Abdeldjalil Madani

Jeremy N Ridenour

Benjamin P Martin

Rishi R Paudel

Anosha Abdul Basir

Vincent Le Moigne

Jean-Louis Herrmann

Stéphane Audebert

Luc Camoin

Laurent Kremer

Christopher D Spilling
email: spillingc@msx.umsl.edu

Stéphane Canaan

Jean-François Cavalier
email: jfcavalier@imm.cnrs.fr

Cyclipostins and Cyclophostin Analogues as Multitarget Inhibitors That Impair Growth of Mycobacterium abscessus

Keywords: Total synthesis, drug susceptibility, activity based-protein profiling, proteomics analysis

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Produced by Streptomyces species, [START_REF] Kurokawa | Cyclophostin, acetylcholinesterase inhibitor from Streptomyces lavendulae[END_REF] natural Cyclophostin (CyC1 -Figure 1) is characterized by a unique seven-membered cyclic phosphate triester fused to a butyrolactone ring. This bicyclic core also contains a unique enol phosphate and chiral centers at both phosphorus and C-3a carbon atom. The structurally related natural Cyclipostins (e.g., Cyclipostin P CyC18 -Figure 1), which varied in the nature of the lipophilic chain attached to the phosphate, were described as bacterial growth inhibitors of various mycobacteria. [START_REF] Seibert | Treating mycobacterial infections with cyclipostins[END_REF] Together, compelling evidence suggests the great potential for cyclic enolphosphate and phosphonate analogs of Cyclophostin (and the Cyclipostins) as anti-mycobacterial agents.

To explore this hypothesis, we have reported the synthesis of Cyclipostins and Cyclophostin analogs (namely the CyCs -Figure 1). [START_REF] Bandyopadhyay | Synthesis and Biological Evaluation of a Phosphonate Analog of the Natural Acetyl Cholinesterase Inhibitor Cyclophostin[END_REF][START_REF] Malla | The First Total Synthesis of (±)-Cyclophostin and (±)-Cyclipostin P: Inhibitors of the Serine Hydrolases Acetyl Cholinesterase and Hormone Sensitive Lipase[END_REF][START_REF] Point | Synthesis and kinetic evaluation of cyclophostin and cyclipostins phosphonate analogs as selective and potent inhibitors of microbial lipases[END_REF][START_REF] Vasilieva | Rat hormone sensitive lipase inhibition by cyclipostins and their analogs[END_REF] We first demonstrated that this first series of 26 CyCs (Figure 1) was able to efficiently inhibit the growth of Mycobacterium tuberculosis H37Rv, in vitro as well as in infected macrophages, with very low toxicity towards the host cells. [START_REF] Nguyen | Cyclipostins and Cyclophostin analogs as promising compounds in the fight against tuberculosis[END_REF] These studies also strongly support the assumption that CyC compounds are multi-target inhibitors impairing various mycobacterial Ser-or Cys-containing enzymes involved in important physiological processes via the formation of a covalent bond between the enol-phosphorous atom and the catalytic residue. [START_REF] Malla | The First Total Synthesis of (±)-Cyclophostin and (±)-Cyclipostin P: Inhibitors of the Serine Hydrolases Acetyl Cholinesterase and Hormone Sensitive Lipase[END_REF][START_REF] Point | Synthesis and kinetic evaluation of cyclophostin and cyclipostins phosphonate analogs as selective and potent inhibitors of microbial lipases[END_REF][START_REF] Nguyen | Cyclipostins and Cyclophostin analogs as promising compounds in the fight against tuberculosis[END_REF][START_REF] Nguyen | Cyclipostins and Cyclophostin analogs inhibit the antigen 85C from Mycobacterium tuberculosis both in vitro and in vivo[END_REF][9] The efficiency/selectivity of these CyCs was investigated further towards three slow-growing species (i.e., M. marinum, M. bovis BCG, and M. tuberculosis) and various mycobacterial clinical isolates and bacteria responsible for nosocomial infections, including Gram-negative and Gram-positive bacteria as well as rapidly-growing mycobacteria (RGM) belonging to the Mycobacterium chelonaeabscessus clade. [START_REF] Nguyen | Cyclophostin and cyclipostins analogs, new promising molecules to treat mycobacterial-related diseases[END_REF] Remarkably, the inhibitory activity of these CyCs was exclusively restricted to mycobacteria. [START_REF] Nguyen | Cyclophostin and cyclipostins analogs, new promising molecules to treat mycobacterial-related diseases[END_REF] Notably, the most potent inhibitor (CyC17) exhibited minimal inhibitory concentrations (MIC) values comparable to those of most classical antibiotics used to treat M. tuberculosis and M. abscessus infections. [START_REF] Nguyen | Cyclipostins and Cyclophostin analogs as promising compounds in the fight against tuberculosis[END_REF][START_REF] Nguyen | Cyclophostin and cyclipostins analogs, new promising molecules to treat mycobacterial-related diseases[END_REF] Collectively, these results emphasized the attractiveness of this new family of compounds as valid drug candidates to be exploited in future therapeutic developments against mycobacterial-associated infections, especially against M. abscessus the most drug-resistant mycobacterial species. 10 Cyclipostins (CyC11-14;17). CyC6-10 and CyC13 were obtained as two diastereoisomers, being either in a trans (α-isomer) or cis (β-isomer) conformation between the OMe on the phosphorus and the H-substituent on the C-5 carbon atom. Adapted from [START_REF] Nguyen | Cyclipostins and Cyclophostin analogs as promising compounds in the fight against tuberculosis[END_REF] .

Over the last decade, non-tuberculous mycobacterial (NTM) infections have increased worldwide, leading to an emerging public health problem, particularly in industrialized countries, sometimes surpassing tuberculosis. [START_REF] Chou | A spatial epidemiological analysis of nontuberculous mycobacterial infections in Queensland, Australia[END_REF] Among them, M. abscessus represents the most important rapidly growing mycobacteria (RGM). [START_REF] Ryan | Mycobacterium abscessus: Shapeshifter of the Mycobacterial World[END_REF] M. abscessus is indeed responsible for a wide spectrum of clinical syndromes ranging from skin infections to severe pulmonary infections in humans with compromised natural defenses, such as patients with cystic fibrosis (CF). [START_REF] Wu | NTM drug discovery: status, gaps and the way forward[END_REF][START_REF] Osmani | Mycobacterium abscessus infections in lung transplant recipients: 15-year experience from a single institution[END_REF] The M. abscessus complex, along with Mycobacterium avium complex, represents over 90% of all reported NTMpulmonary infection cases in CF patients. [START_REF] Ryu | Diagnosis and Treatment of Nontuberculous Mycobacterial Lung Disease: Clinicians' Perspectives[END_REF][START_REF] Van Ingen | Poor adherence to management guidelines in nontuberculous mycobacterial pulmonary diseases[END_REF] Reports also show that M. abscessus isolated from CF patients ranged from 16% and up to 68% of all isolated NTM. [START_REF] Pierre-Audigier | Age-related prevalence and distribution of nontuberculous mycobacterial species among patients with cystic fibrosis[END_REF][START_REF] Roux | Multicenter study of prevalence of nontuberculous mycobacteria in patients with cystic fibrosis in France[END_REF][START_REF] Lim | Profiling non-tuberculous mycobacteria in an Asian setting: characteristics and clinical outcomes of hospitalized patients in Singapore[END_REF] In addition, the presence of M. abscessus infection prior to lung transplantation represents a risk factor for developing lung infection or a disseminated disease after transplantation. [START_REF] Osmani | Mycobacterium abscessus infections in lung transplant recipients: 15-year experience from a single institution[END_REF] M. abscessus displays rough (R) and smooth (S) colony morphotypes. [START_REF] Roux | The distinct fate of smooth and rough Mycobacterium abscessus variants inside macrophages[END_REF][START_REF] Gutierrez | Glycopeptidolipids, a Double-Edged Sword of the Mycobacterium abscessus Complex[END_REF] The S morphotype is related to the abundance of the surface-associated glycopeptidolipids (GPL), which is associated with sliding motility and biofilm formation. [START_REF] Roux | The distinct fate of smooth and rough Mycobacterium abscessus variants inside macrophages[END_REF] In contrast, a remarkable reduction in GPL amount in the cell wall of the R variant was correlated with cord formation. [START_REF] Sanchez-Chardi | Demonstration of cord formation by rough Mycobacterium abscessus variants: implications for the clinical microbiology laboratory[END_REF] In addition, S and R variants can be considered as two representatives of the same isolate, which may coexist and/or evolve differently in response to host immunity, resulting in different fates for the mycobacteria in its host. [START_REF] Roux | The distinct fate of smooth and rough Mycobacterium abscessus variants inside macrophages[END_REF] Several studies have confirmed the correlation between colony morphology and virulence. The S variants are less virulent than R variants, [START_REF] Nessar | Deletion of the mmpL4b gene in the Mycobacterium abscessus glycopeptidolipid biosynthetic pathway results in loss of surface colonization capability, but enhanced ability to replicate in human macrophages and stimulate their innate immune response[END_REF] which are frequently associated with severe pulmonary infections [START_REF] Catherinot | Hypervirulence of a rough variant of the Mycobacterium abscessus type strain[END_REF][START_REF] Catherinot | Acute respiratory failure involving an R variant of Mycobacterium abscessus[END_REF] and are able to persist for years especially in infected CF patients. [START_REF] Jönsson | Molecular epidemiology of Mycobacterium abscessus, with focus on cystic fibrosis[END_REF] In the light of these findings, M. abscessus is known to transition from a smooth (S) morphotype with cell surface-associated GPL to a rough (R) morphotype lacking GPL. Accordingly, M. abscessus is notorious for being one of the most drug-resistant mycobacterial species, refractory to standard antimicrobials used for the treatment of Gram-positive and Gramnegative bacteria. [START_REF] Luthra | The Role of Antibiotic-Target-Modifying and Antibiotic-Modifying Enzymes in Mycobacterium abscessus Drug Resistance[END_REF] In addition, most antitubercular agents (i.e., rifampin, isoniazid, and ethambutol) are also ineffective against M. abscessus, referred to as an "antibiotic nightmare". [START_REF] Nessar | Mycobacterium abscessus: a new antibiotic nightmare[END_REF] This intrinsic polyresistance to drugs results from multiple factors, [START_REF] Ripoll | Non mycobacterial virulence genes in the genome of the emerging pathogen Mycobacterium abscessus[END_REF] including the presence of a highly impermeable cell envelope preventing the penetration of drug, multiple-drug efflux pumps, [START_REF] Richard | Mechanistic and Structural Insights Into the Unique TetR-Dependent Regulation of a Drug Efflux Pump in Mycobacterium abscessus[END_REF][START_REF] Richard | Mutations in the MAB_2299c TetR Regulator Confer Cross-Resistance to Clofazimine and Bedaquiline in Mycobacterium abscessus[END_REF] inability to convert prodrugs into their active metabolites, [START_REF] Rominski | Intrinsic rifamycin resistance of Mycobacterium abscessus is mediated by ADP-ribosyltransferase MAB_0591[END_REF] expression of numerous enzymes which can modify the drug-target or inactivate the drug itself, [START_REF] Luthra | The Role of Antibiotic-Target-Modifying and Antibiotic-Modifying Enzymes in Mycobacterium abscessus Drug Resistance[END_REF] and the acquisition of mutations in the antibiotic targets. Hence, the development of new therapeutic approaches and/or discovery of new chemical entities to fight this pathogen are needed.

Herein, we have synthesized 12 new analogs, i.e. CyC19 to CyC30, by varying the lipophilicity of the R 3 chain located on the 7-membered enolphosphorus ring in our phosphate and phosphonate analogs. This structural modification is also supported by the isolation of the anti-malarial Salinipostins, [START_REF] Schulze | Long-Chain Bicyclic Phosphotriesters as a Potent and Selective Antimalarial Chemotype[END_REF][START_REF] Zhao | Total Synthesis of Marine Cyclic Enol-Phosphotriester Salinipostin Compounds[END_REF] which are essentially Cyclipostins with variations in the enol alkyl substituent.

Their respective antibacterial activity was further assessed against a panel of pathogenic mycobacteria, including the M. abscessus R and S variants. Importantly, the MIC of all (26+12) CyCs determined against either extracellular or intracellular M. abscessus growth, revealed the efficacy of eight new candidates. The potential targets of CyC17 and CyC26, the two most potent inhibitors against extracellularly-growing bacteria, were identified via an activity-based protein profiling (ABPP) approach.

RESULTS AND DISCUSSION

Synthesis of new Cyclophostin phosphate and phosphonate analogs. We incremented the already available library comprising 26 CyC compounds (i.e., CyC1-18 including the cis-() and trans-() isomers), [START_REF] Nguyen | Cyclipostins and Cyclophostin analogs as promising compounds in the fight against tuberculosis[END_REF][START_REF] Nguyen | Cyclophostin and cyclipostins analogs, new promising molecules to treat mycobacterial-related diseases[END_REF] by synthesizing 12 additional analogs (CyC19-30) from various 3-keto esters following previously described synthetic routes (Scheme 1). [START_REF] Bandyopadhyay | Synthesis and Biological Evaluation of a Phosphonate Analog of the Natural Acetyl Cholinesterase Inhibitor Cyclophostin[END_REF][START_REF] Malla | The First Total Synthesis of (±)-Cyclophostin and (±)-Cyclipostin P: Inhibitors of the Serine Hydrolases Acetyl Cholinesterase and Hormone Sensitive Lipase[END_REF][START_REF] Point | Synthesis and kinetic evaluation of cyclophostin and cyclipostins phosphonate analogs as selective and potent inhibitors of microbial lipases[END_REF][START_REF] Vasilieva | Rat hormone sensitive lipase inhibition by cyclipostins and their analogs[END_REF] The general approach previously used for the synthesis of phosphonate analogs CyC4-10 was applied (Scheme 1A) to the synthesis of cyclic phosphonates with variation in the enol alkyl substituent (CyC19-22). The palladium-catalyzed substitution reaction of methyl or ethyl acetoacetate derivatives (2) [START_REF] Oikawa | Meldrum's acid in organic synthesis. 2. A general and versatile synthesis of .beta.-keto esters[END_REF] with the allylic carbonate (1) gave the vinyl phosphonates (3) as an E/Z mixture in good yields (35-63%). The E and Z isomers could be separated by silica gel chromatography, but were generally taken into the next reaction as mixture. Quantitative hydrogenation of (3a-d) led to the saturated phosphonates (4a-d), which after selective demethylation and cyclization gave the racemic forms of monocyclic enolphosphonates (5a-d) corresponding to compounds CyC19 to CyC22 (17-62%, 2 steps).

To optimize the potent observed antibacterial activity of compound CyC17, [START_REF] Nguyen | Cyclipostins and Cyclophostin analogs as promising compounds in the fight against tuberculosis[END_REF][START_REF] Nguyen | Cyclophostin and cyclipostins analogs, new promising molecules to treat mycobacterial-related diseases[END_REF] the synthesis of various monocyclic phosphate analogs has also been completed. Following a literature procedure 36- 37 β-ketoesters (8a-d) were prepared by alkylation of t-butyl acetoacetate derivatives (7a-d) with iodide (6) [START_REF] Herb | Total Synthesis of Salicylihalamides A and B[END_REF] (Scheme 1B). The tert-butyl ester was chosen so as to minimize the risk of lactonization upon deprotection of the alcohol function. Reaction of compounds (8a-d) with dimethyl chlorophosphite followed by oxidation of crude material with I2 and methanol gave the enolphosphates (9a-d) in modest yield along with recovered starting material (21-49%, 49-81% BRSM). The 4-methoxybenzyl ether (PMB) protecting group was then removed with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) under standard conditions to afford alcohols (10a-d).

Demethylation and subsequent cyclization using 1-mesitylene-sulfonyl-3-nitrotriazole (MSNT) gave the monocyclic phosphate tert-butyl esters CyC27 to CyC30 (11a-d). Cleavage of the tert-butyl moiety with TFA in anhydrous conditions followed by TMSCHN2 treatment resulted in the formation of the corresponding racemic cyclic enolphosphate methyl esters CyC23 to CyC26 (12a-d) in good yields (79-100%).

Scheme 1. Synthesis of novel monocyclic enol-phosphonate and -phosphate analogs of Cyclophostin.

Antibacterial activity of the new CyC19-CyC30 compounds. The antimicrobial activity of the 12 newly synthesized derivatives was evaluated as previously reported. [START_REF] Nguyen | Cyclophostin and cyclipostins analogs, new promising molecules to treat mycobacterial-related diseases[END_REF] Briefly, a preliminary screen on solid medium (30 µM final concentration) was performed on a selected panel of four pathogenic mycobacterial species (i.e., M. marinum, M. bovis BCG, M. tuberculosis mc 2 6230, and both S/R variants of M. abscessus) and 2 Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa). Following CFU count, 5 out of the 12 CyCs inhibited growth in the range of 50-100% relative to the positive growth control (i.e., bacteria without antibiotics). Moreover, and as previously observed with CyC1 to CyC18, [START_REF] Nguyen | Cyclophostin and cyclipostins analogs, new promising molecules to treat mycobacterial-related diseases[END_REF] this inhibitory activity was restricted to the four mycobacteria tested, while growth of E. coli and P. aeruginosa was not impacted (see Supplementary Material Table S1). The MIC of these selected CyC21, CyC22, CyC25, CyC26 and CyC30 towards each mycobacterial strain were next determined using the resazurin microtiter assay (REMA) [START_REF] Nguyen | Cyclipostins and Cyclophostin analogs as promising compounds in the fight against tuberculosis[END_REF][START_REF] Nguyen | Cyclophostin and cyclipostins analogs, new promising molecules to treat mycobacterial-related diseases[END_REF] (Table 1). Among the abovementioned CyCs, only CyC26 inhibited growth of M. marinum, M. bovis BCG and M. tuberculosis, with MIC50 values ranging from 1.8 to 26.4 µM (Table 1). Interestingly, CyC21 and CyC22 exhibited the best activity (MIC50 5.6-8.7 μM) against M. tuberculosis mc 2 6230. These latter compounds represent two new potential candidates against this bacterium, therefore extending the first series of CyC growth inhibitors previously identified. [START_REF] Nguyen | Cyclipostins and Cyclophostin analogs as promising compounds in the fight against tuberculosis[END_REF][START_REF] Nguyen | Cyclophostin and cyclipostins analogs, new promising molecules to treat mycobacterial-related diseases[END_REF] Despite the fact that only CyC25 and CyC26 were active against M. abscessus on solid medium (Table 1), we also determined the respective MIC50 values of all CyCs including the first series of 26 analogs against each S/R variant of M. abscessus to investigate deeper their efficiency/selectivity towards this pathogen (see Supplementary Material Table S2). Among the 38 tested compounds including the 12 new synthesized analogs, the best growth inhibitors against M. abscessus S variant still remained CyC17 and CyC18 which displayed similar MIC50 values (mean MIC50 = 11.7 ±0.94 µM) [START_REF] Nguyen | Cyclophostin and cyclipostins analogs, new promising molecules to treat mycobacterial-related diseases[END_REF] comparable to that of amikacin (AMK) (Table 1 S2). By contrast, nearly all CyCs were found more active against M. abscessus R variant as compared to the S variant.

Although most of the CyCs exhibited quite weak MIC50 in the range 100 to >200 µM; eight analogs displayed moderate activities (mean MIC50 = 51.7 ±7.4 µM for CyC6;8;9;18;21-22;29-30) (see Supplementary Material Table S2). CyC25 (MIC50 = 13.9 µM) and CyC26 (MIC50 = 6.9 µM) were confirmed as very good antimycobacterial molecules, in addition to CyC17 (MIC50 = 0.37 µM) and CyC18 (MIC50 = 8.9 µM). [START_REF] Nguyen | Cyclophostin and cyclipostins analogs, new promising molecules to treat mycobacterial-related diseases[END_REF] Of interest, these latter four best inhibitors of M. abscessus growth were all phosphate esters bearing a long lipophilic C10 / C16 alkyl chain either at the R 1 or R 3 position (Figure 1 and Scheme 1).

M. abscessus surface-exposed GPL drives the antibacterial potency of CyC compounds. From

these results, M. abscessus R appeared to be 6.1-to 34-times more sensitive than M. abscessus S to CyC25, CyC26 and CyC17 (Table 1). As previously mentioned, the major difference between these two variants resides in the loss of surface-associated glycopeptidolipids (GPL) in the R form. [START_REF] Roux | The distinct fate of smooth and rough Mycobacterium abscessus variants inside macrophages[END_REF][START_REF] Nessar | Deletion of the mmpL4b gene in the Mycobacterium abscessus glycopeptidolipid biosynthetic pathway results in loss of surface colonization capability, but enhanced ability to replicate in human macrophages and stimulate their innate immune response[END_REF][START_REF] Pawlik | Identification and characterization of the genetic changes responsible for the characteristic smooth-to-rough morphotype alterations of clinically persistent Mycobacterium abscessus[END_REF] It may be inferred that the GPL covering the surface of M. abscessus S acts as a protective shield towards the CyCs, although the natural Cyclipostin P CyC18β was not strongly affected by the GPL profile.

To confirm the possible relationship between CyC potency and GPL production, drug susceptibility was assessed using the GPL-deficient mmpL4b mutant generated in an S background of the type strain CIP104536 T , and its complemented counterpart. [START_REF] Medjahed | Construction of Mycobacterium abscessus Defined Glycopeptidolipid Mutants: Comparison of Genetic Tools[END_REF][START_REF] Roux | Overexpression of proinflammatory TLR-2-signalling lipoproteins in hypervirulent mycobacterial variants[END_REF][START_REF] Bernut | Mycobacterium abscessus cording prevents phagocytosis and promotes abscess formation[END_REF] The mmpL4b gene encodes for the MmpL4b membrane protein, which allows translocation of GPL across the plasma membrane. [START_REF] Medjahed | Construction of Mycobacterium abscessus Defined Glycopeptidolipid Mutants: Comparison of Genetic Tools[END_REF][START_REF] Ripoll | Genomics of glycopeptidolipid biosynthesis in Mycobacterium abscessus and M. chelonae[END_REF] Mutations in this gene result in the loss of GPLs and acquisition of a R morphotype. [START_REF] Nessar | Deletion of the mmpL4b gene in the Mycobacterium abscessus glycopeptidolipid biosynthetic pathway results in loss of surface colonization capability, but enhanced ability to replicate in human macrophages and stimulate their innate immune response[END_REF][START_REF] Medjahed | Construction of Mycobacterium abscessus Defined Glycopeptidolipid Mutants: Comparison of Genetic Tools[END_REF][START_REF] Roux | Overexpression of proinflammatory TLR-2-signalling lipoproteins in hypervirulent mycobacterial variants[END_REF] Both the wild-type M. abscessus S and the mmpL4b complemented strain responded similarly to CyC17, CyC25 and CyC26, with a more pronounced resistance level to these compounds as compared to the GPL-deficient mmpL4b mutant, which behaved like the wild-type R variant (Figure 2 and Table 2). Accordingly and as observed with M. abscessus S vs. R variant, the MIC50 of CyC25, CyC26 and CyC17 reached on the mmpL4b complemented strain were 4.8-to 32-times higher than those determined on the mmpL4b deletion mutant (Table 2). In contrast, there was a low influence of the GPL content in the activity of CyC18β, as judged by the obtained MIC50 values against different strains (Table 2). A significant reduced susceptibility to CyC17 has been reported previously in a M. smegmatis_lipg mutant strain in which GPLs were overexpressed due to disruption of lipG, resulting in a change vs. WT in MIC50 value of ×15.7 fold. [START_REF] Santucci | LipG a bifunctional phospholipase/thioesterase involved in mycobacterial envelope remodeling[END_REF] Both results are also in agreement with a study [START_REF] Khoo | Altered expression profile of the surface glycopeptidolipids in drug-resistant clinical isolates of Mycobacterium avium complex[END_REF] reporting that M. avium GPL-positive serovar was less sensitive to ethambutol than GPL-negative one, presumably because hydrophobic GPL may alter cell-wall fluidity/permeability and correlate with antibiotic tolerance. [START_REF] Khoo | Altered expression profile of the surface glycopeptidolipids in drug-resistant clinical isolates of Mycobacterium avium complex[END_REF][START_REF] Sarathy | The role of transport mechanisms in mycobacterium tuberculosis drug resistance and tolerance[END_REF] Together, these findings underline the importance of the surfaceexposed GPL in the CyC-dependent inhibition of M. abscessus growth. Remarkably, CyC18β, which differs from CyC17 by the presence of a butyrolactone ring fused to the seven-membered cyclic phosphate triester (Figure 1), failed to show a high GPL-dependent activity profile. Accordingly, the presence of the lactone may be responsible for this behavior, different from the other monocyclic CyC analogs, towards M. abscessus variants.

Activity-based protein profiling (ABPP) approach for targets identification. In light of our previous study demonstrating the strong affinity of the CyCs for (Ser/Cys)-enzymes bearing a catalytic serine or cysteine residue, 4-9, 37, 47-48 we presumed that these inhibitors would also target/impair the activity of enzymes participating in a wide range of physiological processes of the M. abscessus life cycle, resulting in bacterial growth inhibition, as previously shown in M.

tuberculosis. [START_REF] Nguyen | Cyclipostins and Cyclophostin analogs as promising compounds in the fight against tuberculosis[END_REF] Hence, to gain access to their putative targets, activity-based protein profiling (ABPP) [START_REF] Nguyen | Cyclipostins and Cyclophostin analogs as promising compounds in the fight against tuberculosis[END_REF][START_REF] Tallman | Small Molecule Probes Reveal Esterases with Persistent Activity in Dormant and Reactivating Mycobacterium tuberculosis[END_REF][START_REF] Lehmann | An Antibacterial beta-Lactone Kills Mycobacterium tuberculosis by Disrupting Mycolic Acid Biosynthesis[END_REF] was employed to capture and identify candidate target enzyme(s) impacted by CyC17 and CyC26, the two best hits acting against extracellular M. abscessus.

In the first approach, a crude lysate of M. abscessus R was incubated with either a CyC inhibitor (or DMSO as a control) and subjected to a competitive probe labeling/enrichment assay with a Desthiobiotin-FP probe (see Supplementary Material Figure S1A). [START_REF] Nguyen | Cyclipostins and Cyclophostin analogs as promising compounds in the fight against tuberculosis[END_REF] The enriched mixtures were then digested with trypsin and the resulting peptides analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), followed by subsequent label free quantification analysis. The proteins that were also found in the control experiment (i.e., DMSO alone for unspecific binding to streptavidin-magnetic beads) were discarded. At first, proteins identified with a permutation false discovery rate (pFDR) up to 10% were selected, leading to a panel of 806 distinct protein candidates in the case of CyC17, which was narrowed down to 30 when applying a pFDR of 1%, as compared to only 4 with CyC26 (14 at a pFDR of 10%) (see Supplementary Material Tables S3-S8).

Given the fact that orthologs typically perform equivalent functions in the respective organisms, [START_REF] Koonin | Orthologs, Paralogs, and Evolutionary Genomics[END_REF] S4-S5). This was substantiated with CyC26 for which 8 out of Interestingly the histidinol-phosphate aminotransferase (i.e., MAB_2669c or HisC1), possibly involved in the histidine pathway and annotated as an essential enzyme in M. tuberculosis, [START_REF] Griffin | High-Resolution Phenotypic Profiling Defines Genes Essential for Mycobacterial Growth and Cholesterol Catabolism[END_REF][START_REF] Sassetti | Genes required for mycobacterial growth defined by high density mutagenesis[END_REF] was also uncovered with CyC26. All target-proteins of CyC26 were also identified with CyC17, thus confirming this latter as a broader multi-target inhibitor. Given the putative mechanism of action of HisC1 (MAB_2669c) together with the recent resolution of the 3D structure of its M. tuberculosis ortholog Rv1600 in complex with its natural substrate, i.e. pyridoxal-5'-phosphate (PDB id: 4R8D), [START_REF] Nasir | Crystal structures of Mycobacterium tuberculosis HspAT and ArAT reveal structural basis of their distinct substrate specificities[END_REF] one can hypothesize that the two phosphate analogs CyC17 and CyC26 may bind to the active site, thus acting as substrate-like adducts.

In the second approach, the penetration/diffusion of CyC17 inhibitor through the mycobacterial cell wall was taken into account in order to reduce the list of potential targets. Thus, a similar experiment was performed on living bacterial cells. [START_REF] Nguyen | Oxadiazolone derivatives, new promising multi-target inhibitors against M. tuberculosis[END_REF] M. abscessus R cells were grown to log phase and incubated with CyC17 (or DMSO as a control). After cell lysis, part of the lysate was processed as described above with Desthiobiotin-FP probe and streptavidin magnetic beads (see Supplementary Material Figure S1B). In parallel, the CyC17-treated lysate was also incubated with TAMRA-FP to reveal by in-gel fluorescence on an SDS-PAGE scanning, the (Ser/Cys)-enzyme candidates that presumably had reacted with this inhibitor [START_REF] Nguyen | Cyclipostins and Cyclophostin analogs as promising compounds in the fight against tuberculosis[END_REF] (see Supplementary Material Figure S1C). At this stage, around 20 distinct bands labeled by TAMRA-FP were detected in the fluorescence readout and also visible by Coomassie staining after release of the enzymes captured by Desthiobiotin-FP (see Supplementary Material Figure S1C-D). In contrast, pre-treatment with CyC17 resulted in a strong decrease in fluorescence intensity of all visible bands; the CyC17-enzyme complex being unable to react with the TAMRA-FP probe. Tryptic digestion followed by tandem mass spectrometry analysis led to the identification of lower numbers of protein candidates as compared to a total lysate; i.e., 39 vs. 208 at a pFDR of 5%, respectively (Supplementary Material Table S6). It is noteworthy that among these 39 potential hits, 24 were previously detected in the CyC17-treated total lysate (see Supplementary Material Tables S3-4): 16 at a pFDR of 10%, 1 at a pFDR of 5%, and 7 at a pFDR of 1%; thus implying that 15 proteins had not been detected in the previous treated M. abscessus total lysate, or at least for a pFDR > 10%.

Consistent with previous work involving CyC17, [START_REF] Nguyen | Cyclipostins and Cyclophostin analogs as promising compounds in the fight against tuberculosis[END_REF] a variety of hydrolases were detected, including one amidase AmiC (MAB_2181c), two Lip-family members LipI (MAB_2814) and LipT (MAB_3336c), two Cutinase-like proteins Cut1 (MAB_3272c) and Cut3 (MAB_3765), a possible hydrolase (MAB_3034), and MAB_176 (Ag85A) and MAB_177 (Ag85-A/B/C precursor) two members of the antigen 85 (Ag85) complex, which catalyzes the formation of trehalose dimycolate and adds mycolic acids to arabinogalactan 9, [START_REF] Sacchettini | The mycobacterial antigens 85 complex--from structure to function: response[END_REF][START_REF] Backus | The Three Mycobacterium tuberculosis Antigen 85 Isoforms Have Unique Substrates and Activities Determined by Non-active Site Regions[END_REF] (see Supplementary Material Table S6).

Interestingly, 9 out of the 39 identified proteins are annotated as essential enzymes in M. tuberculosis genome (Table 3). [START_REF] Griffin | High-Resolution Phenotypic Profiling Defines Genes Essential for Mycobacterial Growth and Cholesterol Catabolism[END_REF][START_REF] Sassetti | Genes required for mycobacterial growth defined by high density mutagenesis[END_REF] Among them, MAB_0172 (i.e., Rv3807c) & MAB_3612 (i.e., Rv3265c) are possibly involved in the arabinogalactan biosynthesis; [START_REF] Cai | Prokaryotic Expression, Identification and Bioinformatics Analysis of the Mycobacterium tuberculosis Rv3807c Gene Encoding the Putative Enzyme Committed to Decaprenylphosphoryl-d-arabinose Synthesis[END_REF][START_REF] Mills | Inactivation of the mycobacterial rhamnosyltransferase, which is needed for the formation of the arabinogalactan-peptidoglycan linker, leads to irreversible loss of viability[END_REF] and MAB_0944 (i.e., Rv0896) is a probable citrate synthase required for the tricarboxylic acid cycle. 60 Of particular interest, the Ag85A/B/C proteins from M. tuberculosis have been recently validated as real targets of CyC17. [START_REF] Nguyen | Cyclipostins and Cyclophostin analogs as promising compounds in the fight against tuberculosis[END_REF]9 Herein, we confirm that the recombinant M. abscessus Ag85C protein, which shares nearly 60% amino acid sequence identity with its M. tuberculosis ortholog and possesses the same conserved catalytic triad (i.e., Ser 124 -Glu 228 -His 260 ), is also inactivated by CyC17.

Ag85CMabs (50 µM) was incubated in its native form with 1 mM (i.e. enzyme/inhibitor molar ratio of Quantification of labeling normalized to protein load revealed that pre-incubation with CyC17 resulted in a significant decrease in fluorescence intensity by around 70% as compared with the nontreated protein. This implies that reaction with the TAMRA probe is strongly impaired in the Ag85CMabs-CyC17 adduct, resulting in a significant loss of fluorescence emission.

Activity of CyC analogs against intracellular M. abscessus. Similar to M. tuberculosis, following

lung infection M. abscessus can grow and survive intracellularly inside macrophages. [START_REF] Wu | NTM drug discovery: status, gaps and the way forward[END_REF][START_REF] Roux | The distinct fate of smooth and rough Mycobacterium abscessus variants inside macrophages[END_REF] However, MIC values determined in broth medium do not always correlate with the activity of the compounds against intracellular bacteria. Such discrepancy between extra-and intracellular activity has been reported in the case of M. tuberculosis with the CyCs 7 as well as another family of inhibitors, the Oxadiazolone-core derivatives. [START_REF] Nguyen | Oxadiazolone derivatives, new promising multi-target inhibitors against M. tuberculosis[END_REF] From these findings, all 38 CyC analogs were further tested for their antibacterial activity against M. abscessus growth inside infected macrophages.

The cytotoxicity of the 12 new compounds was first assessed using murine Raw264.7 macrophages. [START_REF] O'brien | Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity[END_REF] Except CyC26 (CC50 = 50 µM), all other inhibitors CyC19-25;27-30 showed very low toxicity towards the cells with CC50 > 100 μM, similarly to AMK (CC50 ≥150 μM). [START_REF] Christophe | High content screening identifies decaprenyl-phosphoribose 2' epimerase as a target for intracellular antimycobacterial inhibitors[END_REF] Taking into account our previous toxicity results obtained with CyC1-18, 7 the 38 synthesized analogs exhibited low cytotoxic towards host mammalian cells.

Due to its high clumping capability, infection with M. abscessus R results in the lysis of nearly all macrophages at 24 h post-infection, making it very difficult to quantify in a reliable manner the intracellular effect of the CyCs. This is, however, not the case with M. abscessus S which does not aggregate like the R form, thus enabling the preparation of homogenous and dispersed suspensions.

The intracellular growth of M. abscessus S was next assessed following a 24 h exposure of infected Raw264.7 cells to the CyC compounds at a final concentration of 30 µM. [START_REF] Rodrigues Felix | Selective Killing of Dormant Mycobacterium tuberculosis by Marine Natural Products[END_REF] To avoid growth of extracellular mycobacteria, cells were extensively washed and treated with amikacin (200 µg/mL = 340 µM; 87 × MIC50) prior to treatment with the CyC analogs. Imipenem (IMP; 80 µg/mL = 267 µM; 67 × MIC50) was used as positive control for this intracellular killing assay. In each case, the viability of infected macrophages was checked by addition of trypan blue [START_REF] Strober | Trypan Blue Exclusion Test of Cell Viability[END_REF] phosphate (i.e., CyC17) chemical groups have been reported for M. tuberculosis. [START_REF] Nguyen | Cyclipostins and Cyclophostin analogs as promising compounds in the fight against tuberculosis[END_REF] In addition, the fact that only CyC7(α,β) and CyC8(α,β) showed a clear preference against intracellularly-replicating mycobacteria may imply that the intracellular mode of action of these CyCs differs from that of CyCs acting exclusively on extracellularly-replicating bacilli. Another hypothesis is that accessibility and/or vulnerability of their corresponding target(s) may be more apparent and crucial during the intracellular lifestyle of M. abscessus. Moreover, a specific stringent response of the macrophage, such as possible host cell metabolism, stimulated by the action of these latter compounds and leading to bacterial clearance cannot, however, be excluded.

To confirm this screen, M. abscessus S infected macrophages were subjected to a dose-response assay using the latter selected CyC7(α,β) and CyC8(α,β) analogs or IMP (Figure 3), taken as reference intracellular drug, in order to determine their respective intracellular MIC50Raw values (Table 4). Both

CyC7α and CyC7β displayed a moderate activity against intracellular M. abscessus S (Figure 3A) 3C). Regarding CyC8β, a plateau value corresponding to 64.7 ±3.8% bacterial killing was reached when the infected cells were treated with the compound at a 5-30 µM concentration range.

with
Remarkably, MIC50Raw values of CyC8α (7.9 µM) as well as CyC8β (2.0 µM) towards intramacrophagic bacilli were 3.6-and 14-fold lower than that of IMP (28.3 µM) (Table 4). These

MIC50Raw are of the same order of magnitude than those previously obtained with CyC8(α,β) acting against intracellular M. tuberculosis (i.e., MIC50Raw_Mtb around 4-12 µM). [START_REF] Nguyen | Cyclipostins and Cyclophostin analogs as promising compounds in the fight against tuberculosis[END_REF] The selectivity index (SI = CC50/MIC50Raw) of the four best inhibitors on intracellular M. abscessus vs. Raw264.7 cells was thus found to be in a range from 1.1 and up to 35 for CyC8β. Overall, these results indicate that the (α) and (β) isomers of CyC7 and CyC8 could enter the macrophages and arrest bacterial replication without exhibiting significant toxicity for the host cell, with comparable effects to IMP. in addition to CyC17 and CyC18β, as active inhibitors of R and S variants. We demonstrated that the absence of surface-exposed GPL in the R variant was responsible for the higher susceptibility to the CyCs as compared to the S strain. The absence of GPL may induce some changes in the cell-wall fluidity/permeability, possibly favoring the penetration/action of these inbhibitors. Putative enzymes targeted by CyC17 or CyC26, the two best extracellular inhibitors, were identified using an ABPP approach. As anticipated, identified proteins were mainly serine or cysteine enzymes involved in mycobacterial lipid metabolism, among them 11 were annotated as essential enzymes in M.

tuberculosis genome for in vitro growth or in infected macrophages. When tested against intracellular bacteria, CyC7(α,β) and CyC8(α,β) appeared as very potent and promising inhibitors with comparable or even lower MIC50Raw values than that of the standard antibiotics imipenem. Of major interest, such inhibitors were selectively and efficiently acting on major pathogenic mycobacteria inside macrophages, including M. tuberculosis [START_REF] Nguyen | Cyclipostins and Cyclophostin analogs as promising compounds in the fight against tuberculosis[END_REF] and now also M. abscessus. In addition to their low toxicity towards host cells, the CyC inhibitors potentially act as multi-target compounds, confirming their potential against one of the most drug-resistant mycobacterial species. It is very likely that blocking simultaneously enzymes involved in various lipid and/or in cell wall biosynthetic pathways would lead to extracellular and/or intracellular inhibition of M. abscessus growth by the selected CyCs. This dual activity of the CyCs is of major importance as it may affect the different stages of the infection process. Moreover, their association with current antibiotics to investigate their potential synergetic activity against resistant M. abscessus strains is currently in progress, and could open the way for improving the current treatments against these mycobacterial infections.

EXPERIMENTAL SECTION

Chemistry -synthesis of compounds CyC19 to CyC30

The synthesis of natural Cyclophostin CyC1, [START_REF] Malla | The First Total Synthesis of (±)-Cyclophostin and (±)-Cyclipostin P: Inhibitors of the Serine Hydrolases Acetyl Cholinesterase and Hormone Sensitive Lipase[END_REF] its phosphonate analogs CyC2 and CyC2, [START_REF] Bandyopadhyay | Synthesis and Biological Evaluation of a Phosphonate Analog of the Natural Acetyl Cholinesterase Inhibitor Cyclophostin[END_REF] the monocyclic enolphosphonates CyC3-4 [START_REF] Dutta | Synthesis and kinetic analysis of some phosphonate analogs of cyclophostin as inhibitors of human acetylcholinesterase[END_REF] and the trans-() and cis-() diastereoisomers CyC5-10; [START_REF] Point | Synthesis and kinetic evaluation of cyclophostin and cyclipostins phosphonate analogs as selective and potent inhibitors of microbial lipases[END_REF] as well as the trans-() and cis-() Cyclipostin P CyC18 4 and the corresponding monocyclic phosphonate CyC11-13, [START_REF] Point | Synthesis and kinetic evaluation of cyclophostin and cyclipostins phosphonate analogs as selective and potent inhibitors of microbial lipases[END_REF][START_REF] Nguyen | Cyclipostins and Cyclophostin analogs as promising compounds in the fight against tuberculosis[END_REF] difluorophosphonate CyC14-15 and phosphate CyC16-17 [START_REF] Vasilieva | Rat hormone sensitive lipase inhibition by cyclipostins and their analogs[END_REF][START_REF] Martin | Synthesis and comparison of the biological activity of monocyclic phosphonate, difluorophosphonate and phosphate analogs of the natural AChE inhibitor cyclophostin[END_REF] analogs have already been reported. The 12 new analogs (CyC19-30) where synthesized in racemic form from various 3-keto esters following already described synthetic routes (See Supplementary Material for detailed procedures and the full chemical characterization of each new compounds), [START_REF] Bandyopadhyay | Synthesis and Biological Evaluation of a Phosphonate Analog of the Natural Acetyl Cholinesterase Inhibitor Cyclophostin[END_REF][START_REF] Malla | The First Total Synthesis of (±)-Cyclophostin and (±)-Cyclipostin P: Inhibitors of the Serine Hydrolases Acetyl Cholinesterase and Hormone Sensitive Lipase[END_REF][START_REF] Point | Synthesis and kinetic evaluation of cyclophostin and cyclipostins phosphonate analogs as selective and potent inhibitors of microbial lipases[END_REF][START_REF] Vasilieva | Rat hormone sensitive lipase inhibition by cyclipostins and their analogs[END_REF] and have purity of ≥ 95% as determined by HPLC analysis as reported previously. [START_REF] Point | Synthesis and kinetic evaluation of cyclophostin and cyclipostins phosphonate analogs as selective and potent inhibitors of microbial lipases[END_REF] The HPLC data were supported by careful analysis of the 1 H, [START_REF] Wu | NTM drug discovery: status, gaps and the way forward[END_REF] C and particularly the [START_REF] Richard | Mutations in the MAB_2299c TetR Regulator Confer Cross-Resistance to Clofazimine and Bedaquiline in Mycobacterium abscessus[END_REF] displaying an R morphotype [START_REF] Medjahed | Construction of Mycobacterium abscessus Defined Glycopeptidolipid Mutants: Comparison of Genetic Tools[END_REF] and its complemented counterpart (M. abscessus S_mmpL4b::C), which stably expresses MmpL4b under the control of the hsp60 promoter, were also used as previously reported in [START_REF] Roux | The distinct fate of smooth and rough Mycobacterium abscessus variants inside macrophages[END_REF] .

Antibiotics. Amikacin was purchased from Euromedex (Souffelweyersheim, France), and imipenem was provided by Mylan (Saint-Priest, France). Stock solution in water were freshly prepared for each experiment and filtered through a sterilized 0.22-µm-pore-size polycarbonate syringe filter (Millipore, Saint-Quentin-en-Yvelines, France).

Drug susceptibility testing on solid medium. This was performed in 24-well suspension culture plates (Greiner bio-one) as described previously. [START_REF] Blanco-Ruano | Antimicrobial Susceptibility Testing for Mycobacterium sp[END_REF] aeruginosa. Each well was spotted with 10 µL of a bacterial culture at 5 × 10 5 cells/mL. Colonies were counted after 1 day to 2 weeks of incubation at 37 °C, depending on the strain tested, to check bacterial viability. The CyC compounds leading to a minimum of 50% growth inhibition from CFU count were selected for subsequent MIC determination using the REMA assay.

Resazurin microtiter assay (REMA) for MIC determination.

The concentrations of compound leading to bacterial growth inhibition were first determined using the resazurin microtiter assay (REMA). [START_REF] Nguyen | Cyclipostins and Cyclophostin analogs as promising compounds in the fight against tuberculosis[END_REF][START_REF] Nguyen | Cyclophostin and cyclipostins analogs, new promising molecules to treat mycobacterial-related diseases[END_REF][START_REF] Nguyen | Oxadiazolone derivatives, new promising multi-target inhibitors against M. tuberculosis[END_REF] Briefly, log-phase bacteria were diluted to a cell density of 5 × 10 6 cells/mL and 100 µL of this inoculum was grown in a 96-well plate in the presence of serial dilutions of compounds.

After 7-14 days incubation, 20 µL of a 0.025% (w/v) resazurin solution was added to each well (200 µL) and incubation was continued until the appearance of a color change (from blue to pink) in the control well (bacteria without antibiotics). Fluorescence of the resazurin metabolite resorufin (excitation, 530 nm; emission, 590 nm) was then measured, [START_REF] Nguyen | Cyclipostins and Cyclophostin analogs as promising compounds in the fight against tuberculosis[END_REF][START_REF] Nguyen | Cyclophostin and cyclipostins analogs, new promising molecules to treat mycobacterial-related diseases[END_REF][START_REF] Nguyen | Oxadiazolone derivatives, new promising multi-target inhibitors against M. tuberculosis[END_REF] and the concentration leading to 50% growth inhibition was defined as the MIC50. See Supplementary Material for detailed protocol.

Determination of cytotoxic activity for new CyC19-CyC30 analogs (resazurin assay).

The cytotoxicity of compounds against eukaryotic cells was measured based on the reduction of resazurin [START_REF] O'brien | Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity[END_REF][START_REF] Rodrigues Felix | Selective Killing of Dormant Mycobacterium tuberculosis by Marine Natural Products[END_REF] Experiments were done three times independently.

CyC17 and CyC26 target enzymes identification. Protein identification and quantification. Protein extract were loaded and stacked on a NuPAGE gel (Life Technologies). Stained bands were submitted to an in-gel trypsin digestion. [START_REF] Shevchenko | Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels[END_REF] Peptides extracts were reconstituted with 0.1% trifluoroacetic acid in 4% acetonitrile and analyzed by liquid chromatography (LC)-tandem mass spectrometry (MS/MS) using a LTQ-Orbitrap Velos Mass Spectrometer (Thermo Electron, Bremen, Germany) online with a nanoLC Ultimate 3000 chromatography system (Dionex, Sunnyvale, CA). Protein identification and quantification were processed using the MaxQuant computational proteomics platform, version 1.5.3.8 [START_REF] Cox | MaxQuant enables high peptide identification rates, individualized p.p.b.range mass accuracies and proteome-wide protein quantification[END_REF]

Activity-based protein profiling (ABPP)

Figure 1 .

 1 Figure 1. Chemical structure of natural Cyclipostins and Cyclophostin, and related CyC analogs. Natural Cyclophostin (CyC1), Cyclipostin P (CyC18β) and its trans diastereoisomer (CyC18α), Cyclophostin phosphonate analog (CyC2), monocyclic enolphosphorus analogs to either Cyclophostin (CyC3-10;15-16) or

Figure 2 .

 2 Figure 2. Dose-response activity of CyC17 and CyC26 against M. abscessus S, mmpL4b mutant (M. abscessus S_mmpL4b), mmpL4b complemented (M. abscessus S_mmpL4b::C), and M. abscessus R strains replicating in broth medium, expressed as normalized relative fluorescence units (RFU%). For each concentration, data are means ± SD of at least two independent assays performed in duplicate (CV% <5%).

  14 identified proteins are (Ser/Cys)-based enzymes. These included the putative β-lactamase MAB_2833 (i.e., Rv1367c), possibly involved in cell wall biosynthesis; the D-amino acid aminohydrolase MAB_2605c (i.e., Rv2913c) and the probable peptidase MAB_4130 (i.e., Rv0457c); three members of the lipase family Lip, LipH (MAB_2039), LipN (MAB_3270c) and LipW (MAB_0826c); and two Cutinase-like proteins, Cut1 (MAB_3272c) and Cut4 (MAB_3809c).

1: 20 )

 20 CyC17 and then treated with 10 µM TAMRA-FP fluorescent probe for 1 h. Equal amounts of proteins (2 µg) were separated by 12% SDS-PAGE and visualized by Coomassie staining or in-gel fluorescence for TAMRA detection (see Supplementary Material Figure S2 for details).

  before cell lysis and plating for CFU counting. Nearly all compounds, including the CyC19-30 new series, failed to show an effect at the concentration investigated. In agreement with previous work with M. tuberculosis, 7 both (α) and (β) isomers of CyC7 and CyC8, which are not active against extracellular bacilli, showed a 40-50% and 63-65% decrease in intramacrophagic CFUs 24 h post infection in the presence of CyC7(α,β) and CyC8(α,β), respectively. Interestingly, although the phosphate analogs CyC17, CyC18β, CyC25 and CyC26 exclusively impaired extracellular growth of M. abscessus, they however remained fully inactive towards infected macrophages. In contrast, the phosphonate analogs CyC7-8 were found active only against intramacrophagic M. abscessus. Similar properties between the phosphonate (i.e., CyC7-8) vs.

Figure 3 .

 3 Figure 3. Intracellular activity of (A) CyC7α & CyC7β, and (B) CyC8α & CyC8β as compared to (C) imipenem (IMP). The activity of selected CyCs on intracellular M. abscessus was tested in Raw264.7 murine macrophages. Cells were infected at a multiplicity of infection (MOI) of 10 with M. abscessus S variant and treated with various concentrations of each inhibitor or IMP for 24 h. Then, surviving bacteria were enumerated by plating serial dilutions of macrophage lysates. Untreated infected macrophages were used as control representing 100% of bacterial viability. Results are shown as mean ± standard error of the mean (SEM) of three independent assays performed in triplicate. SC, solvent control (DMSO). **, p-value < 0.01. *, pvalue < 0.05. Statistical analysis was done using a Student's t-test.

  7 macrophages were infected by M. abscessus S at a MOI of 10, and further treated with each CyC or IMP for 24 h. The viable mycobacteria were quantified using the agar plating method. Untreated infected macrophages were used as control representing 100% of bacterial viability. MIC50Raw were calculated from curve fitting of CFU% as a function of the inhibitor concentration. Data are expressed as mean values of three independent assays performed in triplicate. IMP, imipenem. SI, selectivity index: SI = CC50/MIC50Raw. CONCLUSION Herein, we have extended our first series of 26 CyC analogs by synthesizing 12 new phosphonate (i.e., CyC19-22) and phosphate (i.e., CyC23-30) compounds in which we varied the R 3 lipophilic chain located on the 7-membered enolphosphorus ring. Evaluation of their antibacterial activity first highlighted CyC21 and CyC22 as two potential candidates against M. tuberculosis. With respect to M. abscessus, the MIC determination of this set of 38 CyCs provided CyC25 and CyC26,

  Homogeneous bacterial suspension of M. abscessus R in 7H9-S was adjusted at an OD600 of 40 and then incubated with the selected CyC17 or CyC26 inhibitor (400 µM final concentration) or DMSO (control) at 37 °C for 2-3 h. under gentle shaking at 75 rpm. Bacteria were then washed 3 times with PBS containing 0.05% Tween 80, resuspended in PBS buffer at a 1:1 (w/v) ratio and then lysed by mechanical disruption on a BioSpec Beadbeater.Both CyC-treated M. abscessus and DMSO-control lysate samples (750 µL -0.75 mg total proteins) were labeled with 2 µM Desthiobiotin-FP probe for 90 min at room temperature. Samples were enriched for biotinylated proteins using Nanolink streptavidin magnetic beads 0.8 µm (Solulink), according to the manufacturer's instructions. The resulting captured biotinylated proteins solution was mixed with 5X Laemmli reducing sample buffer, and heated at 95 °C for 5 min. The released denatured proteins were subjected to tryptic digestion, peptide extraction, and LC-MS/MS analysis as described below. Alternatively, the CyC-treated M. abscessus and DMSO-control lysate samples (100 µL -100 µg total proteins) were incubated with 2 μM ActivX TAMRA-FP probe (Thermo Fisher Scientific) for 90 min at room temperature and in absence of light. The reaction was stopped by adding 4X Laemmli reducing sample buffer and boiling at 95 °C for 5 min. The labeled proteins were further separated by SDS-PAGE electrophoresis. TAMRA fluorescence (TAMRA: λex 557 nm, λem 583 nm) was detected using a ChemiDoc MP Imager (Bio-Rad).Detailed Material and Methods regarding ABPP experiments is given in Supplementary Material.

for CyC1-4;6-9;11;16;19;21;27-28), or a moderate (MIC50 = 45-97 µM for CyC15;18;20;22;25-26;29-30) antibacterial activity (see Supplementary Material Table

	). In all other cases, MIC50 were indicative
	either of a poor (MIC50 ≥117 µM

Table 1 .

 1 Antibacterial activities of the selected active CyC compounds against four pathogenic mycobacterial strains a

				MIC50 (µM)
	Cpds	M. marinum ATCC BAA-535/M	M. bovis BCG	M. tuberculosis mc 2 6230	M. abscessus CIP 104536 T
					S variant	R variant
	AMK	1.1 ±0.05	0.54 ±0.03	0.42 ±0.02	3.9 ±0.19	7.4 ±0.26
	INH	20.5 ±0.71	0.71 ±0.03	1.5 ±0.07	>100	>100
	CyC17 b	1.5 ±0.04	0.54 ±0.01	0.55 ±0.02	12.7 ±0.26	0.37 ±0.01
	CyC18β b	23.1 ±1.07	6.0 ±0.29	1.6 ±0.07	10.8 ±0.43	8.9 ±0.09
	CyC21	>100	13.8 ±0.69	8.7 ±0.28	139.0 ±4.80	63.0 ±0.91
	CyC22	>100	24.9 ±1.16	5.6 ±0.25	65.0 ±2.19	51.0 ±2.20
	CyC25	>100	>100	>100	85.0 ±0.86	13.9 ±0.56
	CyC26	1.8 ±0.08	12.4 ±0.44	26.4 ±0.60	60.0 ±2.50	6.9 ±0.16
	CyC30	>100	14.2 ±0.60	>100	62.6 ±2.39	57.0 ±2.70

a MIC50 corresponding to the concentration leading to 50% growth inhibition as determined by the REMA assay, are expressed as mean values of two independent assays performed in triplicate (CV% <5%). b Data for CyC17 and CyC18β are from

[START_REF] Nguyen | Cyclophostin and cyclipostins analogs, new promising molecules to treat mycobacterial-related diseases[END_REF]

. AMK, amikacin. INH, isoniazid.

Table 2 .

 2 Variation

of MIC50 (µM) of CyC17, CyC18β, CyC25 and CyC26 against both R & S variants of M. abscessus as well as mmpL4b mutant strains a MIC50 (

µM) Fold change in MIC50

		M. abscessus S	M. abscessus R	M. abscessus S_mmpL4b	M. abscessus S_mmpL4b::C	R → S	mmpL4b → mmpL4b::C
	CyC17	12.7 ±0.26	0.37 ±0.01	0.60 ±0.03	19.2 ±0.62	×34.3	×32.0
	CyC18β	10.8 ±0.43	8.9 ±0.09	3.3 ±0.16	5.4 ±0.26	×1.2	×1.6
	CyC25	85.0 ±0.86	13.9 ±0.56	15.7 ±0.73	74.9 ±3.4	×6.1	×4.8
	CyC26	60.2 ±2.50	6.9 ±0.16	7.2 ±0.28	67.3 ±3.1	×8.7	×9.3
	a MIC50 corresponding to the concentration leading to 50% growth inhibition as determined by the REMA
	assay, are expressed as mean values of two independent assays performed in triplicate (CV% <5%).

  the corresponding orthologs in M. tuberculosis H37Rv genome of the various identified proteins were reported, adding reliable predictions of the gene functions in terms of essentiality, genomic location and activity. With each inhibitor, except for a few uncharacterized proteins, most of the identified hits were (Ser/Cys)-based enzymes participating in mycobacterial lipid metabolism (see Supplementary Material Tables

Table 3 .

 3 CyC17 target proteins identified in M. abscessus R culture by LC-ESI-MS/MS analysis and annotated as essential a for M. tuberculosis orthologs.

	Protein Ids	Mol. Weight [kDa]

a from

[START_REF] Griffin | High-Resolution Phenotypic Profiling Defines Genes Essential for Mycobacterial Growth and Cholesterol Catabolism[END_REF][START_REF] Sassetti | Genes required for mycobacterial growth defined by high density mutagenesis[END_REF]

.

b CF: Culture filtrate; CW: Cell wall; M: Membrane fraction; WCL: Whole cell lysate. c IM/R: Intermediary metabolism/respiration; CW/CP: cell wall/cell processes; LM: Lipid metabolism; V/D/A: Virulence, detoxification, adaptation.

Table 4 .

 4 Antibacterial activities of the most active CyC analogs against M. abscessus S infected macrophages a

	Compounds	CC50 (µM)	MIC50Raw (µM)	SI
	IMP	ND	28.3 ±4.7	-
	CyC7α	>100	29.3 ±2.9	>3.4
	CyC7β	>70	65.3 ±6.2	>1.1
	CyC8α	>100	7.9 ±0.23	>12.7
	CyC8β	>70	2.0 ±0.91	>35
	CyC17	>100	no effect	-
	CyC18β	>100	no effect	-
	CyC25	>100	no effect	-
	CyC26	>50	no effect	-

a Experiments were performed as described in Experimental Section. CC50: compound concentration leading to 50% Raw264.7 macrophages toxicity. MIC50Raw: minimal compound concentration leading to a 50% decrease in CFU count as compared to untreated cells. Raw264.

  P NMR spectra (see Supplementary Material). Stock solutions (10 mM) in which the CyC compounds were found to be completely soluble in dimethyl sulfoxide (DMSO), were prepared prior to drug susceptibility testing.

	Antibacterial evaluation.
	Bacterial strains and growth condition. M. marinum ATCC BAA-535/M, M. bovis BCG
	Pasteur, M. abscessus CIP104536 T with either a smooth (S) or rough (R) morphotype, and M.
	tuberculosis mc 2 6230 (H37Rv RD1 panCD 65 ) strains were routinely grown in Middlebrook 7H9
	broth (BD Difco, Le Pont de Claix, France) supplemented with 0.2% glycerol, 0.05% Tween 80
	(Sigma-Aldrich, St. Quentin Fallavier, France) and 10% oleic acid, albumin, dextrose, catalase
	(OADC enrichment; BD Difco) (7H9-S). In the case of M. tuberculosis mc 2 6230, 24 µg/mL D-
	pantothenate (Sigma-Aldrich) was also added in the 7H9-S medium. All cultures were kept at 37 °C
	without shaking, except M. marinum which was grown at 32 °C. Escherichia coli DH10B and
	Pseudomonas aeruginosa PA01 were grown at 37 °C in LB Broth Base medium (ThermoFisher
	Scientific, Illkirch, France). A M. abscessus S mmpL4b KO (M. abscessus S_mmpL4b) mutant

  E. coli and P. aeruginosa were grown on LB agar medium at 37 °C. All mycobacteria were grown at either 32 (M. marinum) or 37 °C (M. abscessus, M. bovis BCG and M. tuberculosis mc 2 6230) on Middlebrook 7H10 agar (BD Difco) supplemented with 10% OADC, and 24 µg/mL D-pantothenate (M. tuberculosis mc 2 6230). The wells were filled with 1 mL of the appropriate medium containing each of the CyC19-CyC30 analogs at a single 30 µM final concentration. Each screening plate contained negative (DMSO) and positive(50 μM

antibiotics) controls, as well as one well for sterility control (i.e., medium alone). For the 100% inhibition control we used 50 µM amikacin (Sigma Aldrich) for M. marinum, M. abscessus, M. bovis BCG, M. tuberculosis mc 2 6230 and E. coli; and 50 µM carbenicillin (Sigma Aldrich) for P.

  DMEM then refed with DMEM FBS supplemented with 200 μg/mL amikacin for 1 h. at 37 °C and 5% CO2 to kill all extra-cellular bacteria; washed again three times with DMEM prior to the addition of 2-fold dilutions of CyC compounds or imipenem (IMP) in DMEM FBS supplemented with 50 µg/mL (i.e., 85.4 µM) amikacin (200 µL final volume). In each plate, negative controls consisting of amikacin (50 µg/mL) and 1% DMSO (i.e., infected macrophages only); as well as positive controls containing amikacin (50 µg/mL) plus 80 μg/mL (i.e., 267 IMP were also included. Plates were incubated for 24 h at 37 °C, 5% CO2. Cells were washed three times with PBS and lysed by adding 200 μL of 0.1% Triton X-100. Serial dilutions of each culture were then plated at least in duplicate on 7H9 agar medium. Colonies were counted after 4 to 5 days of incubation at 37 °C to check intracellular bacterial viability following treatment with each compound concentration. DMSO-treated infected macrophages corresponded as control representing 100% of bacterial viability. Intracellular MICRaw values were determined by fitting the CFU% sigmoidal doseresponse curves in Kaleidagraph 4.2 software (Synergy Software). The lowest compound concentration inhibiting 50% of intracellular bacterial growth was defined as the MIC50Raw.

	as a value of cellular viability by metabolic activity. Murine (Raw264.7) macrophages
	(American Type Culture TIB-71) were cultured from a freezer stock in Dulbecco's
	modified Eagle medium (DMEM; Gibco) supplemented with 10% heat-inactivated fetal calf serum
	(FBS, Invitrogen) (DMEM FBS ). Cells were grown at 37 °C and 5% CO2 to subconfluent
	concentrations, then 1 × 10 5 cells/well were seeded in 96-well flat-bottom Nunclon Delta Surface
	microplates with lid (ThermoFisher Scientific, ref. 167008) in a final volume of 200 µL per well and
	Intramacrophage killing assay. Murine (Raw264.7) macrophages (American Type Culture
	Collection TIB-71) were grown in DMEM FBS medium at 37 °C and 5% CO2 to subconfluent
	concentrations, then 5 × 10 4 cells/well were seeded in 96-well flat-bottom Nunclon Delta Surface
	microplates with lid (ThermoFisher Scientific, ref. 167008) in a final volume of 200 µL per well and
	cultured for additional 24 h. The cells were infected with M. abscessus S at a multiplicity of infection
	(MOI) of 1:10 and incubated at 37 °C in the presence of 5% CO2 for 3 h. Cells were then washed
	three times with

cultured for additional 24 h. The medium was removed by aspiration, and 200 µL of serial two-fold dilution of each compound (CyC19 to CyC30) in DMEM FBS were then added to each well. After 24 h incubation, 20 µL of a 0.025% (w/v) resazurin solution was added to each well. Fluorescence was measured following a 4-h incubation at 37 °C and 5% CO2 in the dark, by excitation at 530 nm and emission at 590 nm as described above, leading to relative metabolic activities. Addition of DMSO was used as 100% viability reference and addition of 0.2% Triton X-100 solution served as negative standard (0% viability). All experiments were performed as two independent triplicates.

  using a UniProt M. abscessus ATCC 19977 (Taxon 561007) database (date 2018.01; 4940 entries). The statistical analysis was done with Perseus program (version 1.5.6.0). Differential proteins were detected using a two-sample t-test at 0.01 and 0.05 permutation based FDR. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (www.proteomexchange.org) 69 via the PRIDE partner repository with the dataset identifier PXD014255. Detailed Material and Methods is given in Supplementary Materials.

ACKNOWLEDGEMENTS

This work was supported by the CNRS and Aix Marseille University. A. Madani was supported by a PhD fellowship from the Association Grégory Lemarchal and Vaincre la Mucoviscidose (projet n°RF20160501651). Proteomics analyses were supported by the Institut Paoli-Calmettes and the Centre de Recherche en Cancérologie de Marseille. Proteomic analyses were done using the mass spectrometry facility of Marseille Proteomics (marseille-proteomique.univ-amu.fr) supported by IBISA (Infrastructures Biologie Santé et Agronomie), the Cancéropôle PACA, the Provence-Alpes-Côte d'Azur Region, the Institut Paoli-Calmettes, and the Centre de Recherche en Cancérologie de Marseille. The authors wish to thank Saroj Kafle and Dr. Bruce Hamper for performing HPLC analysis of CyC compounds. ABBREVIATIONS USED ABPP, activity-based protein profiling; AMK, amikacin; CC50, compound concentration leading to 50% of cell cytotoxicity; CF, cystic fibrosis; CFU, colony-forming units; CyC, Cyclipostins & Cyclophostin analogs; GPL, glycopeptidolipids; IMP, imipenem; INH, isoniazid; MIC50, minimal inhibitory concentration leading to 50% of growth inhibition; NTM, non-tuberculous mycobacterial; pFDR, permutation false discovery rate; REMA, resazurin microtiter assay; RFU%, relative fluorescence units.

ANCILLARY INFORMATION.

Supporting Information: The Supporting Information is available free of charge on the ACS Publications website.

Detailed protocols, full chemical characterization, NMR spectra and HPLC chromatograms of all new CyC compounds; detailed protocols regarding the MIC determination and targets identification; S1 (antimicrobial susceptibility testing of the new series of 12 CyCs by the agar plate method) and Table S2 (Extracellular antibacterial activities of all CyC analogs against both R & S variants of M.

abscessus). (PDF)

Tables S3-S8: CyC17 and CyC26 target proteins identified in M. abscessus R total lysate and in culture cell by LC-ESI-MS/MS analysis, and full dataset from the proteomics analysis. (XLSX)

AUTHOR INFORMATION

Corresponding Author Information

* JFC: e-mail: jfcavalier@imm.cnrs.fr. ORCID 0000-0003-0864-8314 * CDS: e-mail: SpillingC@msx.umsl.edu.

Author Contributions

Notes

The authors declare no competing financial interest.


Laure Brayer -Some thoughts on Experimentation as a Way to Implement Translation

1 -Some Thoughts on Experimentation as a Way to Implement Translation. From the second workshop: "Interdisciplinary reading, measurement and notation of ambiances", Montréal 2015. -Quelques réflexions sur l'expérimentation comme mise en oeuvre de la traduction. Retours sur le 2 nd séminaire du GDRI I would like to share with you a small text I wrote after the 2 nd workshop of Montreal. It has, in fact, more to do with a collection of thoughts than a set of "lessons" -like I saw it was written in the program.

It's a reflection from my own point of view of what happen there, during the different experimentations what we made within my group and the questions that these experiments could raise regarding the translation of ambiances.

The idea behind this text was to share it with the other members of my group or some other people who took part of this seminar, and propose to them to do the same: writing their own journal, in order to debate on what happen during these experiments that could lead to a better understanding of the translation of ambiance. Because we did many things during this workshop, but in my opinion we didn't really have the time to speak about them.

So here are some thoughts on experimentation as a way to implement translation of ambiances.

First of all, just a little precision about the composition of our group: we were 5 persons, all women, coming from a broad range of places (North America, South America, Maghreb and Europe). But our fields of work were not so diverse, since we were 4 persons associated to the field of architecture and urban design and only one artist. And I think that this detail is important if we stress the fact that the accent of the second workshop was put on the topic of interdisciplinary translation.

In our group 4 people proposed an experiment and we did the 4 of them during the time we had: so only a very short time was dedicated to each experiment. For example, I conceive my proposition thinking what we could spend at least half a day on it, but finally we had only 30 minutes to do so.

And if I'm saying that, it is because from the beginning this time issue oriented the way we worked 2 together: some people didn't want to spend too much time on trying to build an experiment from the combinations of the different proposals, but they prefer to try to do all of them quickly. So we briefly exposed to each other the different experiments and we went on with them. So, the 4 experiments we did in that frame, and that were regarding the site of La Place des Arts in Montréal, can be summarized by these 4 actions:

1 -Telling the words of the others through some kind of a performed vocal choreography 2 -Mapping our own perceptions and appreciations of the place 3 -Addressing the colors that we perceived in the place

-Comparing different videographic views of the place

For the first experiment proposed by Sophie, she had collected on the Internet different images of Place des Arts, and reframed them. She asked us to describe them and tell what these fragments called to mind. Our comments were recorded. And from these recordings Sophie edited a sonic piece that we then had to perform on stage during the final restitution in a sort of choreography: each of us were listening to the sonic piece via earphones, and we had to repeat some edited fragments of speech that we said about the pictures. This performance was composed of repetitions and echoing between the voices of the participants. It seams to me that this experiment dealt with the question of repetitions, series, and shifting between what as been said and repeated, between the images and the real site, between the reframing and the first frame. And it made me wonder what Sophie was looking for in this experiment: was it the translation of the words of others? Or their interpretation? What is to tell the words of others through an apparatus of sound recording and sound diffusion?

The first question about translation that this experiment made me formulate was: does translating have to do with an act of performing a rewriting? For the second experiment, which was about mapping our own perceptions and appreciations of the place, Cristiane had prepared a map of the Place des Arts and a set of color pencil. The idea was to follow a path and to fill the map with indications of open spaces vs./ constraining spaces; pleasant / unpleasant noises; cold or warm places; good and bad smell (and maybe I'm forgetting 1 or 2 entries). These different entries were corresponding to colors and pictograms and we had to draw the limits of the zones for each items. I followed the instruction of this experiment but, at the same time, I twisted them a little bite because I had the feeling that no space fits perfectly into a category. So, for example, I added contradictions in my drawing because the Place des Arts is full of contradictions: from one perspective the space can seams to be open, but if we look at it from another point of view it will seams closed and constrained. Or sometimes I painted the same area with 2 colors. It was the case of the musical swings that were on site: if at first there sound can be enjoyable, if you're living near by I think you can soon become very mad about these sounds! So, my thoughts about this experiment were about the possibility of complexifying the categories.

And it led to another question regarding the translation, which is: do we have to simplify in order to translate? And how can we do, in the process of translating, in order not to loose the complexity, nay sometimes the contradictory aspect of places regarding ambiances?

The third experiment was about the colors that we perceived on Place des Arts. The only indication that Anne gave us was : to "Browse the field of study by walking wherever you want. When a prominent color catches your attention, take a picture of it and describe briefly your perception". So I walked on site trying to focuss my attention only on colors, on their impact on my perception of the space, but also on my way of walking. And I did felt that this experiment made me see the place differently, I was particularly attentive to the colors of lights (the reflections on the windows, but also the articial lights). For the restitution, Anne picked some recurrent color effects from the pictures we took, and she composed some families of images around these color effects. Then she photoshoped some of the pictures in order to amplify the effects. And finally, she made a map of the color effects that we perceived on site.

The translation proposed in this experiment seams to me to raise the question of the necessary articulation of different registers of images. Different technics and means of expression like photography and digital manipulation of the pictures, written descriptions, and maps were use to translate our perception of colors. This multiplication of methods and means of representation raise the question of the materials with which we work -the "rawness" of the recordings or their manipulations -and the means of mediation that can be suitable for the translation of ambiance.

Unlike these 4 first experiments, the one I proposed about the comparison of different videographic views of the Place des Arts didn't have strict indications or protocole to follow. On the contrary, I thought what we would build together filmic sequences combining 2 contrasted ways to read the same place : 1/ an objective and distant take of the place and 2/ a subjective and engaged take of the same place. The goal was to exchange between us (on the field and then by looking at the recordings) about how these different ways of filming can expose or translate something of the ambiance of the place. But with the time issue I mentionned earlier, we didn't have the time for this debate.

So I just edited a little film to show during the final restitution. This experiment, in my opinion, wanted to address the question of the construction of representations in order to translate the ambiance, and wanted to point out the impact on the understanding of ambiance by a spectator that can have a specific relation to the world (or point of view) embedded in the representations.

The last though about this workshop was about the question of the restitution of the experiments.

In my opinion, it is less important to be able to show some final productions at the end of the workshop than to be able to debate on what happen during the experiments, on what are the different stakes or limits of the experiments regarding the question of translation. And this collective exchange is important particularly if we want work on the question of translation from a disciplinary or an interdisciplinary point of view. And I think that such a debate did actually take place after the presentation of Samuel Tronçon about the network representation of the different experiments that he proposed. And it made me wonder if this happen because this type of representation is quite unusual for the majority of the people that were there (people coming more from architecture, urban design and art, and being not very familiar with "system complex" and mathematics logic).

And this raised a last set of questions about our ability to interrogate the modes of representation that are obvious for us, that we use all the time without thinking much about them: how to question our habits, our usual disciplinary tools? Just like linguistic translation is questionning as much our own language that the foreign language with which we are trying to communicate, can we think that disciplinary translation requires as well to rethink our own methods and ways of working? So, this is the extent of my thoughts after Montreal.

Présentation dans le cadre du 3ème séminaire du GDRI, à Volos, 20/09/2016

Présentation dans le cadre du 3ème séminaire du GDRI, à Volos, 20/09/2016 5 And the proposition I would like to make is that if some of you would be interested to do the same about the experiments in which they took part, maybe we could try to write a collective text that could work as a synthesis of this workshop. So, let me know if you are interested. And thank you.