Christoph Binkert

Pharmacology of Macitentan, an Orally Active Tissue-Targeting Dual Endothelin Receptor Antagonist

Macitentan, also called Actelion-1 or ACT-064992 [N-[5-(4-bromophenyl)-6-(2-(5-bromopyrimidin-2-yloxy)ethoxy)-pyrimidin-4-yl]-N′-propylaminosulfonamide], is a new dual ETA/ETB endothelin (ET) receptor antagonist designed for tissue targeting. Selection of macitentan was based on inhibitory potency on both ET receptors and optimization of physicochemical properties to achieve high affinity for lipophilic milieu. In vivo, macitentan is metabolized into a major and pharmacologically active metabolite, ACT-132577. Macitentan and its metabolite antagonized the specific binding of ET-1 on membranes of cells overexpressing ETA and ETB receptors and blunted ET-1-induced calcium mobilization in various natural cell lines, with inhibitory constants within the nanomolar range. In functional assays, macitentan and ACT-132577 inhibited ET-1-induced contractions in isolated endothelium-denuded rat aorta (ETA receptors) and sarafotoxin S6c-induced contractions in isolated rat trachea (ETB receptors). In rats with pulmonary hypertension, macitentan prevented both the increase of pulmonary pressure and the right ventricle hypertrophy, and it markedly improved survival. In diabetic rats, chronic administration of macitentan decreased blood pressure and proteinuria and prevented end-organ damage (renal vascular hypertrophy and structural injury). In conclusion, macitentan, by its tissue-targeting properties and dual antagonism of ET receptors, protects against end-organ damage in diabetes and improves survival in pulmonary hypertensive rats. This profile makes macitentan a new agent to treat cardiovascular disorders associated with chronic tissue ET system activation.

The Discovery of N-[5-(4-Bromophenyl)-6-[2-[(5-bromo-2-pyrimidinyl)oxy]ethoxy]-4-pyrimidinyl]-N′-propylsulfamide (Macitentan), an Orally Active, Potent Dual Endothelin Receptor Antagonist

Starting from the structure of bosentan (1), we embarked on a medicinal chemistry program aiming at the identification of novel potent dual endothelin receptor antagonists with high oral efficacy. This led to the discovery of a novel series of alkyl sulfamide substituted pyrimidines. Among these, compound 17 (macitentan, ACT-064992) emerged as particularly interesting as it is a potent inhibitor of ET(A) with significant affinity for the ET(B) receptor and shows excellent pharmacokinetic properties and high in vivo efficacy in hypertensive Dahl salt-sensitive rats. Compound 17 successfully completed a long-term phase III clinical trial for pulmonary arterial hypertension.

2-Imino-thiazolidin-4-one Derivatives as Potent, Orally Active S1P1 Receptor Agonists

Sphingosine-1-phosphate (S1P) is a widespread lysophospholipid which displays a wealth of biological effects. Extracellular S1P conveys its activity through five specific G-protein coupled receptors numbered S1P(1) through S1P(5). Agonists of the S1P(1) receptor block the egress of T-lymphocytes from thymus and lymphoid organs and hold promise for the oral treatment of autoimmune disorders. Here, we report on the discovery and detailed structure-activity relationships of a novel class of S1P(1) receptor agonists based on the 2-imino-thiazolidin-4-one scaffold. Compound 8bo (ACT-128800) emerged from this series and is a potent, selective, and orally active S1P(1) receptor agonist selected for clinical development. In the rat, maximal reduction of circulating lymphocytes was reached at a dose of 3 mg/kg. The duration of lymphocyte sequestration was dose dependent. At a dose of 100 mg/kg, the effect on lymphocyte counts was fully reversible within less than 36 h. Pharmacokinetic investigation of 8bo in beagle dogs suggests that the compound is suitable for once daily dosing in humans.

Bosentan, Sildenafil, and Their Combination in the Monocrotaline Model of Pulmonary Hypertension in Rats:

The dual endothelin receptor antagonist, bosentan, and the phosphodiesterase inhibitor, sildenafil, are efficacious in experimental and clinical pulmonary hypertension (PHT). The effects of bosentan, sildenafil, and their combination were evaluated in rats with monocrotaline (MCT)-induced PHT. A first group consisted of control rats with no MCT injection. Four other groups of rats received MCT subcutaneously and were assigned to receive no treatment, 300 mg/kg/day bosentan as food admix, 100 mg/kg/day sildenafil in drinking water, or their combination for 4 weeks. The doses of bosentan and sildenafil were the maximally effective doses based on a dose-range–finding study. Mortality was 0%, 53%, 11%, 11%, and 0%, respectively, in the five different groups. Bosentan and sildenafil significantly attenuated the increase in mean pulmonary arterial pressure, and the combination had an additional effect. Similarly, bosentan, sildenafil, and, to a greater extent, their combination significantly reduced right ventricular (RV) hypertrophy. Bosentan, but not sildenafil, decreased norepinephrine and BNP plasma concentrations, reduced kidney weight, and normalized systemic hemodynamics. In conclusion, bosentan and sildenafil are efficacious in rats with chronic PHT, and their combination shows an additional effect for decreasing pulmonary arterial pressure, reducing plasma catecholamines, maintaining body weight, and reducing mortality.

X-ray Structure of Plasmepsin II Complexed with a Potent Achiral Inhibitor

The malaria parasite Plasmodium falciparum degrades host cell hemoglobin inside an acidic food vacuole during the blood stage of the infectious cycle. A number of aspartic proteinases called plasmepsins (PMs) have been identified to play important roles in this degradation process and therefore generated significant interest as new antimalarial targets. Several x-ray structures of PMII have been described previously, but thus far, structure-guided drug design has been hampered by the fact that only inhibitors comprising a statine moiety or derivatives thereof have been published. Our drug discovery efforts to find innovative, cheap, and easily synthesized inhibitors against aspartic proteinases yielded some highly potent non-peptidic achiral inhibitors. A highly resolved (1.6 Å) x-ray structure of PMII is presented, featuring a potent achiral inhibitor in an unprecedented orientation, contacting the catalytic aspartates indirectly via the “catalytic” water. Major side chain rearrangements in the active site occur, which open up a new pocket and allow a new binding mode of the inhibitor. Moreover, a second inhibitor molecule could be located unambiguously in the active site of PMII. These newly obtained structural insights will further guide our attempts to improve compound properties eventually leading to the identification of molecules suitable as antimalarial drugs.

Design and Preparation of Potent, Nonpeptidic, Bioavailable Renin Inhibitors

Starting from known piperidine renin inhibitors, a new series of 3,9-diazabicyclo[3.3.1]nonene derivatives was rationally designed and prepared. Optimization of the positions 3, 6, and 7 of the diazabicyclonene template led to potent renin inhibitors. The substituents attached at the positions 6 and 7 were essential for the binding affinity of these compounds for renin. The introduction of a substituent attached at the position 3 did not modify the binding affinity but allowed the modulation of the ADME properties. Our efforts led to the discovery of compound (+)-26g that inhibits renin with an IC(50) of 0.20 nM in buffer and 19 nM in plasma. The pharmacokinetics properties of this and other similar compounds are discussed. Compound (+)-26g is well absorbed in rats and efficacious at 10 mg/kg in vivo.

Development of a homogeneous immunoassay for the detection of angiotensin I in plasma using AlphaLISA acceptor beads technology

Plasma renin activity (PRA) is a well-established biomarker for assessing the efficacy of various antihypertensive agents such as direct renin inhibitors, angiotensin receptor blockers, and angiotensin-converting enzyme inhibitors (ACEIs). PRA measurements are obtained through the detection and quantification of angiotensin I (Ang I) produced by the action of renin on its natural substrate angiotensinogen. The most accepted and reproducible method for PRA measurement uses an antibody capture Ang I methodology that employs specific antibodies that recognize and protect Ang I against angiotensinase activities contained in plasma. The amount of Ang I is then quantified by either radioimmunoassay (RIA) or enzyme immunoassay (EIA). In the current report, we describe the optimization of a novel homogeneous immunoassay based on the AlphaScreen technology for the detection and quantification of antibody-captured Ang I using AlphaLISA acceptor beads in buffer and in the plasma of various species (human, rat, and mouse). Ex vivo measurements of renin activity were performed using 10 microl or less of a reaction mixture, and concentrations as low as 1 nM Ang I were quantified. Titration curves obtained for the quantification of Ang I in buffer and plasma gave similar EC(50) values of 5.6 and 14.4 nM, respectively. Both matrices generated an equivalent dynamic range that varies from approximately 1 to 50 nM. Renin inhibitors have been successfully titrated and IC(50) values obtained correlated well with those obtained using EIA methodology (r(2)=0.80). This assay is sensitive, robust, fast, and less tedious than measurements performed using nonhomogeneous EIA. The AlphaLISA methodology is homogeneous, does not require wash steps prior to the addition of reagents, and does not generate radioactive waste.

The Expression of Urotensin II Receptor (U2R) is Up‐regulated by Interferon‐γ

Abstract Urotensin‐II (U‐II) was identified as the natural ligand of the G protein‐coupled receptor GPR14, which has been correspondingly renamed Urotensin‐II receptor (U2R). The tissue distribution of U2R and the pharmacological effects of U‐II suggest a novel neurohormonal system with potent cardiovascular effects. We here report the human rhabdomyosarcoma cell line TE‐671 as the first natural and endogenous source of functional U2R in an immortalized cell line. In TE‐671 cells, U‐II stimulated extracellular signal regulated kinase phosphorylation and increased c‐fos mRNA expression. Furthermore, we demonstrate that the expression of U2R mRNA and functional U‐II high affinity binding sites are serum‐responsive and that they are specifically up‐regulated by interferon γ (IFNγ). We propose that IFNγ contributes to the previously observed increase of U2R density in the heart tissue of congestive heart failure (CHF) patients and we suggest that U2R up‐regulation, as a consequence of an inflammatory response, could lead to a clinical worsening of this disease.

Replacement of Isobutyl by Trifluoromethyl in Pepstatin A Selectively Affects Inhibition of Aspartic Proteinases

Two bis‐trifluoromethyl pepstatin A analogues, carboxylic acid 1 and its methyl ester 2, have been synthesised in order to probe the properties and size of the trifluoromethyl (Tfm) group and compare it to the “bigger” isobutyl that is present in pepstatin A. The results demonstrate that Tfm can effectively replace the isobutyl chain as far as inhibitory activity against plasmepsin II (PM II), an aspartic proteinase from Plasmodium falciparum, is concerned. On the other hand, replacement of isobutyl by Tfm selectively affected activity against other aspartic proteinases tested. Two lines of evidence led to these conclusions. Firstly, compounds 1 and 2 retained single‐digit nanomolar inhibitory activity against PM II, but were markedly less active against PM IV, cathepsin D and cathepsin E. Secondly, the X‐ray crystal structures of the three complexes of PM II with 1, 2 and pepstatin A were obtained at 2.8, 2.4 and 1.7 Å resolution, respectively. High overall similarity among the three complexes indicated that the central Tfm was well accommodated in the lipophilic S1 pocket of PM II, where it was involved in tight hydrophobic contacts. The interaction of PM II with Phe111 appeared to be crucial. Comparison of the crystal structures presented here, with X‐ray structures or structural models of PM IV and cathepsin D, allowed an interpretation of the inhibition profiles of pepstatin A and its Tfm variants against these three enzymes. Interactions of the P1 side chain with amino acids that point into the S1 pocket appear to be critical for inhibitory activity. In summary, Tfm can be used to replace an isobutyl group and can affect the selectivity profile of a compound. These findings have implications for the design of novel bioactive molecules and synthetic mimics of natural compounds.

De Novo design, synthesis, and in vitro evaluation of a new class of nonpeptidic inhibitors of the malarial enzyme plasmepsin II.

Malaria, a life-threatening disease caused by parasites of the genus Plasmodium, affects 500 million people annually, of which more than one million die. The emergence of multi-drugresistant strains of Plasmodium falciparum, the parasite that causes the deadliest form of malaria, exacerbates the situation and necessitates new medicines with novel modes of action. Plasmepsin II (PII ; EC3.4.23.39), a parasitic aspartic protease involved in the hemoglobin degradation process that takes place in an acidic vacuole, has been identified as a potential target for antimalarial therapy. Several groups reported PII inhibitors that mimic the natural substrate and display up to single-digit nanomolar activity. Inhibition of PII is expected to block the life cycle of the parasite. Here we report the synthesis and in vitro evaluation of a new class of nonpeptidic PII inhibitors developed with the help of structure-based de novo design that show up to single-digit micromolar inhibitory activities. A major conformational change around the active site of the human aspartic protease renin (EC3.4.23.15) upon complexation of 3,4-disubstituted piperidines has been observed, which unveils unexpected flexibility of the enzyme. The flap that lies over the catalytic dyad, and a tryptophan side chain of the core domain, move and thereby unlock a new hydrophobic pocket (flap pocket). The high sequence homology between renin and PII prompted us to hypothesize that an induced-fit adaptation such as that of the active site of renin might also be operative [3] H. Zhu, M. Bilgin, R. Bangham, D. Hall, A. Casamayor, P. Bertone, N. Lan, R. Jansen, S. Bidlingmaier, T. Houfek, T. Mitchell, P. Miller, R. A. Dean, M. Gerstein, M. Snyder, Science 2001, 293, 2101. [4] A.-C. Gavin, M. Bˆsche, R. Krause, P. Grandi, M. Marzioch, A. Bauer, J. Schultz, J. M. Rick, A.-M. Michon, C.-M. Cruciat, M. Remor, C. Hˆfert, M. Schelder, M. Brajenovic, H. Ruffner, A. Merino, K. Klein, M. Hudak, D. Dickson, T. Rudi, V. Gnau, A. Bauch, S. Bastuck, B. Huhse, C. Leutwein, M.-A. Heurtier, R. R. Copley, A. Edelmann, E. Querfurth, V. Rybin, G. Drewes, M. Raida, T. Bouwmeester, P. Bork, B. Seraphin, B. Kuster, G. Neubauer, G. Superti-Furga, Nature 2002, 415, 141. [5] M. Mann, R. C. Hendrickson, A. Pandey, Annu. Rev. Biochem. 2001, 70. [6] A. Dongre, J. K. Eng, J. R. Yates, Trends Biotechnol. 1997, 15, 418. [7] M. R. Wilkins, K. L. Williams, R. D. Appel, D. F. Hiochstrasser, Proteome Research: New Frontiers in Functional Genomics, Springer, Berlin, 1997. [8] Y. Ho, A. Gruhler, A. Heilbut, G. D. Bader, L. Moore, S.-L. Adams, A. Millar, P. Taylor, K. Bennett, K. Boutilier, L. Yang, C. Wolting, I. Donaldson, S. Schandorff, J. Shewnarane, M. Vo, J. Taggart, M. Goudreault, B. Muskat, C. Alfarano, D. Dewar, Z. Lin, K. Michalickova, A. R. Willems, H. Sassi, P. A. Nielsen, K. J. Rasmussen, J. R. Andersen, L. E. Johansen, L. H. Hansen, H. Jespersen, A. Podtelejnikov, E. Nielsen, J. Crawford, V. Poulsen, B. D. S ̆rensen, J. Matthiesen, R. C. Hendrickson, F. Gleeson, T. Pawson, M. F. Moran, D. Durocher, M. Mann, C. W. V. Hogue, D. Figeys, M. Tyers, Nature 2002, 415, 180. [9] L. Wang, A. Brock, B. Herberich, P. G. Schultz, Science 2001, 292, 498. [10] L. Wang, A. Brock, P. G. Schultz, J. Am. Chem. Soc. 2002, 124, 1836. [11] J. W. Chin, S. W. Santoro, A. B. Martin, D. S. King, L. Wang, P. G. Schultz, J. Am. Chem. Soc. 2002, 124, 9026. [12] J. C. Kauer, S. Erickson-Viitanen, H. R. Wolfe, W. F. DeGrado, J. Biol. Chem. 1986, 261, 10695. [13] J. W. Chin, A. B. Martin, D. S. King, L. Wang, P. G. Schultz, Proc. Natl. Acad. Sci. USA 2002, 99, 11020. [14] G. Dorman, G. D. Prestwich, Biochemistry 1994, 33, 5661. [15] D. A. Fancy, K. Melcher, S. A. Johnston, T. Kodadek, Chem. Biol. 1996, 3, 551. [16] Y. Maru, D. E. Afar, O. N. Witte, M. Shibuya, J. Biol. Chem. 1996, 271, 15353. [17] M. A. McTigue, D. R. Williams, J. A. Tainer, J. Mol. Biol. 1995, 246, 21. [18] W. A. Houry, D. Frishman, C. Eckerskorn, F. Lottspeich, F. U. Hartl, Nature 1999, 402, 147. [19] The near-UV irradiation per unit area (fluence) at 365 nm used in these studies for a five-minute exposure was approximately 10 ± 30 kJm . Classic studies on the effects of bacterial exposure at this wavelength have shown that lethal effects of such radiation on bacterial cells require fluences one to two orders of magnitude greater than this value. This observation raises the possibility that the phenotypes of live cells may be altered by photocrosslinking between protein surfaces in vivo. [20] B. Alberts, Cell 1998, 92, 291.