J. Med. Chem. 2010, 53, 2695–2708 2695 DOI: 10.1021/jm901294u
Urotensin-II Receptor Modulators as Potential Drugs Bruce E. Maryanoff*,† and William A. Kinney‡ †
Johnson & Johnson Pharmaceutical Research & Development, Welsh and McKean Roads, Spring House, Pennsylvania 19477-0776 and Pennsylvania Biotechnology Center, 3805 Old Easton Road, Doylestown, Pennsylvania 18902
‡
Received August 28, 2009
Introduction Urotensin-II (U-IIa) represents a series of cyclic peptides ranging in size from 8 to 14 amino acids across different animal species, from frogs to fish to humans (Figure 1).1 Amino acid residues 5-10 of human U-II are contained in a disulfidelinked cyclic array, [CFWKYC], which is highly species-conserved, whereas the N-terminal region from other species differs in length and constitution (Figure 1). The first U-II peptide (a 12-mer) was isolated from the urophysis of the goby fish (Gillichthys mirabilis) as a smooth muscle contractile substance.2 Subsequently, various U-II peptides were identified in different species on the basis of their precursor protein sequences.1 For example, the rat U-II sequence was originally indicated to be the 14-mer shown in Figure 1.3 However, the only U-II immunoreactive substance found in the rat brain is a structurally related 8-mer, referred to as urotensin-related peptide (URP), which is proposed as the endogenous U-II peptide substance in rats, and it may even exist in humans.4 The U-II peptides have been found to be among the most potent vasoconstrictors known.1,5,6 There is now a keen appreciation that U-II peptides activate the urotensin-II receptor (UT), which is a seven-transmembrane G-protein-coupled receptor (GPCR) present in a wide assortment of species.7 This receptor, originally classified as orphan receptor GPR14, is expressed in many tissues, including blood vessels, heart, liver, kidney, skeletal muscle, and lung.7,8 As a consequence of the sizable knowledge base in this area, U-II and its receptor have been associated with cardiorenal and metabolic diseases,1 including hypertension,9 heart failure,10 chronic renal failure,10c,d,11 diabetes,10c,12 and atherosclerosis.10e,13 Five years after the first isolation of goby U-II,14 its cardiovascular and renal pressor effects were identified in eels.15 Purified cyclic peptide Ala-Gly-Thr-Ala-Asp-[Cys-Phe-TrpLys-Tyr-Cys]-Val exhibited various pharmacological effects in fish, such as smooth muscle contraction and osmoregulation,2,16 but was not originally considered to be relevant to
Figure 1. Primate U-II along with other U-II peptides (AA = amino acid).
*To whom correspondence should be addressed. Phone: 267-9803512. Fax: 858-784-2798. E-mail:
[email protected], bmaryano@ scripps.edu. a Abbreviations: CHF, congestive heart failure; CHO, Chinese hamster ovary; Cpa, 4-chlorophenylalanine; CNS, central nervous system; Dba, 2,4-diaminobutyric acid; ET, endothelin; FLIPR, fluorometricimaging plate reader; GFR, glomerular filtration rate; GPCR, Gprotein-coupled receptor; Hcy, homocysteine; HLM, human liver microsomes; HTS, high-throughput screening; hUT, human urotensin-II receptor; Nal, naphthylalanine; Pal, 3-pyridinylalanine, Pen, penicillamine; PK, pharmacokinetics; Pyrm, pyrimidin-2-yl; RBF, renal blood flow; SAR, structure-activity relationship; Th, 2-thienyl; TM, transmembrane; U-II, urotensin-II; URP, urotensin-related peptide; UT, urotensin-II receptor.
mammals. Nearly 20 years later, the human gene for the U-II precursor protein was found8a and the U-II receptor was characterized.8b,17 Experiments on the actions of U-II, its levels in different disease states, and its receptor distribution have sometimes provided conflicting information. In most reports U-II has functioned as a potent vasoconstrictor in both animals6b and humans.6a However, its effects can be species and tissue specific,18 such as for a particular vascular bed. As an example of conflicting effects for U-II, whereas anesthetized cynomolgus monkeys exhibited a pressor response, conscious rats exhibited frank vasodilation.19 The complexity of U-II’s behavior has even surfaced in in vitro experiments with rat aortic strips.20 Although elevated plasma levels of U-II are associated with heart21,22 and renal failure in humans,11a,23 suggesting potential therapeutic utility for a UT antagonist, U-II has also been noted to be cardioprotective in end-stage renal disease.22 In fact, high levels of U-II can predict better patient outcomes and survival in acute myocardial infarction24 and chronic kidney disease.25 The causative role of increased levels of U-II and its receptor in atherosclerosis has been less controversial.13b,26 The broad distribution of the receptor in renal, cardiovascular, endocrine, and CNS tissues has contributed to the challenge of confidently interpreting the physiological and pathophysiological roles of U-II.8b,d,10c,11a,27 Nevertheless, one can come to appreciate that there is potential for employing U-II receptor modulators, particularly receptor antagonists, in the treatment of various cardiovascular and renal disorders. To a certain degree, interest in U-II drug discovery has been inspired by the success with the drug target endothelin (ET), another vasoactive cyclic peptide. Endothelin-1 (ET-1), a 21amino-acid cyclic peptide originally isolated from endothelial cell cultures,28 and its congeners ET-2 and ET-3 are also
r 2009 American Chemical Society
Published on Web 12/31/2009
pubs.acs.org/jmc
2696 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Chart 1. Advanced U-II Receptor Antagonist Molecules
among the most potent vasoconstrictors known.29 The endothelins are produced by diverse cell types, including endothelial cells, vascular smooth muscle, cardiomyocytes, and neurons,30 while ET-1 is only expressed in vascular endothelium and accounts for nearly all circulating endothelin. The endothelin-responsive GPCRs, ETA, ETB1, and ETB2, which are widely expressed in different cell types,30 are instrumental in regulating the cellular actions.31 The potent vasoconstrictor actions of endothelin are mediated primarily by ETA receptors on vascular smooth muscle cells,32 and these receptors modulate vascular and cardiac hypertrophy,33 as well as cardiac contractile function.34 ETB receptors mediate vasodilation (endothelial cells), vasoconstriction (vascular smooth muscle cells), and sodium/water reabsorption (renal tubular epithelial cells).35 Three endothelin receptor antagonists, bosentan, sitaxentan, and ambrisentan, are currently on the market and used principally for the treatment of pulmonary arterial hypertension.36 Considering this favorable scenario for endothelin receptor antagonists, a general question that has percolated through the minds of drug discovery scientists is the following: Could U-II and its receptor deliver drug candidates that can emerge as successful drug products? In this review we will endeavor to present the current state of knowledge surrounding U-II, U-II peptide analogues, and U-II receptor modulators (agonists and antagonists), with an emphasis on recent progress toward the discovery of drug candidates. This discourse will encompass the structure and function of U-II peptides and the efforts to identify potent receptor antagonists, especially non-peptide UT antagonists. From a drug discovery standpoint, we will reflect on the properties of UT antagonists in animal models and relate that information to the potential for such agents as future drugs. The most advanced development milestone attained by a U-II receptor antagonist to date is phase 2b clinical trials, which occurred with palosuran (1, ACT-058362) under the auspices of Actelion Pharmaceuticals Ltd. (Chart 1).37 Compounds from GlaxoSmithKline, such as SB-706375 (2a), SB-657510 (2b), and related structures, have been extensively investigated pharmacologically (Chart 1).38 Recently, some important reviews on non-peptide U-II receptor modulators39 have appeared that complement our discussion in this Perspective. From this collection of information, one should be able to gain a reasonably clear picture of the state of the art in the U-II field and the potential of UT antagonists in pharmacotherapy. Urotensin-II Peptide Agonists and Antagonists Structure-Function Studies. Human U-II is an 11-mer cyclic peptide, Glu-Thr-Pro-Asp-[Cys-Phe-Trp-Lys-Tyr-Cys]Val, which contains a cyclic hexapeptide motif that is conserved throughout the different species (Figure 1). The corresponding goby U-II is a 12-mer with the sequence AlaGly-Thr-Ala-Asp-[Cys-Phe-Trp-Lys-Tyr-Cys]-Val. When we
Maryanoff and Kinney Table 1. Agonist Activity for Truncated and Alanine-Scan Peptides peptide
FLIPR EC50 (nM)
AGTAD[CFWKYC]V (goby U-II) GTAD[CFWKYC]V TAD[CFWKYC]V AD[CFWKYC]V AA[CFWKYC]V AD[CAWKYC]V AD[CFAKYC]Va AD[CFWAYC]Va AD[CFWKAC]Va AD[CFWKYC]A D[CFWKYC]V [CFWKYC]V [CFWKYC]
0.17 ( 0.05 0.29 ( 0.18 0.11 ( 0.05 0.16 ( 0.05 0.60 ( 0.10 6.5 ( 1.5 >1000 >1000 67% at 1000 0.40 ( 0.20 0.10 ( 0.04 0.76 ( 0.65 1.6 ( 0.9
a
Inactive as an antagonist at 10 000 nM.
began our studies to discover U-II receptor antagonists in 2000, we thought that it would be a good idea to understand the critical structural features of U-II peptide agonists that were responsible for receptor binding and functional action. That information could be helpful in the rational design of new agonist and antagonist molecules. We planned to ultimately apply a campaign of high-throughput screening (HTS) to the problem of discovering potent UT antagonists. The positive HTS results could then benefit from the structural concepts derived from work with U-II peptide analogues. Consequently, we carried out a structure-function analysis around U-II and assembled a ligand/receptor working model.40 From prior research, it was known that the cyclic portion of U-II is essential for biological activity and that capping of the N- and C-termini is tolerated.41 Our peptide structurefunction work pinpointed the importance of the Trp-LysTyr (WKY) motif,40 which was confirmed by related contemporaneous work from another group.42 Our biological evaluation of synthetic derivatives of goby U-II yielded other structural features that are required for stimulating the U-II receptor. The 6-mer cyclic core turned out to be the minimum size for a truncated U-II peptide with robust agonist potency. By installing unnatural amino acids for key U-II residues, we found that tyrosine could be replaced with L-(2-naphthyl)alanine (2-Nal) with a marked increase in rat UT affinity. Subsequently, we employed the high-affinity agonist ligand Ac-[CFWK(2-Nal)C]-NH2 (3) in bioassays and in an HTS protocol involving a fluorometric-imaging plate reader (FLIPR) (vide infra). The accumulated structure-function information was helpful for constructing a plausible molecular model of the U-II receptor/ligand complex.40 NMR spectroscopy studies with the ligand42 and our receptor homology model docking work40 led to two hypothetical, three-dimensional agonist pharmacophores corresponding to unbound and receptor-bound U-II, respectively. In our structure-function study, we initially evaluated synthetic derivatives of goby U-II in a FLIPR-based calcium mobilization assay involving rat UT expressed in Chinese hamster ovary (CHO) cells to gauge agonist activity.40,43 Residues were removed systematically from the N-terminus of goby U-II, from the 12-mer to the 7-mer, and then Val was deleted from the C-terminus (Table 1). Although the 6-mer exhibited a 10-fold reduction in agonist potency, it still resided in the singledigit nanomolar range. An alanine scan on the 9-mer established which amino acids are the most important for UT stimulation. Thus, W, K, and Y were found to be very important amino acid residues for potent agonist activity.
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7 2697
Perspective
A binding assay based on displacement of rat [125I]U-II from rat UT in CHO cells was also established. Replacement of the two cysteines with serines to give an acyclic 6-mer (viz. Ac-SFWKYS-NH2) demonstrated the importance of the cyclic architecture for UT binding and activation. Further truncation, end-capping, and installation of D-amino acids confirmed the crucial cyclic motif, [CFWKYC], and its inherent stereochemistry (Table 2). Also, the comparison of Ac-[CFWKYC]-NH2 with [CFWKYC] indicated that the N-terminal amino and C-terminal acid groups are unnecessary. Although inversion of amino acid stereochemistry has been known to convert an agonist into an antagonist for certain cyclic peptides,44 that did not occur in this series of derivatives. L-2-Naphthylalanine (2-Nal) was investigated as a replacement for Trp and for Tyr. Although a 20-fold loss in potency occurred in the Trp position, 2-Nal in the Tyr position was helpful for attaining strong agonist potency and high receptor affinity (Table 3). This result, along with our UT homology model,40 suggested the importance of hydrophobic interactions at the Y position. Cyclic 6-mer 3 had 3-fold better functional potency and 10-fold better affinity (cf. entries 1 and 5), and full-length congener 4 had nearly 10-fold greater potency than its parent, goby U-II (cf. entries 7 and 8). In fact, the 20 pM binding affinity of 12-mer 4 was quite impressive in the superagonist realm. goby U-II exhibited a high level of vasoconstrictor response (91%) at 30 nM and (2-Nal)-6-mer 3 was reasonably competitive (43%) (Table 3). In 2002, concurrent with our report,40 there was a flurry of disclosures concerning the structure-activity relationships (SARs) of U-II agonist peptides,42,45-47 which confirmed and further characterized the agonist pharmacophore. Flohr et al. explored analogues of human U-II and concluded that the WKY motif is crucial for receptor recognition and activation.42 Coy et al. found that a free N-terminal amino group was unnecessary and that an amidated C-terminus Table 2. End-Capping, Ring Removal, and D-Amino Acid Scanning of 6-Mer peptidea
FLIPR EC50 (nM)
binding Ki (nM)b
[CFWKYC] Ac-[CFWKYC]-NH2 Ac-SFWKYS-NH2 Ac-FWKY-NH2 Ac-WKY-NH2 Ac-[CfWKYC]-NH2 Ac-[CFwKYC]-NH2 Ac-[CFWkYC]-NH2 Ac-[CFWKyC]-NH2
1.6 ( 0.9 1.6 ( 0.5
