Miroslava Zoldakova

Zsuzsanna Kornyei

Andreas Brown

Bernhard Biersack

Emília Madarász

Rainer Schobert
email: rainer.schobert@uni-bayreuth.de

Effects of a combretastatin A4 analogous chalcone and its Pt-complex on cancer cells: a comparative study of uptake, cell cycle and damage to cellular compartments

Keywords: Chalcones, platinum, microtubules disrupting agents, melanoma 518A2 cells, organic cation transporters, Golgi apparatus

The combretastatin A4 analogous chalcone (2E)-3-(3-hydroxy-4-methoxyphenyl)-1- (3,4,5trimethoxyphenyl)prop-2-en-1-one 1 and its dichloridoplatinum(II) (6-aminomethylnicotinate) complex 2 were previously found to be highly active against a variety of cancer cell lines while differing in their apoptosis induction and long-term regrowth retardation (Schobert R et al. J Med Chem 2009;52:241-6) [1]. Further differences were identified now.

The cellular uptake of complex 2, like that of oxaliplatin, occurred mainly via organic cation transporters (OCT-1/2; ~32%) and copper transporter related proteins (Ctr1; ~24%), whereas that of chalcone 1 was dependent on endocytosis (~80%). Complex 2 was more tumourspecific than 1 concerning neural cells. This was apparent from the ratios of IC 50 (48 h) values against primary astrocytes versus human astroglioma cells U87 ( > 7000 for complex 2; 55 for compound 1). In tubulin-rich neurons and 518A2 melanoma cells complex 2 disrupted microtubules and actin filaments. Cancer cells treated with 2 could repair the cytoskeletal damage but ceased to proliferate and perished. Complex 2 was particularly cytotoxic against P-gp-rich cells. It acted as a substrate for ABC-transporters of types BCRP, MRP3, and MRP1 and so was less active against the corresponding cancer cell lines. Complex 2 arrested the cell cycle of the melanoma cells in G 1 and G 2 /M phases. A fragmentation of their Golgi apparatus was observed by TEM for incubation with complex 2 but not with 1. In conclusion, unlike chalcone 1, its platinum complex 2 is highly cell line specific, is taken up via cellcontrolled transporters and induces apoptosis by triggering multiple targets.

Introduction

Angiogenesis plays an essential role in the development and growth of solid tumours. The tumoural vascular network is characterised by peculiar irregularities and thus has been identified early on as a promising drug target. Vascular disrupting agents (VDAs) have been intensively investigated and some already went on clinical trials, mostly as combination regimens with DNA-targeting drugs [START_REF] Kanthou | Microtubule depolymerizing vascular disrupting agents: novel therapeutic agents for oncology and other pathologies[END_REF][START_REF] Lippert | Vascular disrupting agents[END_REF]. The majority of VDAs show a pronounced affinity for tubulin and interfere with the delicate mitotic machinery of dynamic microtubule formation and destruction. The microtubular cytoskeletal fibres are crucial to mitosis and cell division, for the maintenance of cell shape and in the transport of vesicles [START_REF] Vantard | Cell cycle regulation of the microtubular cytoskeleton[END_REF]. Moreover, they are essential structural components of dendrites. In the brain, the formation of dendrites is dependent on neural activity and is mediated in part by the modulation of mictrotubule stability [START_REF] Vaillant | Signaling mechanisms underlying reversible, activity-dependent dendrite formation[END_REF]. A good deal of the recently published VDAs are synthetic variants of longknown natural tubulin binders, e.g., combretastatin A4 (CA4) which specifically binds to the colchicine binding site of tubulin and exhibits distinct anticancer activity in vitro and in vivo [START_REF] Hinnen | Vascular disrupting agents in clinical development[END_REF]. Structurally related are trimethoxyphenyl chalcones [START_REF] Lawrence | Effects of alpha-substitutions on structure and biological activity of anticancer chalcones[END_REF]. The molecular mechanism of their interference with microtubules and their vascular disrupting effect is not fully understood, yet. Some studies even invoked a neuroprotective effect of chalcones [START_REF] Hv | Neuroprotective effects of chalcones from Myracrodruon urundeuva on 6-hydroxydopamine-induced cytotoxicity in rat mesencephalic cells[END_REF][START_REF] Takano | A dibenzoylmethane derivative protects dopaminergic neurons against both oxidative stress and endoplasmic reticulum stress[END_REF].

Previously we disclosed a comparison of the effects of chalcone 1 and its platinum complex 2 on twenty-one tumour cell lines [1]. Now, we present a study of their mechanisms of action and effects in living cells.

((Figure 1 here))

Materials and Methods

Reagents, materials and antibodies

Chemicals: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; ABCR, Karlsruhe, Germany), Pluronic F127, propidium iodide, calcein-AM, fumitremorgin C, Antibodies: anti-goat polyclonal OCT-1 (N-12) (Santa Cruz Biotechnology; sc-19809), antigoat polyclonal OCT-2 (C-13) (Santa Cruz Biotechnology; sc-19814), anti-mouse monoclonal alpha-tubulin (clone 12G10 from hybridoma supernatant), bovine anti-goat HRP (Santa Cruz Biotechnology; sc-2378), anti-mouse (Fab specific)-peroxidase antibody produced in goat (Sigma; A9917), glial fibrillary acidic protein (GFAP; Invitrogen), streptavidin-conjugated horseradish peroxidase (HRP; Sigma A8924), phalloidin-biotin, anti-mouse IgG Alexa 594 (A11005), anti-rabbit Alexa 594 chicken, and 4'-6-diamidino-2-phenylindole (Invitrogen), anti-mouse neuron specific III-tubulin (SDL3D10) and goat anti-mouse streptavidin AlexaFluor 488 conjugate (S32354) (Sigma). Cell cultures: Human 518A2 melanoma cells were obtained from the department of oncology and hematology of the Martin-Luther-University, Halle, and cultured in D-MEM with 10% FBS, 0.55% antibiotic-antimycotic solution and 0.3% gentamycin. Cervix carcinoma (KBv1 +Vbl ) and breast carcinoma (MCF-7 +Top ) cells were a gift from the department of pharmacy, University of Regensburg. The KBv1/KBv1 +Vbl cells were used from 90 th -118 th passage and cultured in D-MEM with 10% FBS, 0.55% antibiotic-antimycotic solution and 0.3% gentamycin, without/with 340 nM vinblastine [START_REF] Kuhnle | Potent and selective inhibitors of breast cancer resistance protein (ABCG2) derived from the p-glycoprotein (ABCB1) modulator tariquidar[END_REF]. The MCF-7/MCF-7 +Top cells (160 th -180 th passage) were grown in E-MEM supplemented with 5% FBS, without/with 550 nM topotecane [START_REF] Kuhnle | Potent and selective inhibitors of breast cancer resistance protein (ABCG2) derived from the p-glycoprotein (ABCB1) modulator tariquidar[END_REF]. The human colon adenocarcinoma cells (HT-29/HT-29 +Colc ; 11 th -16 th passage) were cultured in RPMI medium supplemented with 5% FBS, without/with 62.5 nM colchicine. Neural cells used: NE-4C neuroectodermal stem cells NE-4C, primary mouse astrocytes [START_REF] Schlett | In vitro pattern formation during neurogenesis in neuroectodermal progenitor cells immortalized by p53-deficiency[END_REF][START_REF] Kornyei | Humoral and contact interactions in astroglia/stem cell co-cultures in the course of glia-induced neurogenesis[END_REF], glioma cell lines: rat glioma C6 [ATCC No.: CCL-107] and human astroglioma U87 [ATCC No.: HTB-14], non-neural mouse fibrosarcoma cells WEHI 164 [START_REF] Huszti | Histamine (HA) suppresses the production of tumor necrosis factor alpha (TNFalpha) in cultured astroglial cells[END_REF]. Cells were maintained under a moisture-saturated atmosphere (95% humidity, 5% CO 2 ) at 37 °C in 75 mL culture flasks (Nunc, Germany). They were serially passaged using 0.05% trypsin / 0.02% EDTA (PAA Laboratories, Cölbe, Germany).

Media and cell cultures

Inhibition of cancer cell growth (MTT assay)

MTT was used to identify viable cells which reduce it to a violet formazan [START_REF] Mosmann | Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays[END_REF]. The cells (110

Cellular uptake of drugs into 518A2 melanoma cells

Routes of uptake of the test compounds were assessed by measuring their cytotoxicity against 518A2 melanoma cells in the presence of specific inhibitors by MTT assays. The cells (5x10 5 cells/mL) were grown on 96-well filtration plates. After 24 h inhibitors were applied to final concentrations of 0.5 mM cimetidine, or 0.1 mM tetraethlyammonium (TEA), or 10 nM wortmannin, or 14 µM chlorpromazine, or 10 µM amiloride, or 5 µM ouabaine. The 518A2 cells were pre-incubated with these inhibitors for 15 min and then treated with 100-200 µM of chalcones 1 or 2 or reference compounds. After 3.5 h cell vitality was measured by MTT tests. The insoluble formazan crystals were washed with distilled water, dissolved in DMSO and the absorbance of the resulting filtered solution was measured. The difference of absorbances at 570 and 630 nm was calculated relative to controls of untreated cells and cells treated with inhibitors. The cytotoxicity of the test compounds was validated by comparison with their effects in cells not treated with an inhibitor and with the net effect of the respective inhibitor in cells not exposed to the test compounds. and incubated for 15 min. Calcein-AM was added to achieve a concentration of 1 µM. After incubation (10 min, 37 °C, 5% CO 2 ), and centrifugation (800 rpm, 5 min, 4 °C) a cell pellet was obtained which was rinsed once with ice-cold PBS and re-suspended in 0.5 mL of Cabuffer. The fluorescence of calcein (λ Ex = 485 nm, λ Em = 535 nm) was measured in a black microplate on a TECAN Infinite F200 plate reader and the data were analysed and calculated relative to verapamil as a positive control.

Western blot analysis of organic cation transporters

Mitoxantrone assay for BCRP activity of MCF-7/MCF-7 +Top cells

The assay was carried out according to a modified literature procedure [START_REF] Kuhnle | Potent and selective inhibitors of breast cancer resistance protein (ABCG2) derived from the p-glycoprotein (ABCB1) modulator tariquidar[END_REF]. MCF-7 +Top cells were incubated with 20 µM mitoxantrone in 70% ethanol and briefly vortexed. 10 µM of test compounds 1 or 2 in DMF were added to allow maximal mitoxantrone uptake into the cells over a period of 30 min at 37 °C. After centrifugation (800 rpm, 5 min, 4 °C) the cells were washed twice with PBS, pipetted into a black microplate together with the positive control fumitremorgin C (FTC) and the negative control DMF. The plates were immediately placed in

Time-lapse microscopy

Cells were grown on IBIDI dishes in a custom-made microscope stage incubator (37 °C, 5%

CO 2 ). Time-lapse recording was performed with a computer-controlled Zeiss Axiovert 200M microscope equipped with 10-and 20-fold objectives and an AxioCam MRm digital camera.

Immunocytochemistry

The cells (518A2 melanoma, astroglia) grown on poly-L-lysine-coated glass cover slips were fixed with 4% paraformaldehyde in PBS for 20 min at room temperature and then permeabilised with Triton X-100 (0.1% v/v in PBS; 5 min). Nonspecific antibodies were blocked by incubation with 3% FBS in PBS (room temperature, 1 h). a-GFAP antibody (rabbit, DAKO) was used in a dilution of 1:2000 and was visualized by anti-rabbit IgG Alexa 594 (1:1000). Phalloidin-biotin was applied in 1:1000 dilution followed by avidin-Alexa 488

(1:800). Fluorescence images were captured manually on a Zeiss Axiovert 200M microscope fitted with 20-60 fold zoom and a Zeiss AxioCam MRm digital camera.

Cell cycle analysis

518A2 melanoma cells (10 6 ) were treated with the test compounds, positive (250 µg/mL nocodazole) / negative (DMF) controls and then harvested at certain time intervals. The cells were washed, re-suspended in 200 µL PBS, and incubated with 8 mL of 70% ice-cold ethanol for fixation. They were centrifuged, washed with PBS, and re-suspended in 1 mL of a PBS solution of 3.8 mM sodium citrate and 50 µg/mL propidium iodide with freshly added RNase [START_REF] Krishan | Rapid flow cytofluorometric analysis of mammalian cell cycle by propidium iodide staining[END_REF]. After 1 h incubation at 37 °C the concentration of double-stranded DNA in the samples A c c e p t e d M a n u s c r i p t 9 was determined by measuring the emission (570 nm) of its fluorescent adducts excited at 488 nm, using a Beckman CYTOMICS FC 500 cytometer.

Transmission electron microscopy

518A2 melanoma cells were plated on pieces of aclar film, grown for 24 h, exposed for 20 min to 100 µM of either compound 1 or 2 and flat-embedded for transmission electron microscopy as follows. Cells were fixed in 0.1 M HEPES, 4 mM CaCl 2 containing 2.5% glutaraldehyde, pH 7.2, for 4 h at room temperature. After three rinses with 0.1 M HEPES, 4 mM CaCl 2 , pH 7.2, cells were postfixed with 1% osmium tetroxide in distilled water for 45 min at 4 °C, rinsed three times in distilled water and incubated in 1% uranyl acetate in distilled water for 1 h at 4 °C. Dehydration of the samples in an ethanol series, infiltration with Epon, and flat embedding was performed according to standard procedures. Ultra-thin sections (60-70 nm) were cut and mounted on pioloform-coated copper grids. Sections were stained with uranyl acetate and lead citrate [START_REF] Reynolds | The use of lead citrate at high pH as an electron-opaque stain in electron microscopy[END_REF] and viewed with a Zeiss CEM 902 transmission electron microscope at 80 kV. Micrographs were taken using EMS EM film.

Results

Cytotoxicity studies with selected cell lines

Neural cells. Chalcones target tubulin and microtubules [START_REF] Jordan | Microtubules as a target for anticancer drugs[END_REF]. We analysed the relative cytotoxicities of chalcones 1 and 2 against tubulin-rich cells of neural origin (Table 1; grey rows). Normal mouse astroglia were not particularly sensitive to chalcone 1 and virtually insensitive to platinum complex 2 during the whole 48 h incubation period. However, human U87 glioma cells responded well upon prolonged exposure to both compounds, and especially well to the Pt-complex 2 at an IC 50 (48h) ~ 140 nM. Primary neurons were also sensitive to a prolonged treatment. Both chalcones were strongly cytotoxic against C6 rat glioma cells with IC 50 (48 h) ~ 150-170 nM and also against NE-4C cells derived from the fore-and midbrain vesicles of p53-deficient 9-day-old mouse embryos [START_REF] Schlett | Retinoic acid induced neural differentiation in a neuroectodermal cell line immortalized by p53 deficiency[END_REF] after exposure for 48 h. WEHI 164 ABC-transporter-rich tumour cells. ABC-transporters pump drugs out of cancer cells and are responsible for chemoresistance. Their interference with compounds 1 and 2 was investigated by using various non-neural cancer cells rich in these transporters such as MCF-7 breast, KBv1 cervix and HT-29 colon carcinomas (Table 1; bottom half). These cells had been grown in the presence of specific substrates of their transporters to stimulate their overexpression.

Human 518A2 melanoma cells served as reference cells not overexpressing any ABCtransporters. By long-term exposure to topotecane resistant MCF-7 +Top cells overexpressing exclusively BCRP (breast cancer resistance protein) were obtained. Chalcone 1 was cytotoxic in these MCF-7 +Top cells with IC 50 (24 h/48 h) ~ 12 / 1 µM. It showed the same IC 50 value in the wild type MCF-7 cells after 24 h, which remained constant for up to 48 h. In comparison with chalcone 1 its Pt-complex 2 exhibited a distinctly lower activity in both MCF-7 cell lines. Resistant KBv1 +Vbl cells overexpressing P-gp were obtained by treating the wild type cells with vinblastine. The chalcones 1 and 2 were highly and comparably efficacious against both cell lines with a slight edge for the latter. HT-29 cells feature a high amount of multiresistance protein 3 (MRP3) and a low amount of MRP1. When treated with colchicine HT-29 cells shift this ratio by overexpressing MRP1 and cutting back on MRP3 [START_REF] Kok | Differential expression of sphingolipids in MRP1 overexpressing HT29 cells[END_REF]. Both test compounds were little active in either HT-29 cell line but distinctly more so in the HT-29 +Colc cells. A similar trend was observed also for cisplatin (data not shown). According to literature [START_REF] Kok | Differential expression of sphingolipids in MRP1 overexpressing HT29 cells[END_REF] colchicine-induced overexpression of MRP1 is accompanied by a changed sphingolipid composition.

((Table 1 here))

Cellular uptake of chalcones 1 and 2

A c c e p t e d M a n u s c r i p t

11

In order to ascertain whether the chalcone 1 or its platinum complex 2 enter the cell via specific transporters or by endocytosis, a simple temperature-dependent experiment was performed. The process of endocytosis is known to be virtually halted at temperatures below 16 °C [START_REF] Punnonen | At reduced temperature, endocytic membrane traffic is blocked in multivesicular carrier endosomes in rat cardiac myocytes[END_REF][START_REF] Stirling | Dual roles for RHOA/RHO-kinase in the regulated trafficking of a voltage-sensitive potassium channel[END_REF]. Almost twice as many 518A2 melanoma cells survived exposure to 100 µM of 1 at 4 °C when compared to 37 °C (cf. Supplementary Data), which is an indication of this chalcone being taken up mainly via endocytosis. In contrast, the paused endocytosis process did not affect the cytotoxicity of platinum complex 2, which left ca 40% cells alive at either temperature. Alternative ways for the entry of platinum complexes into cells involve copper related transport proteins (Ctr1) and organic cation transporters (OCT-1/2). Gust et al. studied their relevance by switching off individual transporters with specific inhibitors [START_REF] Kapp | Dinuclear alkylamine platinum(II) complexes of [1,2-bis(4fluorophenyl)ethylenediamine]platinum(II): influence of endocytosis and copper and organic cation transport systems on cellular uptake[END_REF]. Earlier on, the accumulation of cisplatin by a high-affinity copper transporter Ctr1 had been found to compete with that of copper [START_REF] Ishida | Uptake of the anticancer drug cisplatin mediated by the copper transporter Ctr1 in yeast and mammals[END_REF]. The OCT convey positively charged substances and may be inhibited by cimetidine (inhibitor) or by tetraethylammonium chloride (TEA; competitor).

Endocytotic processes depending on a sodium gradient can be slowed down by adding ouabaine, an inhibitor of the Na + /K + pump [START_REF] Ivanov | Endocytosis of epithelial apical junctional proteins by a clathrin-mediated pathway into a unique storage compartment[END_REF]. Macropinocytosis, a distinct form of endocytosis, depends on a Na + /H + exchanger that can be blocked by amiloride [START_REF] Kapp | Dinuclear alkylamine platinum(II) complexes of [1,2-bis(4fluorophenyl)ethylenediamine]platinum(II): influence of endocytosis and copper and organic cation transport systems on cellular uptake[END_REF][START_REF] Meier | Adenovirus triggers macropinocytosis and endosomal leakage together with its clathrinmediated uptake[END_REF]. The enzyme phosphatidylinositol 3-kinase that plays a central role in the down-stream signalling cascade associated with macropinocytosis by F-actin microfilament re-arrangement, can be inhibited by the fungal metabolite wortmannin [START_REF] Zhang | PI3-K/Akt pathway contributes to IL-6-dependent growth of 7TD1 cells[END_REF]. Clathrine-mediated endocytosis can be blocked by chlorpromazine [START_REF] Nawa | Interference in Japanese encephalitis virus infection of Vero cells by a cationic amphiphilic drug, chlorpromazine[END_REF]. We now examined the influence of all these inhibitors on the uptake of chalcones 1 and 2 into 518A2 melanoma cells. To this end, the cells were preincubated with non-toxic concentrations of the respective inhibitors for 15 min before the chalcones were added and their cytotoxicities were measured by the usual MTT assay. Figure 2 illustrates the effects of the individual inhibitors on the cytotoxicity of chalcone 1 (grey bars) and complex 2 (black bars) against 518A2 cells and so indirectly also the share that the ((Figure 2 here))

Western blot analysis of organic cation transporters

Evidence for the presence in 518A2 melanoma and KBv1 +Vbl cervix carcinoma cells of organic cation transporters hOCT-1 and hOCT-2 that belong to the solute carrier subfamily SLC22A of membrane transport proteins was provided by westernblotting. More than one band were visible upon staining with a primary antibody for hOCT-1 (= SLC22A1) since it recognises the N-terminus of this protein which is a highly conserved segment also present in OCT-2. In addition, several isoforms of hOCT-1 are known. We could assign the band corresponding to a protein of size ~54 kDa to hOCT-1. Staining of the membrane with an antibody specific for the C-terminus of hOCT-2 (= SLC22A2), which differs considerably from the C-terminus of hOCT-1, rendered a single intense band for this protein of size ~30 kDa. This band had also been visible upon staining with the less specific OCT-1 antibody.

Although the abundance of hOCT-1/2 in tumour cells in general had been frequently

proposed [START_REF] Koepsell | Polyspecific organic cation transporters: structure, function, physiological roles, and biopharmaceutical implications[END_REF], this is the first experimental evidence for their occurrence in the said two cancer cell lines.

((Figure 3 here))

Interference with ABC transporters

We investigated whether chalcones 1 and 2 interact with drug transport proteins of types ABCB1 (P-gp) [START_REF] Simstein | Apoptosis, chemoresistance, and breast cancer: insights from the MCF-7 cell model system[END_REF][START_REF] Smith | P-glycoprotein efflux at the blood-brain barrier mediates differences in brain disposition and pharmacodynamics between two structurally related neurokinin-1 receptor antagonists[END_REF][START_REF] Hyde | Structural model of ATP-binding proteins associated with cystic fibrosis, multidrug resistance and bacterial transport[END_REF] or BCRP [START_REF] Borst | Do cMOAT (MRP2), other MRP homologues, and LRP play a role in MDR?[END_REF] in a substrate-or inhibitor-like fashion. P-gp overexpressing KBv1 +Vbl cervix carcinoma cells [START_REF] Schwab | Comparison of in vitro Pglycoprotein screening assays: recommendations for their use in drug discovery[END_REF] alongside their wild type parent cells were treated with chalcones 1 and 2 in the presence of calcein-AM (Fig. 4a). The inhibition of P-gp allows for calcein-AM taken up by the cells to get cleaved with formation of fluorescent calcein. As a positive control, the fluorescence intensity of KBv1 +Vbl cells treated with 50 µM to be competitive substrates for the BCRP proteins with the free chalcone 1 having a slightly greater binding affinity than its complex 2.

((Figure 4 here))

Time-lapse microscopy

Neurons are particularly rich in tubulin and so are well suited to test agents affecting microtubule organisation. Their regular growth requires microtubules being transported plus ends first from the cell body all the way down into the axon [START_REF] Hasaka | Role of actin filaments in the axonal transport of microtubules[END_REF]. The effect of chalcones 1 and 2 on the structure, development and movement of a network of rat neurons was now showed a retraction of their processes back to the soma within 30 minutes (Fig. 5, top). The cells got closer to each other with their bodies swelling and aggregating. Most of the neural network lost its integrity and processes were disconnected from the cell bodies of adjacent neurons. However, exposure to Pt-complex 2 caused only an incomplete disruption of the networks. An analogous experiment was performed with 518A2 melanoma cells, which were initially tightly attached as a monolayer to the surface of the mini Petri dish. Overall, dynamics and motility of untreated cells was slow as they underwent merely negligible morphological changes. Only a few cells detached from the monolayer within the first two hours. In contrast, exposure of 518A2 cells to chalcone 1 led to an immediate rounding and an enhancement of motility and cell shrinking dynamic (Fig. 5, bottom). A very similar behaviour of 518A2 cells, delayed by 2 min, was observed upon incubation with complex 2.

The cells readily dissociated from the monolayer with their form turning from flat to spherical (cf. movies as supplementary files in the online version).

((Figure 5 here))

Immunocytochemistry of actin filaments in astrocytes and 518A2 cells

An immunocytochemical analysis of the morphological changes of the actin filaments of astrocytes initiated by the chalcones 1 and 2 was carried out and compared with the behaviour of 518A2 melanoma cells. Antibodies to astrocyte-specific GFAP intermediate filaments (glial fibrillary acidic protein) were employed to visualise the astroglial cells and phalloidin labelling was used to identify actin filaments. The micrographs showed that both types of filaments, actin (in green) and GFAP (in red), were disintegrated upon treatment with the test chalcones, most pronouncedly the actin filaments near the cell membrane. The GFAP filaments shrank only in the vicinity of the cell nuclei, which remained unaffected. Figure 6 shows typical effects of chalcone 1 on astrocytes. Results with complex 2 were similar.

A c c e p t e d M a n u s c r i p t 16

Likewise, fluorescence micrographs of immunostained 518A2 cells revealed a significant breakdown of the actin network when treated with chalcones 1 or 2 (Fig. 7).

((Figures 6 and7 here))

Cell cycle analysis

While cisplatin is known to generally arrest cells in the G1 phase due to cross-link interaction with DNA [START_REF] Wang | Cellular processing of platinum anticancer drugs[END_REF] chalcone, 1,3-diphenylpropenone, arrests cells of the human T24 and HT-1376 bladder cancers in the G2/M phase [START_REF] Shen | Chalcone arrests cell cycle progression and induces apoptosis through induction of mitochondrial pathway and inhibition of nuclear factor kappa B signalling in human bladder cancer cells[END_REF]. We now assessed the cell cycle progression of melanoma 518A2 cells treated with chalcones 1 and 2 by FACS analysis after DNA staining with propidium iodide (Fig. 8 and Supplementary Data). 63% of untreated 518A2 cells were in G1 phase, 24% in S phase and 13% in G2/M phase after 18 h. 518A2 cells treated with 250 µg/L nocodazole, a synthetic analogue of taxol, were mainly in sub-G1 phase after 18 h, a shorter 12 h exposure led to 76% of the cells being in G2/M phase. 10 µM of chalcone 1 had a similar effect arresting 61% in G2/M phase after 18 h incubation. 518A2 cells treated with 10 µM of Pt-complex 2 had their cell cycle mainly arrested in G2/M phase (59%) as is typical for chalcones, but atypically numbers of cells in G1 phase increased as well (19%). 22% of the cells were in the S phase. To dismiss a conceivable hydrolytic cleavage of 2 during incubation, we also proved that 100 µM of dichlorido(6-aminomethylnicotinic acid)platinum(II), the assumed hydrolysis product of ester 2, had no effect on the cell cycle at all. As a positive control, treatment with cisplatin led to 69% of 518A2 cells being arrested in G1 phase.

((Figure 8 here))

Transmission electron microscopy

For a visualisation of morphological effects 518A2 melanoma cells were treated with 100 µM 1 or 2 for 15 min, kept under medium for another hour, then embedded and finally viewed with a transmission electron microscope (TEM). While untreated control cells appeared prolate and undifferentiated with filopodia for cell attachment on the rim, the cells incubated A c c e p t e d M a n u s c r i p t with the chalcones 1 or 2 adopted a rounded shape. A similar morphological change had been reported for H460 non-small cell lung cancer cells treated with CA4 [START_REF] Vitale | Combretastatin CA-4 and combretastatin derivative induce mitotic catastrophe dependent on spindle checkpoint and caspase-3 activation in non-small cell lung cancer cells[END_REF]. In addition, the treated 518A2 cells had blebs emerging on their surface (cf. Supplementary Data). Stress fibres were also observed in the treated cells (Fig. 9h). Multivesicular bodies and autophages (Fig. 10b) with double membranes were visible in most of the analysed cells (Fig. 9f). Control cells and treated cells also differed with respect to the state of their mitochondria, centrioles and Golgi apparatus (GA). Mitochondrial populations consist mainly of short and long tubules, which constantly migrate along microtubule tracks [START_REF] Chan | Mitochondrial fusion and fission in mammals[END_REF]. Any disruption of the microtubules/actin skeleton, e.g., by treatment with chalcones 1 or 2 should severely affect the mitochondrial trafficking and cause mitochondrial disorganisation. We found that mitochondria of cells treated with the chalcones were highly condensed and localised mainly at the apical side of the cells. They frequently displayed dislocated inner membranes (Fig. 9e).

Only a small percentage of the untreated control cells were found in G2/M phase with visible centrioles. After treatment with the chalcones, many cells had visible pro-centrioles (Fig. 9c),

which is an indication of them being arrested in G2/M phase. The GA undergoes irreversible fragmentation during apoptosis in part as a result of caspase-mediated cleavage of several Golgi-associated proteins. A fragmentation of the GA was observed mainly in 518A2 cells treated with complex 2 (Fig. 10b) but not with chalcone 1.

((Figures 9 and 10 here))

Discussion

The in vitro cytotoxicities of the chalcones 1 and 2 vary by a factor of 200 for the different cell lines tested. Moreover, individual cell lines show different sensitivities to the chalcones 1 and 2 suggesting distinct and incongruent mechanisms by which these compounds act. The large spread between the IC 50 (48 h) of non-malignant astroglia (100 µM) and U87 glioma cells (140 nM) in the case of Pt-complex 2 is pharmacologically interesting and a significant improvement over parent chalcone 1. The platinum complex is also less well expelled by cells overexpressing P-gp when compared to the unmetallated chalcone. The results of the calcein-AM assays showed that it inhibits the efflux functionality of P-gp by acting as a genuine inhibitor or a modulator. As a consequence, complex 2 is more efficacious in multidrug resistant cancer cells featuring this particular ABC-transporter, e.g., KBv1 +Vbl . Despite their similar chemical structures the test compounds are taken up by cells via different pathways.

While chalcone 1 is preferentially accumulated by endocytosis its platinum complex 2 is taken up mainly via the organic cation and copper related transporters. Insofar, complex 2 resembles oxaliplatin which was shown by Lippard et al [START_REF] Zhang | Organic cation transporters are determinants of oxaliplatin cytotoxicity[END_REF] to be an excellent substrate for the human organic cation transporters OCT-1 and OCT-2. Both chalcones 1 and 2 vigorously depolymerised microtubules and actin filaments in neural and cancer cells. They proved equally effective in damaging mitochondria and in arresting cancer cells in G2/M phase.

However, only complex 2 led to an accumulation of cells in G1 phase, reminiscent of the effect of cisplatin. Hence, complex 2 has a dual impact on the cell cycle of 518A2 melanoma cells by combining the effects of Pt-complexes and of chalcones in general. However, the DNA binding activity of 2 was found to be four times lower than that of the good DNAbinder cisplatin [1]. This could explain the lower number of cells arrested in G1 phase upon treatment with 2. Morphologically, both test compounds led to a rounding and detachment of 518A cells and an enhancement of their motility and cell shrinking dynamic. This behaviour is in line with the generally accepted mechanisms of tubulin binding agents (TBAs) [START_REF] Lippert | Vascular disrupting agents[END_REF] involving activation of RhoA/RhoA kinase, an intracellular coordinator of cytoskeletal rearrangement of microtubules and actin. In our experiment with 1 and 2, 518A2 cells were able to repair the shrinkage effects and most of them spread again after three hours. The results from the transmission electron microscopy of 518A2 cells treated with the compounds 1 and 2 are also in keeping with their effects on the cell cycle. The occurrence of centrioles as A c c e p t e d M a n u s c r i p t a result of aberrant chromosome segregation could be due to DNA damage (possibly in tandem with defective checkpoints) or to a destabilisation of the spindle apparatus [START_REF] Vitale | Combretastatin CA-4 and combretastatin derivative induce mitotic catastrophe dependent on spindle checkpoint and caspase-3 activation in non-small cell lung cancer cells[END_REF][START_REF] Castedo | Cell death by mitotic catastrophe: a molecular definition[END_REF].

Maintenance of the microtubule integrity plays a pivotal role in stabilising the pericentriolar matrix. Our observation is consistent with the notion that the microtubule depolymerising effect of both chalcones 1 and 2 leads to a mitotic catastrophe and thus to an arrest in G2/M phase. A similar mitotic failure had been reported for H460 NSCLC cells treated with CA4 [START_REF] Vitale | Combretastatin CA-4 and combretastatin derivative induce mitotic catastrophe dependent on spindle checkpoint and caspase-3 activation in non-small cell lung cancer cells[END_REF]. Moreover, complex 2 but not chalcone 1 initiated the fragmentation of the Golgi apparatus of 518A2 melanoma cells. This fragmentation is an early event that occurs independently of major changes to the actin and tubulin cytoskeleton [48].

Thus, the observed differences in the anticancer activities of the free chalcone 1 vs. its platinum complex 2 could be traced back to distinct differences in the biochemistry of their uptake, clearance and impact on the cell cycle and on cellular components. Complex 2 apparently combines some modes of action of chalcones and platinum complexes and so can address more cancer-relevant targets than parent chalcone 1.

ANTIBIOTICS AND CHEMOTHERAPEUTICS

Effects of a combretastatin A4 analogous chalcone and its Pt-complex on cancer cells: a comparative study of uptake, cell cycle and damage to cellular compartments

Miroslava Žoldáková, Zsuzsanna Kornyei, Andreas Brown, Bernhard Biersack, Emília Madarász, Rainer Schobert

A chalcone and its Pt-complex, while both anticancer active, were found to differ distinctly in uptake routes, selectivity for glioma over normal neural cells, efficiency in multidrug resistant cancer cells, influence on cell cycle progression, and fragmentation of the Golgi apparatus.

A c c e p t e d M a n u s c r i p t 4 cimetidine 2 g

 42 , wortmannin, chlorpromazine, amiloride, ouabaine (CalBiochem, Darmstadt, Germany), PageRuler™ Prestained Protein Ladder (Fermentas, St. Leon-Rot, Germany), mitoxantrone (Ralenova, Germany), RNase 10 mg/L (Qiagen, Hilden, Germany), 18% formaldehyde (Riedel de Haen, Seelze, Germany), glycine, 1% OsO 4 in H 2 O, N-2hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES; Roth), PBS buffer [8 g NaCl, 0.KCl, 1.44 g Na 2 HPO 4 , 0.24 g KH 2 PO 4 per L; pH 7.4], Pb(NO 3 ) 2 (Merck), 25% glutaraldehyde, Glycider 100, propylene oxide, 2,4,6-tris(dimethlyaminomethyl)phenol (Epon; Serva), 0.5% tannic acid (Mallinckrodt), polioform powder, uranyl acetate (Plano, Wetzlar, Germany). The chalcones 1 and 2 were synthesized according to literature [1,10]. Materials: 96-well filtration plates HTS (Millipore), neutral pH 7.4 gradient (Serva, 43221), Fluorobind Membrane, surface PVDF (Serva, 42571), FACS tubes (Becton Dickinson), 1818 mm Aclar-folia (Ted Pella), slot grids (Plano), Maco EM-Film EMS, 8.310.2 cm (Maco).

A c c e p t e d M a n u s c r i p t 5

 5 Media and supplements: MEM and E-MEM (Eagle's minimum essential) media were purchased from Sigma. Ham's 12, D-MEM (Dulbecco's modified eagle medium), NeuroBasal media, Insulin-Transferin-Selenium ITS were bought from Gibco, Invitrogen, B27 supplement, foetal calf serum (FCS), antibiotic-antimycotic solution, gentamycin, 3 mM retinoic acid, 10% polylysine, and glutamine from Gibco-BRL-Life.

A c c e p t e d M a n u s c r i p t 8 a

 8 TECAN fluorescence plate reader and mitoxantrone fluorescence was measured at 620 nm excitation and 670 nm emission wavelengths. Three wells of each plate contained the internal standard FTC (10 µM). Its cellular fluorescence was taken as a measure of 100% BCRP inhibition.

A c c e p t e d M a n u s c r i p t 10 fibrosarcoma

 10 cells, included for reasons of comparison, were equally susceptible to chalcone 1 and its platinum complex 2.

A c c e p t e d M a n u s c r i p t various transport pathways blocked by these inhibitors have in the uptake of 1 and 2 .

 2 Treatment of the cells with 100 µM chalcone 1 in the absence of any inhibitors for 3.5 h left 40% of the cells vital. Pre-incubation with 5 µM ouabaine attenuated the cytotoxicity of 1 by ca. 40% so as to leave 66.5 ± 5% of the cells alive. In other words, ca. 40% of chalcone 1 were accumulated by the cells via Na + /K + -dependent endocytosis. Addition of 10 µM amiloride led to 55 ± 2% of the cells surviving. This corresponds to ca. 24% uptake of 1 via Na + /H + pump driven macropinocytosis. Addition of 14 µM chlorpromazine resulted in ca. 50% of the cells remaining vital, which speaks for 18% of the chalcone being taken up through clathrine-mediated endocytosis. 10 nM wortmannin decreased the percentage of vital cells after exposure to 1 to 48 ± 3 %, indicating 13% contribution from PI3-kinase associated macropinocytosis in the accumulation of chalcone 1. Interestingly, the inhibitory effect of wortmannin was time-dependent and most pronounced at the beginning (1-2 h) of the incubation with 1. All these inhibitors interfere with endocytosis processes. A possible explanation is that 518A2 cells are mTOR positive and that PI3-K/Akt-signalling regulates mTOR activity. Hence, any inhibition of PI3 kinase might postpone the cytotoxic effect of 1.Inhibition of the OCT and Ctr1 had little influence (2-3%) on the cytotoxicity of 1. Complex 2 was accumulated by 518A2 cells differently from the parent chalcone 1. If administered without any inhibitors added, 200 µM concentrations of complex 2 were necessary to reduce the number of viable cells to 40 ± 2% as in the case of 1. Addition of 5 µM of the Na + /K + -ATPase pump inhibitor ouabaine decreased the cytotoxicity of 2 by 24%. Clathrine-dependent endocytosis contributed ca. 14% to the uptake of complex 2 as shown by a reduction of the cytotoxicity to the effect of 50 ± 3 % viable cells upon addition of 14 µM inhibitor chlorpromazine. Uptake of 2 via Na + /H + pump driven macropinocytosis seemed to play no role. Cell entry of complex 2 took place mainly via OCT-1/2 (32%) as shown by an increase A c c e p t e d M a n u s c r i p t 13 of viable cells to ca. 61% upon addition of 500 µM cimetidine and via Ctr1 (20%) as the addition of 8 nM CuCl 2 increased the percentage of viable cells to ca. 55%.

A c c e p t e d M a n u s c r i p t 14 of

 14 the clinically established chemosensitiser verapamil[START_REF] Schwab | Comparison of in vitro Pglycoprotein screening assays: recommendations for their use in drug discovery[END_REF][START_REF] Sharom | The P-glycoprotein efflux pump: how does it transport drugs?[END_REF] was set to 100%. Surprisingly, wild type KBv1 cells under the same conditions reached only ~45% of calcein fluorescence of the resistant KBv1 +Vbl cells. Possibly, they have other ways at their disposal to eliminate calcein-AM or calcein, which is not without precedence[START_REF] Marchan | Multidrug resistance-associated protein 1 as a major mediator of basal and apoptotic glutathione release[END_REF][START_REF] Oku | Permeability change of liposomal membrane induced by interleukin-1 alpha[END_REF]. Calcein fluorescence in KBv1 cells was also relatively low in the absence of verapamil. Treatment with chalcone 1 led to almost the same absolute fluorescence intensity of calcein in either, resistant and wild type cell line. In contrast, Pt-complex 2 was more effective than chalcone 1 in the resistant cells. Relative to the fluorescence in the presence of verapamil, complex 2 caused an identical decrease of calcein fluorescence in KBv1 and KBv1 +Vbl cells. MCF-7 breast carcinoma cells express BCRP drug transporters and when treated with topotecane (550 nM over 40 generations) turn multi-drug resistant overexpressing this protein (MCF-7 +Top cells). The mitoxantrone assay is based on the active export through BCRP of the fluorescent mitoxantrone, accumulated by the cells via diffusion. The effects of chalcones 1 and 2 on the fluorescence of intracellular mitoxantrone in MCF-7 and MCF-7 +Top cells relative to that of the specific BCRP inhibitor fumitremorgine C [39] and to a negative control of cells treated only with mitoxantrone are shown in Fig. 4b. Both compounds gave rise to a doubling of the mitoxantrone fluorescence in either cell line relative to the negative control and so are likely

A c c e p t e d M a n u s c r i p t

  monitored by time-lapse microscopy[START_REF] Hegedus | Locomotion and proliferation of glioblastoma cells in vitro: statistical evaluation of videomicroscopic observations[END_REF]. Neurons treated with 10 µM of the test compounds

u s c r i p t 24 48Fig. 1 .Fig. 2 .Fig. 3 .Fig. 4 .

 241234 Fig. 1. Chalcone 1 and its dichlorido(6-aminomethylnicotinate)Pt(II) complex 2.

Fig. 5 .A c c e p t e d M a n u s c r i p t 29 (Fig. 6 .

 5296 Fig. 5. Top row: left: untreated neurons with prolonged axons (arrowheads); middle: retraction of axons back to soma or decay (arrowheads) upon treatment with 10 µM 1; right: upon incubation with 10 µM 2 some axons (arrowheads) retracted and most somas coagulated (arrows). Bottom row: left: untreated 518A2 melanoma cells (control); middle: rounded cells of increased motility starting to detach after 10 min incubation with 10 µM of 1; right: same effects delayed by 2 min upon treatment with 10 µM of complex 2.

A c c e p t e d M a n u s c r i p t 30 (Fig. 7 .Fig. 8 .A c c e p t e d M a n u s c r i p t 32 (Fig. 9 .

 3078329 Fig. 7. Immunostaining for actin of 518A2 cells untreated (top row), after 24 h incubation with 10 µM chalcone 1 (middle row), and with 10 µM Pt-complex 2 (bottom row).

A c c e p t e d M a n u s c r i p t 33 (Fig. 10 .

 3310 Fig. 10. TEM images of 518A2 cells treated with chalcone 1 (a) or complex 2 (b). Arrows indicate Golgi apparatuses, which are intact in (a) but fragmented in (b). Dashed arrow indicates an autophage.

Fig. 1 .Fig. 2 .

 12 Fig. 1. Chalcone 1 and its dichlorido(6-aminomethylnicotinate)Pt(II) complex 2.

Fig. 3 .Fig. 4 .

 34 Fig.3. Semiquantitative westernblot analysis for organic cation transporters OCT-1 (~56 kDa) and OCT-2 (~30 kDa) in 518A2 melanoma and KBv1 +Vbl cervix carcinoma cells.Samples were probed with an antibody against -tubulin (60 kDa) to show equal loading of lanes.

Fig. 5 .Fig. 6 .Fig. 7 .

 567 Fig. 5. Top row: left: untreated neurons with prolonged axons (arrowheads); middle: retraction of axons back to soma or decay (arrowheads) upon treatment with 10 µM 1; right: upon incubation with 10 µM 2 some axons (arrowheads) retracted and most somas coagulated (arrows). Bottom row: left: untreated 518A2 melanoma cells (control); middle: rounded cells of increased motility starting to detach after 10 min incubation with 10 µM of 1; right: same effects delayed by 2 min upon treatment with 10 µM of complex 2.

Fig. 8 .

 8 Fig. 8. Flow cytometric analysis of 518A2 melanoma cells stained with propidium iodide. The cells were treated with 250 µg/mL nocodazole or with 10 µM of chalcone 1, or chalcone-Pt 2 or cisplatin. Detailed FACS profiles are available in the supplementary data file.

Fig. 9 .

 9 Fig. 9. TEM images of organelles in 518A2 cells. a, d: untreated cells with image a showing the cross and longitude section through the centrioles and image d sound mitochondria with cristae invaginations surrounded by endoplasmic reticulum. b, e, h: cells treated with 1. c, f, g, i: cells treated with 2. Image e shows mitochondria at various stages of inner-membrane destruction, image f the formation of multivesicular bodies (arrowheads). Some mitochondria were highly condensed (i, g stars) and some formed multi-lamellar bodies (arrows). Stress fibres are visible in panel h. Black bar indicates 0.4 µm.

Fig. 10 .

 10 Fig. 10. TEM images of 518A2 cells treated with chalcone 1 (a) or complex 2 (b). Arrows indicate Golgi apparatuses, which are intact in (a) but fragmented in (b). Dashed arrow indicates an autophage.

A c c e p t e d M a n u s c r i p t 37

 37

Figure2 A c c e p t e d M a n u s c r i p t

  Figure2

Figure3 A c c e p t e d M a n u s c r i p t

  Figure3

Figure4A

  Figure4

Figure8A

  Figure8

  SDS) in DMSO containing 0.6% acetic acid. For the neural cells the formazan was dissolved in acidified (0.08 M HCl) isopropanol and measured immediately. The absorbance at 570 nm and 630 nm was measured with an automatic microplate reader (MWG-BIOTECH/BioRad). All experiments were carried out at least in triplicate; the percentage of viable cells was calculated as the mean ± SD relative to controls set to 100%.

5

cells/mL) were cultured for 12-24 h depending on growth progress. Incubation (5% CO 2 , 95% humidity, 37 °C) of the cells following treatment with the test compounds dissolved in DMF (dilution series ranging from 0.01 to 50 µM in medium) was continued for 24 h or 48 h. Blank and solvent controls were treated identically. Then a 1.25 mg/mL stock A c c e p t e d M a n u s c r i p t 6 solution of MTT in phosphate buffered saline was added to a final MTT concentration of 0.125 mg/mL. After 2 h the formazan precipitate was dissolved in a 10% solution of sodium dodecylsulfate (

Acknowledgement

We thank the Deutsche Forschungsgemeinschaft for financial support (grant Scho 402/8-3).

We are grateful to Dr. S. Geimer, D. Scholz and M. Fischer from the Electron Microscopy Laboratory (Biology) for expert help with electron microscopy and to Prof. O. Stemmann from the Genetics department for help with cell cycle analysis.

Supported by the Deutsche Forschungsgemeinschaft DFG (grant Scho 402/8-3).

((classification: antibiotics and chemotherapeutics))

A c c e p t e d M a n u s c r i p t 2


Kenneth J Broadley

Shane C Sykes

Dr Robin H Davies

Towards a Thermodynamic Definition of Efficacy in Partial Agonism: II The Thermodynamics of Efficacy and Ligand Proton Transfer in a G Protein-coupled Receptor of the Rhodopsin Class

Keywords: Thermodynamics, efficacy, agonist, antagonist binding enthalpies, ligand proton transfer, rhodopsin receptors

_____________________________________________________________________

The thermodynamic binding profiles of agonist and antagonist complexes of the 4hydroxypropanolamine partial agonist, prenalterol, on the chronotropic adrenergic response in guineapig right atria were determined over a 15 temperature range. The tissue response was compared with data on the ethanolamine agonist, isoprenaline, given by binding studies in a number of rat tissues.

Utilising the residue conservatism surrounding the known active conformers bound to either of two aspartate residues ( -helices II,III) in both receptors ( 1 , 2 ) and species (guinea-pig, rat and human), no significant deformation in the extended sidechain could be found in prenalterol's agonist binding compared to isoprenaline. Antagonist binding gave a highly favourable entropy contribution at 30.0 C of -4.7 1.2 kcal/mol. The enthalpy change between bound agonist and antagonist complexes, a function of the efficacy alone, was -6.4 ± 1.1 kcal/mol, coincident with the calculated intrinsic preference of a primary/secondary amine-aspartate interaction for a neutral hydrogen-bonded form over its ion pair state, giving values of 6.3 -6.6 kcal/mol with calculations of good quality, a figure expected to be close to that shown within a hydrophobic environment. Delivery of a proton to a conserved aspartate anion ( -helix II) becomes the critical determinant for agonist action with resultant proton transfer stabilisation dominating the enthalpy change.

A proposed monocation-driven ligand proton pumping mechanism within the ternary complex is consistent with the data, delivery between two acid groups being created by the movement of the cation and the counter-movement of the ligand protonated amine moving from Asp 138 ( -helix III) to Asp 104 ( -helix II).

Introduction

An interpretation of partial agonism as the result of two bound complexes related to agonist and antagonist action existing within a G protein-receptor ternary complex was considered in paper I [START_REF] Broadley | Towards a thermodynamic definition in partial agonism[END_REF] and the requisite concentration-response theory for a compound obeying a hyperbolic relation derived using a simple adaptation of the Black and Leff operational model of agonism [2]. A tissue response, (the chronotropic in the guinea-pig right atrium) was selected based on data indicating its dominant control by the 1 -adrenergic receptor under normal conditions of signal amplification (refer also, paper I [START_REF] Broadley | Towards a thermodynamic definition in partial agonism[END_REF]). The advantage of such a description, transposing the observed parameters of binding and efficacy into two binding constants related to agonist and antagonist action, lies in temperature variation and the determination of the thermodynamic profiles of the respective agonist and antagonist complexes.

Temperature studies [START_REF] Weiland | Fundamental difference between the molecular interactions of agonists and antagonists with the beta-adrenergic receptor[END_REF][START_REF] Weiland | Thermodynamics of agonist and antagonist interactions with mammalian beta-adrenergic receptors[END_REF][START_REF] Contreras | Thermodynamic properties of agonist interactions with the beta-adrenergic receptor-coupled adenylate cyclase system. I High and low affinity states of agonist binding to membrane-bound beta-adrenergic receptors[END_REF][START_REF] Contreras | Thermodynamic properties of agonist interactions with the beta-adrenergic receptor-coupled adenylate cyclase sytem. II Agonist binding to soluble beta-adrenergic receptors[END_REF] using isolated membranes for the binding of ligands to G protein-coupled receptors complexes utilising -adrenergic receptor sub-types showed linear plots of unit slope over a 25

C range of temperature offering the prospect of accurate enthalpic changes if sufficient precision were attained with a given receptor. While such data contain binding studies on more than one receptor subtype, it was considered here, more illuminating to select a non-specific partial agonist structurally related to the natural hormone but to choose the tissue response dominated by a single receptor sub-type.

Under normal signalling conditions, the chronotropic response in guinea-pig right atria was shown to be dominated by the 1 -adrenergic receptor with a less than 5% contribution from the 2 -receptor. Data 'in vitro' at 30.0 C on the binding and efficacy of the phenoxypropanolamine partial agonist, prenalterol to this cardiac 1 -adrenergic receptor were given in the first paper [START_REF] Broadley | Towards a thermodynamic definition in partial agonism[END_REF]. The precision was shown to be sufficient for evaluation of the thermodynamics of the respective complexes over a 15 C range of temperature [START_REF] Broadley | Towards a thermodynamic definition in partial agonism[END_REF]. Considerable insight into structural features of ligands using close extra-thermodynamic comparison can be obtained by transforming data to a pure hydrocarbon phase reference using appropriate partitioning methods which can provide additional insight into the polar and distortional changes observable on receptor binding [START_REF] Davies | Drug and Receptors in Molecular Biology[END_REF]. Using this reference, the thermodynamic profile of the agonist binding of prenalterol will be compared with published data for the non-specific 3,4 di-hydroxyphenethanolamine agonist, isoprenaline, determined from -adrenergic receptors in a number of rat tissues. The 1 -receptor residues in guinea-pig, rat and human which surround the known bound ethanolamine conformer within 4.5 Å of its expected 'upper' (Asp138; -helix III) and 'lower' (Asp 104; -helix II) positions within the receptor are all conserved. Of the 19 residues in potential contact in the upper position and eighteen residues in the lower, only two residues in the upper position, Phe 362 (Leu -2 ) and Arg 208 (Asn-2 )

receptors distinguish 1 -from 2 -receptors. While these latter residues are not in immediate contact, their potential influence on signal amplification will be considered in Section 6. Selectivity in receptor binding is thus not expected with the ethanolamine derivative between rat, guinea pig and human while consistency should also be observed in the binding of isoprenaline to 1 -and 2 -receptor derivatives in these three species. It will be later shown that the relevant agonist bound conformer of the phenoxypropanolamine derivative, prenalterol, surprisingly, shows no deformation in its binding to the 1 -adrenergic receptor but its thermodynamic profile is very closely related to the ethanolamine derivative. Finally, as reported earlier, [START_REF] Broadley | The separation of agonist and antagonist components in the gross binding of a partial agonist on the cardiac -adrenoceptor and associated conformer thermodynamics[END_REF] in comparison with data on membrane-binding studies, it is shown that the enthalpy associated with the Black and Leff amplifying parameter, , is negligible, indicative of a membrane diffusion process or ion channel opening.

A c c e p t e d M a n u s c r i p t

Efficacy/Proton transfer_II

The publication of the first mammalian G protein-coupled receptor structure in 2000 [START_REF] Palczewski | Crystal Structure of Rhodopsin: A G Efficacy/Proton transfer_II protein-coupled receptor[END_REF] led to support for a proposed proton shuttle mechanism for the ligand action in an adapted G protein-coupled β 1adrenergic receptor ternary complex with the identification of a series of potential proton relays in a GTP synthase mechanism of action [START_REF] Allen | Monocation-driven proton transfer relays within G protein-coupled receptors of the rhodopsin class and the GTP synthase mechanism[END_REF]. More recently, the adapted receptor structure has been confirmed by the presence of very similar -helical packing in a modified β 2 -adrenergic receptor structure [START_REF] Rasmussen | Crystal Structure of the 2 -adrenergic G protein-coupled receptor[END_REF]. We present a simple physical interpretation of the enthalpic changes observed between agonist and antagonist complexes supporting the view that minimal agonist response is dependent on ligand protonation of the conserved aspartate residue at position 104 on α-helix II. This is consistent with the wider proposed mechanism of the operation of the ligand as a proton shuttle operating on delivering proton transfer through two proton relays under the driving force of a monocation.

2.

Methods

Experimental protocols for determining binding constants and efficacies of the partial agonist, prenalterol [3-(4-hydroxyphenoxy)-1-isopropylamino propan-2-ol, ref. Figure 2], based on the 1adrenoceptor dominating the chronotropic response in guinea-pig right atria were given in Paper I [START_REF] Broadley | Towards a thermodynamic definition in partial agonism[END_REF].

The simple conversion to agonist and antagonist binding complexes was exemplified with data at 30.0 °C.

Results

The conformer binding constants are closely related to the Black and Leff representation and are summarised in [START_REF] Broadley | Towards a thermodynamic definition in partial agonism[END_REF]. The apparent binding constant K B / of the partial agonist B is the sum of the conformer binding constants but where the agonist component incorporates an amplification factor from the signal enhancement of the response. The agonist component may thus be defined by K ijx ( + 1)

where the agonist conformer i interacts with its receptor counterpart j x but there is an additional term K ijx from the signal amplification. The antagonist component is defined by the ligand and receptor

and the appropriate free energy relationships may be defined. As commented earlier, the fact that such bound drug-receptor conformers may have no separate unbound existence does not invalidate their pharmacological definition and thermodynamic functions can obviously be associated with such forms.

The theoretical unbound equivalent forms, however, represent the ideal conformation for maximum interaction with the receptor in the given binding mode. In these simple functions, it is seen in ( 1) that the ratio of the two conformer constants is independent of the apparent binding and is a function of the efficacy alone. Thus on the log scale, differences in these conformer constants plotted as a reciprocal function of the absolute temperature, or more simply changes in the efficacy function alone yield the required enthalpy changes in going from the bound antagonist to the agonist form directly. This is a remarkably useful result in elucidating the contributions to the composite energy requirements for receptor activation.

The respective binding constants at 40.0 °C and 25.0 °C are given in Tables 1 and2. Over the narrow 15 °C temperature range, the Langmuir behaviour is observed, the slopes of the dose-response relations for both the agonist and antagonist responses of prenalterol having slope relations not significantly different from unity. This is also shown for the data at 30.0 °C in the first paper [START_REF] Broadley | Towards a thermodynamic definition in partial agonism[END_REF]. For individual experiments at 40.0 °C and 25.0 °C , there is a slight, but not significant tendency for experiments with lower efficacy to have apparent binding constants (K B /

) slightly greater when determined by antagonist binding than by agonist binding.

The observed binding constants whether determined from the agonist or antagonist response of the partial agonist show no significant differences over the three temperatures (40.0 C 7.16 ± 0.05 [START_REF] Contreras | Thermodynamic properties of agonist interactions with the beta-adrenergic receptor-coupled adenylate cyclase sytem. II Agonist binding to soluble beta-adrenergic receptors[END_REF], Accuracies in binding and efficacy on the log 10 scale are required to within ± 0.03 5 log 10 units for determining the enthalpy of binding between agonist and antagonist conformers to within 1.0 -1.5

kcal/mol over the 15 °C temperature range and are of this order. Plotting of the EC50 binding data against the reciprocal of the absolute temperature with the associated enthalpies of interaction are shown in Figure 1. The data have very slightly tighter error bars compared with the data reported in our 1991 paper [START_REF] Broadley | The separation of agonist and antagonist components in the gross binding of a partial agonist on the cardiac -adrenoceptor and associated conformer thermodynamics[END_REF] but are, in effect, identical to the reported values (±0.1kcal/mol). The earlier thermodynamic enthalpic functions are reproduced in Table 3. The binding constants determined from the blocking action of the partial agonist have tighter error bars but the differences between the conformer binding enthalpies are, unsurprisingly, the same 6.6 ± 1.6-2.7 kcal/mol Direct fitting of e/(1e) against 1/T on the log scale gives an enthalpy of activation of 6.2 ± 1.1 kcal/mol [START_REF] Broadley | The separation of agonist and antagonist components in the gross binding of a partial agonist on the cardiac -adrenoceptor and associated conformer thermodynamics[END_REF]. We take a compromise figure of 6.4 kcal/mol for this activation of agonist action for prenalterol and compare existing data on the thermodynamics of binding of ethanolamine and phenoxypropanolamine agonists and antagonists on the β 1 -adrenergic receptor.

Discussion

3.1

Extrathermodynamic comparison of related derivatives of prenalterol and the use of a reference hydrocarbon phase model

There is a wealth of evidence to suggest that the best insight into comparing binding of ligands within membrane-bound proteins is to employ a mobile reference hydrocarbon phase model. At the membrane level, the relation between observed binding and that predicted by a partitioning effect into a hydrocarbon phase is one of exact unit slope where no change in intrinsic binding takes place [START_REF] Davies | A steady-state model of drug-receptor interaction in vivo applied to antagonists of the -adrenergic receptor[END_REF]. The question arises as to how far extra-thermodynamic changes can be usefully interpreted by such a model when comparing closely related species and their thermodynamic changes bearing in mind the overall accuracy required in observing thermodynamic changes. An early exploration showed that such where the free energy changes indicated that there was flexible freedom for localised groups even when adjacent to expected polar ligand-receptor interactions in mammalian -adrenergic receptors. The data were exemplified by predicting the thermodynamic binding changes of adrenaline from isoprenaline based on simple alkyl differences observed in turkey erythrocyte β-adrenergic receptor data [START_REF] Davies | Drug and Receptors in Molecular Biology[END_REF]. The underlying reason for predicting these changes is based on the incremental thermodynamics of partitioning of the -CH2group where the local interactions in hydrocarbon and water are energy and entropically driven respectively and the data are reproduced in Table 4 [START_REF] Abraham | Free energies enthalpies and entropies of gaseous nonpolar nonelectrolytes in water and nonaqueous solvents. The hydrophobic effect[END_REF]. The overall partitioning change from water to hydrocarbon becomes one of apparent simplicity, the overall effect being one of almost equal favourable enthalpic and entropic (-T S) contributions of -0.45 and -0.49 kcal/mol respectively at blood temperature, with an overall group free energy change of -0.94 kcal/mol. The effective overall group partitioning assumption on the basis of simple additivity appears accurate to within 5% in accordance with conclusions attributable to three body effects in solution in simple non-polar inert gas liquid mixtures [START_REF] Cahill | Rydberg-London potential for diatomic molecules and unbonded atom pairs[END_REF]. A group additivity assumption directly applied to the potency of halogenated hydrocarbons as volatile anaesthetics perturbing an ion channel was accurate to 5-10% [START_REF] Davies | A quantitative interpretation of phase effects in anaesthesia[END_REF][START_REF] Davies | Anaesthetic Potency and the perturbation of positive ion or proton acceptor sites in the nerve membrane[END_REF]. Thus , if there is regional tolerance in the binding of the alkylamino groups in isoprenaline and adrenaline we should expect a free energy increment at 30.0 C of -1.84 kcal/mol with appropriate local enthalpic and entropic contributions.

We use this simple group assumption to check the consistency of earlier reported thermodynamic binding data on isoprenaline and adrenaline on rat β-adrenergic receptors within the cerebral cortex, cerebellum, heart and lung, where the cortex and heart are dominated by 1 -adrenergic receptors and the cerebellum and cortex predominantly by the corresponding β 2 -receptors [2]. The data are confined to those where 300 μM concentrations of GTP were added. As commented in the Introduction, early structural models of the 1 -and 2 -adrenergic receptors based on bovine rhodopsin (refer Section 6)

show that the immediate residues surrounding these two ligands, isoprenaline and prenalterol bound to the two conserved aspartate residues on -helices III and II in the respective binding sites are identical both for rat, guinea-pig and human. Table 5 shows the accurate consistency of these data at the the enthalpy change is reported to be -17.2 ± 1.03 kcal/mol. We have no explanation for why this result for the cardiac receptor should show such a significant difference from the predicted value based on adrenaline in these otherwise excellently consistent data based on binding alone, particularly when simple correlations at the free energy level 'in vitro' on the rat and higher species indicate tolerance of the methyl and isopropyl non-polar groups within the β 1 -receptor site [START_REF] Contreras | Thermodynamic properties of agonist interactions with the beta-adrenergic receptor-coupled adenylate cyclase system. I High and low affinity states of agonist binding to membrane-bound beta-adrenergic receptors[END_REF]. Structurally and experimentally, the ligands, themselves are not expected to show tissue selectivity between 1 -and 2receptor binding nor to exhibit binding differences in common species. The data require further validation but we take the hypothesis that the most consistent values for the thermodynamics of binding of isoprenaline to the cardiac β 1 -receptor are likely to be those given by the mean values in Table 5. This assumption must remain a working hypothesis.

3.2

Binding thermodynamics of the agonist conformer of prenalterol

Thermodynamic data on the agonist binding of the phenoxypropanolamine derivative, prenalterol (refer also Table 5), determined from chronotropic reponses are in very close agreement with those predicted from the mean values of the ethanolamine analogue, isoprenaline, using radioligand binding studies.

This is an unexpected result and we first review any concentration difference expected from extrathermodynamic comparison using a hydrocarbon reference phase. The two compounds are shown in Figure 2. In the agonist conformer [17], the 3-hydroxy group of isoprenaline is expected to have an intramolecular hydrogen bond to the 4-oxygen atom while both compounds have two delocalised aromatic oxygen atoms. For any concentration difference in a hydrocarbon reference phase, this difference on the free energy scale is expected, at most, to be of the order of 0.5-0.9 kcal/mol. We take A c c e p t e d M a n u s c r i p t 10 Efficacy/Proton transfer_II this value to be not significant for comparison purposes. Our first conclusion is that, since the rat thermodynamic data were determined from radioligand membrane binding studies, there is clear evidence in prenalterol activation that the enthalpy change associated with the signalling amplification factor, , is very small. This is indicative, as previously commented, of a diffusion process from ion channel opening rather than a specific activation energy.

The current background to the known binding conformations of phenoxypropanolamine and phethanolamine ligand side chains is reviewed. Much of the development of the understanding of the behaviour of ligands in the β 1 -adrenergic receptor site has come from close molecular comparison and synthetic effort. The agonist conformation of the adrenaline side chain was determined by appropriate cyclisation of the ethanolamine side chain and the resultant identification of the agonist conformer [17]. Cyclisation of the side chain of the appropriate phenoxypropanolamine derivative to fold the amino group back to an ethanolamine position proved inactive in both agonist and antagonist action (Clarkson R, Dowell R. unpublished data ). These data suggested that a possible contraction of the phenoxypropanolamine side chain closer to the given ethanolamine agonist conformation might be the primary cause of agonist action. Some support for this came from a series of 2-substituted phenoxypropanolamine analogues of prenalterol lacking the p-hydroxyl substituent which showed a constant observed binding constant (K B / ± 0.15 kcal/mol) when referenced to the hydrophobic environment but the efficacy varied between 0.13 and 0.51 [START_REF] Wilkinson | Distortion and energetics in the agonist conformation of bound phenoxypropanolamine agents in the 1 -adrenoceptor[END_REF]. The energetics of an in-plane distortion around the 2-substituted phenoxymethylene moiety consistent with a small contraction of little more than 0.5 Å gave a correlation of unit slope when plotted against the hyperbolic efficacy relation on the thermodynamic scale over a predicted 1.2 kcal/mol range. This was consistent with the highest observed agonist activity of a phenoxypropanolamine derivative in the form of a benzdioxepine substituent with the appropriate side chain (see Figure 3). On the free energy scale, the movement from antagonist to almost full agonist over a 5 -95 % efficacy range should be complete over a 2.0 kcal/mol free energy range and these energetics for close comparison were of the right order. Yet we have a large enthalpic change in going from an antagonist to agonist complex within the phenoxypropanolamine agonist complex which is, as yet, unaccountable. An early examination of a contraction in the phenoxypropanolamine side chain of a β ! -adrenergic receptor partial agonist attempted to see whether inhibition of vibrational contributions in the side chain could contribute significantly to the energetics observed for contractions involving 0.2, 0.5 and 1.0 Ǻ respectively in an approximate 1-dimensional contraction of the bound molecule retaining the expected agonist conformer [START_REF] Wilkinson | Vibrational modes in the antagonist and agonist action of ligands on the 1 -adrenoceptor and some receptor site geometry[END_REF]. With a limited basis set, the scale of these unfavourable deformation energies gave 0.4, 1.5 and 4.6 kcal/mol respectively. The vibrational energy differences on contraction were, however, negligible. In assessing the potential contributions, we utilised the observed enthalpy of binding for isoprenaline on the cardiac β 1 -adrenergic receptor taking the previously discussed value of 17.2 kcal/mol and attempted to allocate a compensatory contraction as a component of the value. This The very significant enthalpic difference of -6.4 ± 1.1 kcal/mol observed between agonist and antagonist complexes may now be considered. One might, in general, have expected quite close enthalpic differences between the two complexes for a 50% partial agonist reaching a critical activation energy for achieving a response or just failing to do so unless some critical stabilisation energy was invoked before activation of the receptor became possible. A critical feature of agonist action in the proton pumping mechanism is the delivery of a proton from the basic protonated amino side chain of the ligand to the receptor's aspartate 104 anion to form the more stable neutral hydrogen bonded acidbase with the resultant delivery of the proton to the receptor. The criterion for stimulant action at its most basic level would be, therefore, the delivery of a proton to this residue in the receptor where an automatic stabilisation to the neutral hydrogen bonded acid-base form would occur. This is strongly supported by the intrinsic stabilisation energy between the neutral and ion pair forms of the acid-base interaction [START_REF] Allen | Monocation-driven proton transfer relays within G protein-coupled receptors of the rhodopsin class and the GTP synthase mechanism[END_REF]. In Table 1 of this reference, three extensive calculations including higher level electron and exchange correlation contributions and a density functional comparison (HF 6-31++G** 6.6; +MP2 6.2; B3-LYP 6.3 kcal/mol) showed strong support for this stabilisation energy which in a hydrophobic environment is anticipated to be close to this value. Thus arrival of the protonated amine in a correct orientation for proton transfer to this aspartate 104 anion site automatically creates this additional stabilisation energy on transfer of the proton and would provide the most basic criterion for agonist action, namely that a proton must be resident at Asp 104 for agonist action (with subsequent delivery to the G -protein under the influence of a monocation).

3.3

The ligand-receptor-G protein ternary complex and signal amplification

The development of a monocation-driven proton pump within the ligand-receptor-G protein-ternary complex where the ligand acts as a proton shuttle has developed quite simply from these thermodynamic Efficacy/Proton transfer_II considerations on the binding of the partial agonist, prenalterol and its companion ethanolamine derivative, isoprenaline, taken in conjunction with the adapted β 1 -adrenergic receptor structure and its proposed ternary complex, based on mammalian bovine rhodopsin [START_REF] Allen | Monocation-driven proton transfer relays within G protein-coupled receptors of the rhodopsin class and the GTP synthase mechanism[END_REF]. Prior to the publication of the bovine rhodopsin structure, a steady state model of the ligand-receptor-G protein complex functioning as a GTP synthase in the β 1 -adrenergic receptor complex was explored [START_REF] Broadley | The ligand-receptor G protein ternary complex as a GTP synthase. Steady state proton pumping and doseresponse relationships for -adrenoceptors[END_REF]. While the proposed acidbase-acid shuttle was not correct involving supporting tyrosine residues rather than more readily ionised acid groups, the exploration of the steady state equations using the ternary complex format [START_REF] Weiss | The cubic ternary complex receptoroccupancy model. 1. Model description[END_REF][START_REF] Weiss | The cubic ternary complex receptoroccupancy model[END_REF] had widespread generality. The model could not distinguish between a GTP synthase or a GDP/GTP exchange model on receptor stimulation. The steady state conditions led to a number of potential solutions with forty potential terms in the denominator, but two dominant features leading to a hyperbolic form of the dose-response were cited. The steady state controlling rate was (1) rate limiting due to activation of proton signalling through an initiating acid-base-acid triad or (2) rate controlled by G protein release but there was, also, an activation barrier to proton signalling though the initial residue triad. The terminology is slightly different to that employed here but is closely correspondent. The approximate steady state solutions for (1) and ( 2) are very similar but there is an additional multiplicative rate constant, k 31 , in the latter, being the effective rate of G protein release. The absence of an enthalpic energy associated with the signal amplification factor, indicates 1) that there is a likely diffusion process activating the signal mechanism and possibly, also, for G protein release or 2) the release of the G protein is not rate limiting. But do we expect to see signal amplification in a steady state solution for proton pumping where there is a 1:1 synthesis of GTP from GDP due to proton-activated phosphate release from a glutamate residue at the base of the G 2-helix (at Glu 203) with transfer to form GTP followed by a 1:1 formation of cAMP by activation of the adenylyl cyclase by the energised G s.GTP protein? An early study showed an absence of signal amplification in the production of cAMP from isoprenaline [START_REF] Minneman | The pharmacological specificity of beta-1 and beta-2 adrenergic receptors in rat heart and rat lung 'in vitro[END_REF]. This conclusion should be valid for any common process of signal amplification involving the G s protein. Equilibrium binding constants for isoprenaline based on cAMP accumulation gave mean values -7.13 ± 0.09 on the log 10 scale from 2 laboratories [START_REF] Buckner | Studies on -adrenoceptors mediating changes in mechanical events and adenosine 3 / ,5 / -monophosphate levels, rat atria[END_REF][START_REF] Buckner | Stereoselectivity of isoproterenol-induced changes in cyclic AMP and mechanical events in rat atria and guinea-pig trachea[END_REF][START_REF] Birnbaum | Changes in mechanical events and adenosine 3 / ,5 / -monophosphate levels induced by enantiomers of isoproterenol in isolated rat atria and uteri[END_REF]. Seven binding studies using guinea pig and cat with both atria and ventricle and rat and dog with ventricle alone, gave an average binding constant on this scale of -6.8 3 ± 0.1 3 (omitting two large outliers) [START_REF] Broadley | Cardiac -adrenoceptors[END_REF]. (based on l-adrenaline) produced an average value of -7.0 ± 0.15. Binding constants observed from chronotropic responses, on the other hand, show observed values of -8.9 0 ± 0.1 1 [START_REF] Kaumann | Desensitisation of kitten atria to chronotropic, inotropic, and adenylyl cyclase stimulating effects of (-) isoprenaline[END_REF][START_REF] Broadley | Functional antagonism as a means of determining dissociation constants and relative efficacies of sympathomimetic amines in guinea-pig isolated atria[END_REF], suggesting an amplification factor of 50-100 and a similar result exists for the inotropic response -8.8 7 ± 0.1 8 on the log 10 scale [START_REF] Kaumann | In kitten ventricular myocardium, the inotropic potency of an agonist is determined by both the intrinsic activity for the adenylyl cyclase and its affinity for theadrenoceptors[END_REF]. However, in Table 2, the thermodynamic parameters for prenalterol were determined from the chronotropic response but compared with the values for isoprenaline determined from radioligand binding studies.

The key to this apparent anomaly lies simply in the effective value of for a partial agonist. It may be seen from equation ( 1) that by the definition of the partial agonist, the net product K ijx ( + 1) K rsx when the efficacy is in the region of 0.5 and the overall error in the true binding constant is, at most, within a factor of 2 of its accurate value. Similarly, this effective value of should be defined by the explicit steady state equations for proton pumping [START_REF] Broadley | The ligand-receptor G protein ternary complex as a GTP synthase. Steady state proton pumping and doseresponse relationships for -adrenoceptors[END_REF]. In conditions ( 12) and ( 13) of the paper (condition A1) where there is an activation barrier to proton signalling through the initiating residue triad, there is an explicit activation rate, k act1 following the binding of the agonist conformer K ijx but the terms are, otherwise, closely correspondent. The dominant terms for the linear response may be written

[X*] k act1 K ijx [A] K ijx K A [A] ____ = _____ ______________ = A _____ ______________ (2) [R TOT ] k e 1 + K A [A] K A 1 + K A [A]
where k e is the effective rate of conversion of the activated adenylyl cyclase or proportional to it.

For the hyperbolic response

[ YX*] K ijx [A] K ijx K / A [A] ______ = A ____________________________ = A ______ __________________ (3) [R TOT ] 1 + A K ijx [A] + K A [A] K / A 1 + K / A [A]
A c c e p t e d M a n u s c r i p t 15 Efficacy/Proton transfer_II where K / A = K A + K ijx = K rsx+ + K ijx ( + 1), the apparent binding constant containing the amplification term K ijx . If A is scaled by A + 1 for the hyperbolic response, the efficacy is as before.

The absence of an enthalpic change associated with the factor led to a search for potential data on related ion channels. Evidence that the local opening of a monocation channel on a rabbit atrial cell on stimulation by acetylcholine has been known for the last twenty five years [START_REF] Sakmann | Acetylcholine activation of single muscarinic potassium ion-channels in isolated pacemaker cells of the mammalian heart[END_REF][START_REF] Soejima | Mode of regulation of the Ach-sensitive K-channel by the muscarinic receptor in rabbit atrial cells[END_REF]. The current was

shown by patch pipette methods where the pipette, containing isotonic potassium chloride, holds the rabbit atrial cell in a surrounding bath. When the acetylcholine is in the bath no increase in current is seen (where release of a G protein might migrate to the region of the patch). The current was thus shown to be G protein dependent but no second messenger was involved. It was proposed that a direct modulation of a potassium (Ach) channel was involved in this membrane-delimited signalling, the activated G protein interacting directly with the channel. The presence of sodium ion has, also, been shown essential at Asp 104 on -helix II for agonist action [START_REF] Horstmann | An aspartate conserved among G protein receptors confers allosteric regulation of -2adrenergic receptors by sodium[END_REF][START_REF] Neve | Pivotal role for aspartate-80 in the regulation of dopamine D2-receptor affinity for drugs and inhibition of adenylyl cyclase[END_REF][START_REF] Strader | Conserved aspartic acid residues 79 and 113 of the -adrenergic receptor have different roles in receptor function[END_REF]. The obvious hypothesis is that the A final word on the Black and Leff amplification factor, , is appropriate. It may be seen that, at the receptor level two methods of exploitation of compound selectivity of action are achievable. Firstly a direct exploitation of residue binding differences but secondly small perturbations to the proton transfer The findings of this thermodynamic study on partial agonism may be summarized.

1. An adaptation of the Black and Leff operational model of agonist action in receptor theory where a single parameter, , is sufficient to define an amplifying contribution to any dose-response relationship exhibiting a hyperbolic form has been found useful in a treatment of partial agonism. By transposing efficacy and gross binding into two effective conformer binding components related to agonist and antagonist action, the binding thermodynamics of the two components of the partial agonist, prenalterol on the 1 -adrenergic receptor were determined. kcal/mol, a value highly consistent with the intrinsic stabilisation energy of a neutral hydrogen bonded acid-base over its ion pair form. This calculated value of 6.2-6.6 kcal/mol for the equivalent of a basic primary alkylamino-aspartate interaction, is expected to be close to that in a hydrophobic environment.

The stabilisation energy between agonist and antagonist complexes can, therefore, be consistent, with the delivery of a ligand proton to the aspartate ion at residue 104. defining the minimsl critical determinant of agonist action.

5. Signal amplification expected from the chronotropic response associated with the cardiac receptor used for determining the binding constant 'in vitro' was not found. The apparent absence of amplification when compared with data on radioligand binding studies arises from the nature of partial agonism, there being, in the component conformer representation, equal contributions of the antagonist and amplified agonist contribution when the efficacy is 0.5. Thus any error in the estimate of total binding arising from agonist signal amplification can, at most, be a factor of two only.

6. The high favourable entropy of binding of the antagonist conformer with a -T S contribution of some -4.7 ± 1.2 kcal/mol at blood temperature is consistent with data on other phenoxypropanolamine antagonists on the 1 -adrenergic receptor.

A c c e p t e d M a n u s c r i p t 4 Efficacy

 4 /Proton transfer_IIIn this second paper, the binding and efficacy of prenalterol, are extended to 25.0 and 40.0 C and the thermodynamic profiles of the agonist and antagonist complexes determined. The enthalpic difference between the agonist and antagonist complexes which is a function only of the efficacy, will be shown to have a simple physical interpretation consistent with a basic criterion for agonist action discussed below.

A c c e p t e d M a n u s c r i p t 6 Efficacy

 6 /Proton transfer_II conformers r and s x respectively or some appropriate sum. The observed conformer binding constants are very simply related to the apparent binding constant K B / by the relations ( 1e B ) K / = K rsx e B K / = K ijx ( + 1)

A c c e p t e d M a n u s c r i p t 7 Efficacy

 7 /Proton transfer_II (EC50); 7.26 ± 0.06[START_REF] Contreras | Thermodynamic properties of agonist interactions with the beta-adrenergic receptor-coupled adenylate cyclase sytem. II Agonist binding to soluble beta-adrenergic receptors[END_REF], (F-R) : 25.0 C 7.45 ± 0.03 (7) (EC50); 7.50 ± 0.04 (7) (F-R)) indicating, as expected, a competitive form of signal amplification of the response as exemplified in the first paper.

A c c e p t e d M a n u s c r i p t 8 Efficacy

 8 /Proton transfer_II incremental enthalpic and entropic changes could be followed for simple alkyl substitution on the ligand

A c c e p t e d M a n u s c r i p t 9 Efficacy

 9 /Proton transfer_II membrane binding level when comparing isoprenaline (observed) with isoprenaline (predicted) from adrenaline. The adrenaline lung datum was omitted on account of its large mean error in the binding, being an order of magnitude greater than the average The mean enthalpic change on binding over the four tissues gives -11.7 ± 0.3 6 kcal/mol over six of the experimental and predicted data points. These experimental and predicted estimates provide a convenient test of potential errors as we have omitted the direct experimental evidence for isoprenaline on the dominant cardiac β 1 -adrenergic receptor where

A c c e p t e d M a n u s c r i p t 11 Efficacy

 11 /Proton transfer_IIThe almost exact correspondence of the binding thermodynamics of the extended agonist conformer of the phenoxypropanolamine partial agonist, prenalterol, with those for the phenethanolamine full agonist, isoprenaline, together with the similar reference concentrations anticipated within the mobile reference hydrocarbon phase of the protein, indicate that we cannot argue about further distortion within the bound phenoxypropanolamine agonist conformer. The ligand-receptor interactions from the parahydroxyl group and from the basic side chain of the ligand in agonist action appear very closely similar to those of the ethanolamine derivative and must be very close to optimal with an extended phenoxypropanolamine side chain bound conformer. Any conformational change must, therefore, come from a facile movement of a receptor residue. The obvious movement in the context of agonist action which is later discussed, is that of a dihedral rotation of the conserved aspartate residue (α-helix II) from its expected ethanolamine interaction of -60.0 , to an extended 180.0 giving a minimal increased separation between relevant oxygen atoms of 2.6Å sited along a similar vertical axis across the membrane-bound protein. Here the extended conformer of the phenoxypropanolamine compound can interact through its basic amino group with the aspartate residue but without significant energy loss in the form of deformational energy along the molecular side chain.

A c c e p t e d M a n u s c r i p t 12 Efficacy

 12 /Proton transfer_II hypothesis is now rejected and we retain the mean tissue enthalpic change of -11.7 ± 0.3 6 kcal/mol for the binding of isoprenaline to the rat cardiac 1 -adrenergic receptor as discussed above.

A c c e p t e d M a n u s c r i p t 13

 13

A c c e p t e d M a n u s c r i p t 14 Efficacy

 14 /Proton transfer_II radioligand binding studies for the three experimental values of isoprenaline and three predicted results

  incipient monocation channel associated with receptors of the rhodopsin class could reside within the receptor-G protein complex and be based on acid groups on -helices I,II,III and VII. An attractive feature of the developed ternary complex model was that three acidic residues Asp 212, Cys 209 and Glu 203 of the G s 2-helix could be aligned with conserved acidic residues of the receptor and at similar inter-residue distances in the overall pathway to the cytoplasm's Glu 203, a pathway showing a verticality to the expected membrane surface and closely parallel to the orientation of -helix IV. The precise mechanism based on neutral acid-base interactions and their conversion to ion-pair forms under the influence of the monocation and resultant movement of the protonated base, has already been cited.

Figure 4

 4 Figure 4 reproduces the known agonist conformer of isoprenaline binding in its upper and lower positions to the two aspartate groups on -helices III and II at Asp 138 and 104 respectively.

A c c e p t e d M a n u s c r i p t 16 Efficacy

 16 /Proton transfer_II pathway may also create selective action by perturbing the efficiency of the proton transfer. Both mechanisms imply some variation in residue or selective binding but the latter action may prove relatively indirect.

2 .

 2 The thermodynamic binding profile for the agonist component of the binding of the 4hydroxyphenoxy-propanolamine, prenalterol, to the dominant cardiac 1 -adrenergic receptor determined from chronotropic responses in guinea pig right atria, was almost identical to that shown by the 3,4dihydroxyphenethanolamine derivative, isoprenaline binding but based on radioligand binding studies in a number of tissue receptors in the rat.. Since the amino acids in guinea-pig, rat and human in the immediate propsed binding regions of the two bound agonist ligands are all conserved in both 1 -and 2 -receptors, there is clear evidence that no deformational differences in binding can be present within the agonist bound conformers of the two compounds. The ligand-receptor interactions anticipated for the p-hydroxyl and side chain amino groups are concluded to be effectively identical.. This is only achievable in the expected agonist receptor site by facile movement of a receptor residue, the candidate being the conserved Asp 104 on -helix II, the dihedral rotation from -60.0 to 180.0 giving a minimal increased separation between relevant oxygen atoms of 2.6 Å sited along a similar axis across the membrane-bound protein.

A c c e p t e d M a n u s c r i p t 17 Efficacy/Proton transfer_II 3 . 4 .

 1734 The enthalpic component of the amplifying factor, , was concluded to be negligible, the amplification being suggestive of a diffusion process or ion channel opening. The temperature dependence of the efficacy ratio e / (1-e) on the thermodynamic scale yielding the enthalpic difference between the agonist and antagonist complexes directly, gave a value of -6.4 ± 1.1

Figure 1 .

 1 Figure 1. Enthalpies of binding of agonist and antagonist components of the binding of prenalterol on the guinea-pig cardiac 1 -adrenergic receptor. Plot of agonist and antagonist conformer binding constants on the log 10 scale plotted against 1/T where T is the absolute temperature. The binding constants shown here are determined from the agonist response (EC50 values). The enthalpy of the overall binding constant without separation into components (8.5 ± 1.3 kcal/mol) is also shown. The enthalpic difference between the bound agonist and antagonist complexes fitted as a function of the efficacy, e alone gave a value of 6.4 ± 1.1 kcal/mol.

Figure 2 .

 2 Figure 2. Comparison of the known agonist side-chain conformer of isoprenaline (a) bound to the cardiac 1 -adrenoceptor [17] compared with the expected bound conformation of the prenalterol (b) based on the almost identical enthalpies of binding .Less relevany hydrogen atoms are only indicated directionally.

Figure 3 .

 3 Figure 3. Comparison of intermolecular distances in molecular overlap between the predicted agonist conformers of the benzdioxepine analogue and isoprenaline [18].

Figure 4 (

 4 Figure4(a) CHARMm minimised energy structures[START_REF] Brooks | CHARMM: a programme for macromolecular energy minimisation and dynamics calculations[END_REF] (using a hydrophobic dielectric of 4.0 and hydrogen bond constraints with the protein backbone fixed) for the agonist conformer of the ligand, isoprenaline (1-R [3,4 di-hydroxyphenyl] 2-isopropylamino ethan-1-ol), binding in two possible positions to the aspartate residues on α-helices III and II respectively[START_REF] Allen | Monocation-driven proton transfer relays within G protein-coupled receptors of the rhodopsin class and the GTP synthase mechanism[END_REF]. The G protein and cytoplasm lie to the base of the Figures. (a) In the upper position, the basic ligand (brown) binds to Asp 138 (III). The agonist conformer is dictated by the β-hydroxyl group's interaction with Asn 369 (VII) and is closely maintained on minimisation. This residue, together with Asn 373 moves to a more vertical orientation relative to the membrane. The potential ligand-binding residues are shown in green. (b) Comparison of the CHARMm energy-minimised agonist conformer when bound at Asp 138 (pink) and, in its lower position, when interacting with Asp 104 (purple) on helix II. The agonist conformer in the latter position is again closely maintained on energy minimisation with the β-hydroxyl group now interacting with Asn 373. The dihedral controlling the lower aspartate 104 interaction is close to -

  Fig 2

Table 2

 2

Table 3 .

 3 Comparison of Enthalpic Changes associated with Agonist and Antagonist Conformer Binding of the 4-Hydroxyphenoxypropanolamine Partial Agonist, Prenalterol, to the Guinea-Pig Cardiac 1 -Adrenergic Receptor.

	Determinations utilising Agonist (EC50 values) and Antagonist (Ferguson-
	Robertson) Responses from 40.0 C to 25.0 C. [8].
		EC50 (kcal/mol)	Ferguson-Robertson (kcal/mol)
	H observed	-8.6 ± 1.9	-8.0 ± 0.2
	H rsx	-5.0 ± 0.9	-4.2 ± 0.6
	H ijx	-11.6 ± 2.7	-10.8 ± 1.5

Table 4 .

 4 Incremental Thermodynamics of Partitioning for the -CH 2 -Group[START_REF] Buckner | Studies on -adrenoceptors mediating changes in mechanical events and adenosine 3 / ,5 / -monophosphate levels, rat atria[END_REF].

	Partitioning phases	310 K	kcal/mol
		G	H	-T S
	1. Cyclohexane/gas	-0.76	-1.12	+0.36
	2. H 2 O/gas	+0.18	-0.67	+0.85
	Cyclohexane/H 2 O	-0.94	-0.45	-0.49

Efficacy/Proton transfer_II

Acknowledgements

The authors would like thank Dr J. D. Fitzgerald for his support and scientific encouragement and acknowledge the support given to the work by ICI under the SERC CASE award No. BC/117 (1985).

The authors would like thank Dr J. D. Fitzgerald for his support and scientific encouragement and acknowledge the support given to the work by ICI under the SERC CASE award No. BC/117 (1985).

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