 mM MgCl 2 . Washing medium was discarded and the intact pellet (around 0.05 mL) was suspended in 0-2 mL Tris HCL (100 mM, pH 8.0) containing 5 µM ZnCl 2 and 5 mM MgCl 2 (called throughout buffer A). The protein concentration in MVs was determined by Bradfords[START_REF] Bradford | A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF] was in the range of 0.2 to 8 mg MV protein mL -1 . MVs were freshly prepared for the IR measurements.

Figure 2 .

 2 Figure 2. Steps of differential centrifugations for the in purification of matrix vesicles (MVs)

Figure 3 .

 3 Figure 3. Part of the infrared spectrum indicating Amide I and Amide II bands of proteins in cells.

Figure 4 .

 4 Figure 4. The two catK inhibitors are not cytotoxic. MTT test of CKI-8 and CKI-13 on a) osteoblast-like Saos-2 cells, b) RAW 264.7 and c) mature osteoclasts, respectively, at the indicated concentrations. After 3-day incubations with CKI-8 and CKI-13, the compounds did not induce any significant toxicity on Saos-2 and RAW 264.7 cells up to 1000 nM, while they were not toxic on mature osteoclasts up to 100 nM. For comparison, commercial E64 inhibitor was not toxic in primary osteoclast cells up to 1000 nM. Results are expressed as

Figure 5 .

 5 Figure 5. Effect of the two catK inhibitors on osteoblast-mediated mineralization. Saos-2 cells were incubated for 6 days without ((a) non-stimulated) or with 50 µg ml-1 AA and 7.5 mM β-GP ((b) stimulated). Both sets of cells were stained with AR-S to detect the calcium nodules (the nodules are shown with a white arrow) and then were photographed. Effects of CKI-8 and CKI-13 on the mineralization process induced by osteoblast-like Saos-2 cells and primary osteoblasts as monitored c and e) by AR-S staining and by d and f) TNAP activity at

Figure 6 .

 6 Figure 6. Gelatin zymography assay on catK activity. (a) Left lanes: Human recombinant catK (CatK) as positive control and right lanes: CatK extracted from RAW264.7 cells (OC), was loaded for gelatin zymography and incubated overnight in active buffer of 0.1 M sodium phosphate, pH 6.0, 1 mM EDTA and 2 mM DTT with CKI-8 and CKI-13 at the indicated concentrations. b) CatK (OC) extracted from primary osteoclasts was loaded for gelatin zymography and incubated overnight in the active buffer of 0.1 M sodium phosphate buffer, pH 6.0, 1 mM EDTA 0.1 % DMSO (mL/mL) and 2 mM DTT without inhibitors (Control) and with CKI-8, CKI-13 and E64 at the indicated concentrations. DMSO 0.1 % (mL/mL) has no effect on CatK activity. c) IC 50 of CKI-13 and (d) CKI-8 as determined by zymography (twice repeated) using human CatK.

Figure 7 .

 7 Figure 7. Resorption activity of osteoclasts derived from murine bone marrow on osteo assay plates mimicking bone surface. Osteoclast incubated in the (a) absence and (b) presence of 100 nM CKI-13. c) Resorption index of osteoclasts on Osteo Assay plates. Resorption index of primary osteoclasts with CKI-8, CKI-13 and E-64,

Figure 8 .

 8 Figure 8. Resorption activity of osteoclasts derived from primary osteoclasts on bovine bone slices. a) Resorption index of primary osteoclasts with 0.1 % (mL/mL) DMSO not treated (control) or treated with following inhibitors: E-64, CKI-8 and CKI-13. 0.1 % (mL/mL) DMSO has no effect on the resorption activity b-e) Typical bone resorbed surfaces by primary osteoclasts without and with CKIs, as revealed by three-min toluidine blue staining. Pits are delineated with white contours and yellow arrows. They were formed by osteoclasts resorbing on bovine bone plates without inhibitors (b), with E-64 (c), with CKI-8 (d) and with CKI-13 (e) at 100nM. Smaller pits were observed in the presence of CKI-8 (d) and with CKI-13 (e) as compared with those without inhibitors (b) and with E-64 (c). Quantitative analysis of mean pit area (µm 2 ) on bovine bone slices without and with inhibitors at 100nM (f).

  The anti-resorption property of CKI-8 and CKI-13 was evaluated on Corning Osteo Assay plates at non-toxic concentration up to 100 nM for CKI-13. CKI-13 inhibited the resorption activity of primary osteoclasts on the Corning Osteo Assay plate, in constrast to CKI-8 and E-64 which did not. The Corning Osteo Assay plate consisting mostly of hydroxyapatite coated onto apolystyrene plate, indicated that CKI-13 may interact with other targets than CatK. The increase in bone resorption observed at around 10 nM inhibitor concentrations (CKI-8, CK-13 and E-64) is probably caused by an acidic secretion of osteoclasts unrelated to the Cat-K inhibition property of CKIs, dissolving the hydroxyapatite coated on the Osteo Assay plate.

Figure 9 .

 9 Figure 9. SDS-PAGE of matrix vesicles. (A) Protein profiles analyzed by 12 % SDS-PAGE followed by Coomassie brilliant blue staining of intact pellet 1 (P1); pellet 2 (P2); supernatant (SN1); supernatant 3 (SN3); pellet 3 (MVs).

Figure 10 .

 10 Figure 10. Specific activity of the different fractions in TNAP measured during hydrolysis of p-NPP at pH 10.4.

  6 h, indicating the formation of calcium-Pi complexes. The maximum value was reached after an 8-h incubation (Fig 11. full line). In the medium containing only the SCL buffer with 2 mM Ca 2+ and 3.42 mM P i without MVs (Fig. 11 dots line), there were less mineral formation than in SCL medium under the same conditions with MVs. In the mineralization medium containing MVs without ions, the changes in turbidity were almost negligible over a 16-h incubation time (Figure 11. small dots line). This indicates that MVs are able to initiate mineral formation under the ionic microenvironment .

Figure 11 .

 11 Figure 11. Mineralization induced by MVs: mineralization in SCL containing 2 mM Ca 2+ , 3.42 mM P i , and 10μg of protein/ml MVs. (dots line): control of mineralization in SCL containing 2mM Ca 2+ and 3.42mM P i , but without MV;(small dots line): control of mineralization in SCL containing MVs only without ions; (full line): mineralization process containing MVs with 2 mM Ca 2+ , 3.42 mM P i . The mineral formation was assessed by light scattering at 340 nm.

Figure 12 .

 12 Figure 12. Infrared spectra of mineral deposits by MVs and pure HA. MVs were incubated at 37 °C in mineralization buffer for 8 hours, then the minerals formed were collected, washed and analyzed by infrared spectroscopy. Infrared spectrum of hydroxyapatite as control (HA); Infrared spectrum of minerals formed by matrix vesicles (MV). Infrared spectra of minerals indicated that the minerals formed by MVs were hydroxyapatite. (Typical infrared spectra averaged with two independent measurements).

Figure 13 .

 13 Figure 13. Infrared spectra of 50 mM inorganic phosphate (bottom trace) and of 50 mM pyrophosphate (top trace) in aqueous buffer 50 mM Tris-HCL, pH 8.0, 5 µM ZnCl 2 and 5 mM MgCl 2 .

Figure 14 .

 14 Figure 14. Phosphatase activity in matrix vesicles as determined by infrared spectroscopy. (a) Kinetic recording of 50 mM PP i hydrolysis by 2 mg MV protein mL -1 in medium containing 5 mM MgCl 2 , 5 μM ZnCl 2 and 100 mM Tris-HCL, pH 8.0 (buffer A) at 37 °C. Successive infrared spectra were measured at the indicated times. (b)

Figure 15 .

 15 Figure 15. Absorbance changes during PP i hydrolysis by MVs of PP i band located at 1107 cm -1 (bottom trace, Molar absportion coefficient = 2158 ± 211 M -1 .cm -1 ) and of the two P i bands located at 1076-1080-cm -1 and 990-cm -1 (top traces, Molar absorption coefficients of 1215 ± 131 M -1 .cm -1 and 443 ± 50 M -1 .cm -1 respectively).

Figure 16 .

 16 Figure 16. a) Absorption changes of PP i band at 1107 cm -1 ; b) Absorption changes of P i peak at 990 cm -1 ;

  determine a phosphatase activity. IR spectra of 50 mM phosphatase substrates: AMP, ADP, -NPP), PP i and Pi in buffer A (100 mM Tris-HCl, pH 8.0, 5 µM ZnCl 2 and 5 mM MgCl 2 ) recorded at 37 °C present sufficient differences in band shapes and positions (Fig.17

  ) to be used for analytical application.

Figure 17 .

 17 Figure 17. IR spectra of 50 mM of different physiological substrates and 50 mM P i in aqueous buffer containing 100 mM Tris-HCl, pH 8.0, 5 mM MgCl 2 and 5 µM ZnCl 2 .

Figure 18 .

 18 Figure 18. Infrared spectra of BSA solution in buffer A that served to determine molar absorption coefficient of the amide-II band at 1550 cm -1

  3 min dead time) after mixing Saos-2 cells with 50 mM substrate such as AMP, ADP, ATP, UTP, α-D-glucose 1-phosphate (G-1-P), β-glycerophosphate (β-GP), pNPP and PP i in aqueous Buffer A (Fig.19full lines). After 30 min-incubation in the infrared cell at 37°C, a second IR spectrum was measured (Fig.19 dashed lines).The bottom trace shows the spectrum of 50 mM P i indicating the positions of the two 1080 and 990 cm -1 bands (vertical blue dashed lines). The IR spectrum of buffer A was deducted from the IR spectra of Saos-2 cells with substrate. In all cases (with AMP, ADP, ATP, UTP, G-1P, β-GP), pNPP or PP i ), the intensity of either one or both P i bands at 1076-1080 and 990 cm -1 increased or the intensity of the substrate decreased, indicating the phosphatase activity in the whole cells (Fig.19). It was clearly observed in the successive difference spectra (spectrum measured at the indicated time minus that measured immediately after mixing 50 mM substrate with Saos-2 cells) (Figure. 20). Negative peaks indicated the disappearance of the substrate while positive peaks at 1076-1080 and 990 cm -1 illustrated the formation of a hydrolytic product, namely P i (Figure.20

Figure 20 .

 20 Figure 20. Successive difference spectra of Saos-2 cells with 50 mM substrate (AMP, ADP, ATP, UTP, G-1P, β-GP, p-NPP, PPi and Pi) in buffer A (spectrum measured after a given time minus that measured immediately after mixing the substrate with cells) at 37°C determined at 5, 10, 20 and 30 min. The negative intensities indicated the decline of substrate concentration while the positive intensities of the 1076-1080 and the 990 cm -1 bands indicated the appearance of P i . The bottom trace shows the spectrum of 50 mM P i to indicate the positions of the two 1080 and 990 cm -1 bands (vertical blue dashed lines).

Figure 22 .

 22 Figure 22. IR spectrum of tris buffer A and IR spectrum of DMEM.

Figure 24 .

 24 Figure 24. Infrared difference spectra of 50 mM PPi in Saos-2 cells or in primary cells measured immediately and after the indicated time. a) Successive IR difference spectra of Saos-2 cells (12 mg of Saos-2-cell protein mL -1 ) in buffer A with 50 mM PP i at 37 °C from 5 min to 30 min (spectrum recorded after the indicated time minus spectrum recorded immediately after mixing substrate with cells). b) IR difference spectrum of Saos-2 cells (28 mg of cell protein mL -1 ) with 50 mM PP i in DMEM medium at 37 °C (spectrum recorded after 30 min minus spectrum recorded immediately after mixing substrate with cells). c) IR difference spectrum of primary osteoblast cells (45 mg of

Fig. 25 a

 25 Fig. 25 a) Absorption of PP i in 1120-1100 cm -1 region as a function of incubation time and of Saos-2 cell protein concentration (from 7.5 to 18 mg mL -1 ). b) Absorption of 990 cm -1 band of P i as a function of incubation time and of Saos-2 cell protein concentration. c) Specific activity of PPi hydrolysis by Saos-2 cells at three concentrations from 7.5, 12 and 18 mg mL -1 ). One enzyme unit (1 U) is expressed as 1 nmol hydrolyzed PP i per min at 37 °C. d) Determination of IC 50 of levamisole by measuring inhibition of PP i hydrolysis by Saos-2 cells.

Schéma 1 Figure 1 .

 11 Figure 1. Les inhibiteurs CKI-8 et CKI-13 ne sont pas cytotoxiques. A) Tests MTT sur une lignée des cellules d'origine humaine Saos-2. B) Test MTT sur des cellules de souris RAW 264.7. C) Cellules matures d'ostéoclastes primaires de souris. Les résultats ont été effectués trois fois en triplicata (n=9).

Figure 2 .

 2 Figure 2. Effets de CKI-8 et CKI-13 sur la minéralisation induite par les ostéoblastes. Les Saos-2 ont été incubés pendant six jours sans (a) ou avec 50 µg ml-1 AA et 7.5 mM β-GP ((b) stimulé), puis colorés avec de l'AR-S afin de détecter les complexes de calcium (indiqués par des flèches blanches). Les effets de CKI-8 et de CKI-13 sur la minéralisation induite sur les Saos-2 (c) et sur les ostéoblastes primaires (e) révélés par AR-S ont été quantifiés en fonction de la concentration de CKI-8 et CKI-13 en comparaison avec le contrôle (sans inhibiteur). L'activité de phosphatase alcaline durant la minéralisation induite par Saos-2 (d) ou par les

Figure 3 .

 3 Figure 3. Activité de résorption suivie sur des tranches d'os d'origine bovine. a) Index de résorption d'ostéoclastes primaires le contrôle et les inhibiteurs E-64, CKI-8 and CKI-13. Surfaces résorbées (indiquées par des contours blancs) sans inhibiteur (b), avec E-64 (c), avec CKI-8 (d) et avec CKI-13 (e) à une

  spectres des substrats AMP, ADP, ATP, UTP, α-D-glucose-1-phosphate (G-1P) βglycérophosphate b-GP, para-nitrophenylphosphate (pNPP) sont très différents de celui du produit P i (Fig 4) rendant possible de déterminer la concentration de substrats et de P i .

Figure 4 .

 4 Figure 4. Spectres IR des substrats (50 mM) AMP, ADP, ATP, UTP, α-D-glucose-1-phosphate (G-1P) βglycérophosphate b-GP, para-nitrophenylphosphate (pNPP), PP i et celui du produit Pi. Deux traits verticaux indiquent la position des bandes de Pi situées à 1076-1080 et à 990-991 cm -1 .

Figure 5 .

 5 Figure 5. Spectres IR de 50 mM de PPi en présence de cellules Saos-2 mesurés au temps t=0 (trait plein) et après 30 min (tiret).

Figure 6 .

 6 Figure 6. Spectres IR des substrats (50 mM) AMP, ADP, ATP, UTP, α-D-glucose-1-phosphate (G-1P) β-glycérophosphate, b-GP, paranitrophenylphosphate (pNPP) et celui du produit Pi. Deux traits verticaux indiquent la position des bandes de Pi situés à 1080 et à 990 cm -1 . En présence de cellules Saos-2 (tiret) après 20 min d'incubation et en absence de cellules (trait plein) .

Figure 5 .

 5 Figure 5. Spectres de différence IR des Cellules Saos-2 en présence de substrats (50 mM) AMP, ADP, ATP, UTP, α-D-glucose-1-phosphate (G-1P) β-glycérophosphate β-GP, paranitrophenylphosphate (pNPP) et celui du produit Pi. Deux traits verticaux indiquent la position des bandes de Pi situés à 1080 et à 990 cm -1 . Spectres de différence: spectres mesurés selon le temps indiqué minus spectre mesuré en temps =0.

Table 1 .

 1 The K i values were calculated from the corresponding IC 50 by using the equation K i =IC 50 /(1+[S]/Km).

	Compounds		K i (nM)
		Cat K	Cat B	Cat L	Cat S
	CKI-8	0.124	0.692	0.262	1.54
	CKI-13	0.0031	3.15	0.031	3.29

  Ph D thesis was directed under the Co-supervision of Prof. Yuqin WU (Jilin

	University) and of Dr. Saida MEBAREK and Prof. René BUCHET (University Lyon 1). Half
	part of my work was done at Jilin University, while the other half was performed at university
	Lyon 1. Due to specific regulations requested by the doctoral schools at Jilin University and
	of University Lyon 1 (Ecole Doctorale Interdisciplinaire Sciences et Santé EDISS), the whole
	Ph D thesis is written in Chineese and in English with a substantial French abstract. My Ph D
	thesis is centered on three distinct topics.

  Briefly, a cell extraction buffer (20 nMTris-HCl pH 7.5, 5 mM ethylene glycol tetraacetic acid (EGTA), 150 mM NaCl, 20 mM β-

	glycerol phosphate, 10 mM NaF, 1 mM sodium orthovanadate, 1 % (mL:mL) Triton X-100,
	and 0.1 % (mL/mL) Tween 20) was added to the cells (RAW 264.7 and primary mouse
	osteoclasts). Then lysates were collected and cleared. They contained CatK in extracellular
	medium and in cells. Protein concentrations were determined using the bicinchoninic acid
	(BCA) assay (Pierce). Five times (5×) no reducing loading buffer (0.05 % (g/mL)
	bromophenol blue, 10 % (g/mL) sodium dodecyl sulfate (SDS), 1.5 M Tris, and 50 %
	(mL/mL) glycerol was added to all samples prior to loading. Equal amounts of protein were

resolved by 12.5% SDS-polyacrylamide gels containing 0.2 % (g/mL) gelatin, a CatK substrate, at 4 °C. Gels were removed were washed in 65 mM Tris buffer (pH 7.4) with 20 %

Table 2 .

 2 Composition of gel electrophoresisMVs provide a favorable environment for mineralization in the presence of calcium ions and phosphate ions by enrichment in alkaline phosphatase. UV-spectrophotometer was employed for real time measurement of mineral formation in MVs with the synthetic cartilage lymph (SCL) medium.[START_REF] Genge | Kinetic analysis of mineral formation during in vitro modeling of matrix vesicle mineralization: effect of annexin A5, phosphatidylserine, and type II collagen[END_REF][START_REF] Wu | Effects of analogues of inorganic phosphate and sodium ion on mineralization of matrix vesicles isolated from growth plate cartilage of normal rapidly growing chickens[END_REF]. The composition of SCL buffer pH=7.8 was 1.42 mM NaH 2 PO 4 .H 2 O, 1.83 mM NaHCO 3 , 12.7 mM KCl, 0.57 mM MgCl 2 , 5.55 mM D-glucose, 63.5 mM succrose, 16.5 mM TES, 100mM NaCl, 0.57 mM Na 2 SO4. Three cuvettes with 1 mL solutions were prepared each containing 500 μL of synthetic cartilage lymph (SCL). One contains MVs (10 μL) with 2 mM CaCl 2 and 3.42 mM NaH 2 PO 4 final concentrations, the second contains 2 mM CaCl 2 and 3.42 mM NaH 2 PO 4 final concentrations without MVs and the third contains MVs at the same concentration but without CaCl 2 and NaH 2 PO 4 .

		Separated gel 12 %	Concentrated gel
	Acrylamide 40%	1.5 mL	0.16 mL
	Tris HCl pH=8.8	1.88 mL
	Tris HCl pH=6.8		0.2 mL
	SDS 10%	50 μL	16 μL
	H 2 O	1.54 mL	1.21mL
	APS 10%	25 μL	20 μL
	TEMED	2.5 μL	4 μL

Table 3 .

 3 Enzymatic activity of TNAP in the different fractions during the hydrolysis of p-NPP.

Table 4 .

 4 Specific activities of TNAP in the different concentration of MVs

  ). The hydrolysis of ATP, UTP and ADP in the Saos-2 cells produced P i

	Figure 19. IR spectra of 50 mM substrate
	(one among the substrates: AMP, ADP, ATP,
	UTP, G-1-P, b-GP, pNPP, PP i ) in buffer A
	with Saos-2 cells, recorded immediately after
	mixing the substrate with cells (full lines) and
	after 30 min (dashed lines) at 37 °C. The
	bottom trace indicated the IR spectrum of 50
	mM P i , the product of the hydrolysis of
	substrate. Vertical lines served to guide the
	eye to evidence P i formation.

41st European Calcified Tissue Society Congress, Prague, Czech Republic 17-20 May 2014 RESUME SUBSTANTIEL EN FRANCAIS Petites molécules régulant la résorption osseuse et l'activité enzymatique dans les cellules osseuses

La thèse comprend trois parties : la première partie est centrée sur les inhibiteurs de cathepsine K qui sont des dérivés d'azanitriles. Les effets des CKIs sur la formation de l'os induite par les ostéoblastes et sur la résorption de l'os par les ostéoclastes ont été determinés. La deuxième partie de ma thèse a été centrée sur le développement de mesure de l'activité de la phosphatase alcaline dans les vésicules matricielles au moyen de la spectroscopie IR. Enfin, la troisième partie de ma thèse est une extension de la méthode de mesure d'activité de phosphatase alcaline par IR applicable sur les cellules. Cette méthode pourrait être utilisée pour le criblage des inhibiteurs de phosphatase alcaline selon les conditions physiologiques.

1)

Effets des inhibiteurs de cathepsine K sur la minéralisation et sur la résorption osseuse.

  La cathepsine K est la seule enzyme de mammifère capable de cliver la forme hélicoïdale des fibres de collagène de type 1 dans l'os minéralisé[4]. Le milieu acidique produit par les ostéoclastes à l'intérieur des podosomes, servant à dissoudre l'hydroxyapatite (composant minéral majeur de l'os) et l'activité de la cathepsine K dégradant la matrice organique, contribuent tous les deux à la résorption de l'os. L'ostéoporose est la sixième plus importante maladie et est considérée comme une atteinte socioéconomique majeure[5,6]. L'ostéoporose est caractérisée par une perte osseuse et une détérioration des microstructures osseuses qui résultent d'une fragilité squelettique et un accroissement de risques de fractures d'os. Des connaissances approfondies de la biologie de l'os, plus particulièrement au niveau de la communication entre les ostéoblastes et ostéoclastes, ainsi que les réseaux de signalisation ont conduit à l'identification de plusieurs cibles thérapeutiques potentielles. Actuellement, un large éventail de thérapies est disponible pour traiter la perte osseuse bien qu'aucune d'entre elles ne sont capables de prévenir

	Dans le squelette osseux en bonne santé, l'équilibre entre la formation de la matrice
	osseuse et la résorption de l'os est atteint grâce à une activité coordonnée des cellules
	ostéoblastes responsables de la formation de l'os et des ostéoclastes impliquées dans la
	résorption osseuse [1,2]. Afin de dégrader l'os, les ostéoclastes doivent d'abord créer une
	cavité de résorption. De larges ensembles d'actine F capables d'adhérer à la matrice cellulaire,
	appelés podosomes, sont formés par les ostéoclastes qui contribuent à créer une zone
	basolatérale étanche à la surface de l'os. Cela permet aux ostéoclastes de contrôler la
	résorption de l'os par un rapide assemblage et démontage des podosomes en réponse de

signaux intracellulaires générés par l'intermédiaire des protéines appartenant aux famille des Rho GTPases et des kinases Src

[3]

. Une fois que la cavité de résorption s'est formée, les enzymes telles que les cystéine protéases et la cathepsine K sont secrétées afin de faciliter la dégradation de l'os. complètement l'ostéoporose. En plus de la thérapie à base d'hormone, d'autres traitements impliquent la prise de bisphosphonates, calcitonine, calcium, strontium, et vitamine D.

.cm -1 ) and at 990 cm -1 (Molar absorption coefficient = 493 ± 49 M -1 .cm -1 ) served to measure

Acknowledgements

This thesis was prepared at the University Claude Bernard Lyon 1, UMR 5246 CNRS, UMR 5242 IGFL, in Lyon (France) and at Jilin University, Key Laboratory for Supramolecular Structure and

ENGLISH ABSTRACT-

 --------------------------------------------------------------------------------------- [START_REF] Holton | Subcellular localization of early biochemical transformations in cancer-activated fibroblasts using infrared spectroscopic imaging[END_REF]Bone resorption on bovine slices decreased significantly at 10 nM CKI-13, while 100 nM CKI-8 and 100 nM of the commercial inhibitor E64 were necessary to significantly decrease bone resorption. CKI-8 and CKI-13 treatments produced smaller pits when compared with the larger pits induced by E64 treatment (Fig. 8b), suggesting that CKI-8 and CKI-13 might affect the mobility of osteoclasts in addition to their osteoclast resorption activity. Possible targets of CKI-8 and CKI-13 are F-actin and other proteins involved in osteoclast podosome dynamics [START_REF] Suda | Regulation of osteoclast function[END_REF]. Although the findings look promising, especially the bone-resorption property of CKI-13, further work is needed to ascertain the feasibility of using CatK inhibitors to treat bone resorption diseases, since their side effects on TNAP activity may impair their therapeutic potential, especially for CKI-13. 1550 cm -1 protein band (amide-II band) allowed us to determine directly protein concentration using ε = 3.6 mg -1 mL cm -1 .

Figure 21. IR spectra of Saos-2 cells (12 mg of Saos-2-cell protein mL -1 ) with 50 mM PP i in buffer A recorded immediately after mixing the substrate with cells (full line) and after 30 min incubation at 37 °C (dashed line).

The absorbance changes of 1120-1100 cm -1 band of PP i and the 990 cm -1 band of P i served to determine the pyrophosphatase activity (Table 6). We obtained an apparent specific phosphatase activity in Saos-2 cells of around 116 ± 113 nmol min -1 mg -1 (Table 6). This compares well with the pyrophosphatase activity of 130 nmol min -1 mg -1 in mammalian HeLa cells at pH 8.5 as assayed by the modified method of Fiske and Subbarow [START_REF] Cox | Alkaline Inorganic Pyrophosphatase Activity of Mammalian-Cell Alkaline Phosphatase[END_REF]. Quantitative determinations were also obtained for AMP, G-1-P, β-GP, p-NPP using both P i bands at 990 and 1070-1080 cm -1 (Table 6). The substrate bands were also used to determine enzyme specific activity, as in the case of AMP (977 cm -1 ), p-NPP (980 cm -1 ) and PP i (1107 cm -1 ) (Table 6). Specific activities determined either from P i or from substrate-bands were identical within experimental errors except for G-1-P and β-GP due to strong band overlapping.

We tested the possibility to determine a specific activity of Saos-2 cells in the cellular medium DMEM. IR spectra of Saos-2 cells (28 mg of Saos-2-cell protein mL -1 with 50 mM PP i in DMEM (Fig. 23a) recorded immediately after mixing the substrate with cells (dashed line) and after 30 min incubation at 37 °C (full line). The spectrum of DMEM was deducted from the spectra of Saos-2 cells. Infrared spectra indicated both the decrease of the 1100-cm -1 band of PP i and the increase of the 1076-1080-cm -1 band of P i , confirming the presence of a pyrophosphatase activity (Fig. 23a). Furthermore, the 1550-cm -1 protein band allowed us to determine directly protein concentration using ε = 3.6 mg -1 mL cm -1 .

Figure 23. a) IR spectra of Saos-2 cells (28 mg of Saos-2-cell protein mL -1 with 50 mM PP i in DMEM recorded immediately after mixing the substrate with cells (dashed line) and after 30 min incubation at 37 °C (full line).

The spectrum of DMEM was deducted from the spectra of Saos-2-cells. b) IR spectra of primary osteoblast cells (45 mg of cell protein mL -1 ) in buffer A with 50 mM PP i recorded immediately after mixing PP i with cells (dashed line) and after 30 min incubation at 37 °C (full line). The spectrum of buffer A was deducted from the spectra of osteoblasts. Saos-2 osteoblast-like cells were selected due to their high TNAP activity, making it easier to monitor phosphatase activity in DMEM. Saos-2 cells are cell lines which are easier to cultivate but which are less good models than primary cells. Therefore, primary osteoblasts from mouse calvaria (45 mg of cell protein mL -1 ) were tested for their phosphatase activities. They exhibited also a pyrophosphatase activity both in buffer A (Fig. 23b) and in DMEM (not shown) but it were much lower than those in Saos-2 cells. This was confirmed qualitatively by the magnitude of the difference in IR spectra (spectrum measured after 30 min or the indicated time minus that cm -1 (ε = 2158 ± 211 M -1 .cm -1 ) et celles de P i localisées à 1076 cm -1 (ε = 1346 ± 116 M -1 .cm - 1 ) et à 991 cm -1 (ε = 493 ± 49 M -1 .cm -1 ) ont servis à mesurer les concentrations du substrat et du produit.

Small Molecules regulating Bone Resorption and Enzyme Activity in osseous cells

ABSTRACT

Cathepsin K is among the most potent mammalian collagenase, capable of cleaving the triple helix in type-I collagen. We developed a series of azanitriles (CKI-8 and CKI-13) which are inhibitors of cathepsin K. CKI-8 (an isomer of CKI-13) and CKI-13 did not induce significant toxicity on osteoblasts Saos-2 and RAW 264.7 cells up to 1000 nM, while they were not toxic on mature osteoclasts up to 100 nM. Commercial E64 inhibitor was not toxic in primary osteoclast cells up to 1000 nM. CKI-8 did not affect alkaline phosphatase activity as well the mineralization induced by Saos-2 cells and by primary osteoblasts. CKI-13 decreased by 35% the mineralization induced by Saos-2 cells while it did not on mineralization induced by primary osteoblasts. Addition of CKI-13 decreased alkaline phosphatase activity by around 20% (Saos-2 cells) and 45% (primary osteoblasts). Bone resorption on bovine slices decreased significantly with 10 nM of CKI-13, with 100 nM of CKI-8 and commercial inhibitor E64. Our findings indicated that CKI-8 and CKI-13 inhibited bone resorption and affected the mobility of osteoclast. To monitor directly the PP i hydrolytic activity by alkaline phosphatase, we developed an infrared (IR) assay taking the advantage to use natural substrate under physiological pH in matrix vesicles and in living cells. PP i band located at 1107 cm -1 (ε = 2158 ± 211 M -1 .cm -1 ) and P i bands located at 1076 cm -1 (ε = 1346 ± 116 M -1 .cm -1 ) and at 991 cm -1 (ε = 493 ± 49 M -1 .cm -1 ) served to measure the substrate and the product concentrations.