Structural basis for selectivity and diversity in angiotensin II receptors.

The angiotensin II receptors AT1R and AT2R serve as key components of the renin-angiotensin-aldosterone system. AT1R has a central role in the regulation of blood pressure, but the function of AT2R is unclear and it has a variety of reported effects. To identify the mechanisms that underlie the differences in function and ligand selectivity between these receptors, here we report crystal structures of human AT2R bound to an AT2R-selective ligand and to an AT1R/AT2R dual ligand, capturing the receptor in an active-like conformation. Unexpectedly, helix VIII was found in a non-canonical position, stabilizing the active-like state, but at the same time preventing the recruitment of G proteins or ß-arrestins, in agreement with the lack of signalling responses in standard cellular assays. Structure-activity relationship, docking and mutagenesis studies revealed the crucial interactions for ligand binding and selectivity. Our results thus provide insights into the structural basis of the distinct functions of the angiotensin receptors, and may guide the design of new selective ligands.

Novel oxazolidinone calcitonin gene-related peptide (CGRP) receptor antagonists for the acute treatment of migraine.

In our efforts to develop CGRP receptor antagonists as backups to MK-3207, 2, we employed a scaffold hopping approach to identify a series of novel oxazolidinone-based compounds. The development of a structurally diverse, potent (20, cAMP+HS IC50=0.67 nM), and selective compound (hERG IC50=19 µM) with favorable rodent pharmacokinetics (F=100%, t1/2=7h) is described. Key to this development was identification of a 3-substituted spirotetrahydropyran ring that afforded a substantial gain in potency (10 to 35-fold).

(E)-Alkenes as replacements of amide bonds: development of novel and potent acyclic CGRP receptor antagonists.

A new class of CGRP receptor antagonists was identified by replacing the central amide of a previously identified anilide lead structure with ethylene, ethane, or ethyne linkers. (E)-Alkenes as well as alkynes were found to preserve the proper bioactive conformation of the amides, necessary for efficient receptor binding. Further exploration resulted in several potent compounds against CGRP-R with low susceptibility to P-gp mediated efflux.

[(11)C]MK-4232: The First Positron Emission Tomography Tracer for the Calcitonin Gene-Related Peptide Receptor.

Rational modification of the potent calcitonin gene-related peptide (CGRP) receptor antagonist MK-3207 led to a series of analogues with enhanced CNS penetrance and a convenient chemical handle for introduction of a radiolabel. A number of (11)C-tracers were synthesized and evaluated in vivo, leading to the identification of [(11)C]8 ([(11)C]MK-4232), the first positron emission tomography tracer for the CGRP receptor.

MK-8825: a potent and selective CGRP receptor antagonist with good oral activity in rats.

Rational modification of the clinically tested CGRP receptor antagonist MK-3207 (3) afforded an analogue with increased unbound fraction in rat plasma and enhanced aqueous solubility, 2-[(8R)-8-(3,5-difluorophenyl)-8-methyl-10-oxo-6,9-diazaspiro[4.5]dec-9-yl]-N-[(6S)-2'-oxo-1',2',5,7-tetrahydrospiro[cyclopenta[b]pyridine-6,3'-pyrrolo[2,3-b]pyridin]-3-yl]acetamide (MK-8825) (6). Compound 6 maintained similar affinity to 3 at the human and rat CGRP receptors but possessed significantly improved in vivo potency in a rat pharmacodynamic model. The overall profile of 6 indicates it should find utility as a rat tool to investigate effects of CGRP receptor blockade in vivo.

Targeting a family B GPCR/RAMP receptor complex: CGRP receptor antagonists and migraine.

The clinical effectiveness of antagonizing the calcitonin gene-related peptide (CGRP) receptor for relief of migraine pain has been clearly demonstrated, but the road to the development of these small molecule antagonists has been daunting. The key hurdle that needed to be overcome was the CGRP receptor itself. The vast majority of the current antagonists recognize similar epitopes on the calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1). RAMP1 is a relatively small, single, transmembrane-spanning protein and along with the G-protein-coupled receptor CLR comprise a functional CGRP receptor. The tri-helical extracellular domain of RAMP1 plays a key role in the high affinity binding of CGRP receptor antagonists and drives their species-selective pharmacology. Over the years, a significant amount of mutagenesis data has been generated to identify specific amino acids or regions within CLR and RAMP1 that are critical to antagonist binding and has directed attention to the CLR/RAMP1 extracellular domain (ECD) complex. Recently, the crystal structure of the CGRP receptor ECD has been elucidated and not only reinforces the early mutagenesis data, but provides critical insight into the molecular mechanism of CGRP receptor antagonism. This review will highlight the drug design hurdles that must be overcome to meet the desired potency, selectivity and pharmacokinetic profile while retaining drug-like properties. Although the development of these antagonists has proved challenging, blocking the CGRP receptor may one day represent a new way to manage migraine and offer hope to migraine sufferers.

Novel CGRP receptor antagonists from central amide replacements causing a reversal of preferred chirality.

A previously utilized quinoline-for-N-phenylamide replacement strategy was employed against a central amide in a novel class of CGRP receptor antagonists. A unique and unexpected substitution pattern was ultimately required to maintain reasonable affinity for the CGRP receptor, while at the same time predicting acceptable heterocycle positioning for related analogs. Subsequently, specific quinoline and naphthyridine compounds were prepared which supported these structural predictions by displaying CGRP binding affinities in the 0.037-0.15 nM range.

Identification of potent, highly constrained CGRP receptor antagonists.

A novel series of potent CGRP receptor antagonists containing a central quinoline ring constraint was identified. The combination of the quinoline constraint with a tricyclic benzimidazolinone left hand fragment produced an analog with picomolar potency (14, CGRP K(i)=23 pM). Further optimization of the tricycle produced a CGRP receptor antagonist that exhibited subnanomolar potency (19, CGRP K(i)=0.52 nM) and displayed a good pharmacokinetic profile in three preclinical species.

Pharmacological properties of MK-3207, a potent and orally active calcitonin gene-related peptide receptor antagonist.

Calcitonin gene-related peptide (CGRP) has long been hypothesized to play a key role in migraine pathophysiology, and the advent of small-molecule antagonists has clearly demonstrated a clinical link between blocking the CGRP receptor and migraine efficacy. 2-[(8R)-8-(3,5-Difluorophenyl)-10-oxo-6,9-diazaspiro[4.5]dec-9-yl]-N-[(2R)-2'-oxo-1,1',2',3-tetrahydrospiro[indene-2,3'-pyrrolo[2,3-b]pyridin]-5-yl]acetamide (MK-3207) represents the third CGRP receptor antagonist to display clinical efficacy in migraine trials. Here, we report the pharmacological characterization of MK-3207, a potent and orally bioavailable CGRP receptor antagonist. In vitro, MK-3207 is a potent antagonist of the human and rhesus monkey CGRP receptors (K(i) = 0.024 nM). In common with other CGRP receptor antagonists, MK-3207 displays lower affinity for CGRP receptors from other species, including canine and rodent. As a consequence of species selectivity, the in vivo potency was assessed in a rhesus monkey pharmacodynamic assay measuring capsaicin-induced changes in forearm dermal blood flow via laser Doppler imaging. MK-3207 produced a concentration-dependent inhibition of dermal vasodilation, with plasma concentrations of 0.8 and 7 nM required to block 50 and 90% of the blood flow increase, respectively. The tritiated analog [3H]MK-3207 was used to study the binding characteristics on the human CGRP receptor. [3H]MK-3207 displayed reversible and saturable binding (K(D) = 0.06 nM), and the off-rate was determined to be 0.012 min(-1), with a t(1/2) value of 59 min. In vitro autoradiography studies on rhesus monkey brain slices identified the highest level of binding in the cerebellum, brainstem, and meninges. Finally, as an index of central nervous system penetrability, the in vivo cerebrospinal fluid/plasma ratio was determined to be 2 to 3% in cisterna magna-ported rhesus monkeys.

Discovery of MK-3207: A Highly Potent, Orally Bioavailable CGRP Receptor Antagonist.

Incorporation of polar functionality into a series of highly potent calcitonin gene-related peptide (CGRP) receptor antagonists was explored in an effort to improve pharmacokinetics. This strategy identified piperazinone analogues that possessed improved solubility at acidic pH and increased oral bioavailability in monkeys. Further optimization led to the discovery of the clinical candidate 2-[(8R)-8-(3,5-difluorophenyl)-10-oxo-6,9-diazaspiro[4.5]dec-9-yl]-N-[(2R)-2'-oxo-1,1',2',3-tetrahydrospiro[indene-2,3'-pyrrolo[2,3-b]pyridin]-5-yl]acetamide (MK-3207) (4), the most potent orally active CGRP receptor antagonist described to date.

Novel CGRP receptor antagonists through a design strategy of target simplification with addition of molecular flexibility.

A novel class of CGRP receptor antagonists was rationally designed by modifying a highly potent, but structurally complex, CGRP receptor antagonist. Initial modifications focused on simplified structures, with increased flexibility. Subsequent to the preparation of a less-potent but more flexible lead, classic medicinal chemistry methods were applied to restore high affinity (compound 22, CGRP Ki=0.035 nM) while maintaining structural diversity relative to the lead. Good selectivity against the closely related adrenomedullin-2 receptor was also achieved.

The identification of potent, orally bioavailable tricyclic CGRP receptor antagonists.

A series of tricyclic CGRP receptor antagonists was optimized in order to improve oral bioavailability. Attenuation of polar surface area and incorporation of a weakly basic indoline nitrogen led to compound 5, a potent antagonist with good oral bioavailability in three species.

The discovery of highly potent CGRP receptor antagonists.

Rational modification of a previously identified spirohydantoin lead structure has identified a series of potent spiroazaoxindole CGRP receptor antagonists. The azaoxindole was found to be a general replacement for the hydantoin that consistently improved in vitro potency. The combination of the indanylspiroazaoxindole and optimized benzimidazolinones led to highly potent antagonists (e.g., 25, CGRP K(i)=40pM). The closely related compound 27 demonstrated good oral bioavailability in dog and rhesus.

Examining the binding properties of MK-0974: a CGRP receptor antagonist for the acute treatment of migraine.

Calcitonin gene-related peptide (CGRP) is a neuropeptide that plays a key role in the pathophysiology of migraine headache. MK-0974 (telcagepant) is a potent and selective antagonist of the human and rhesus CGRP receptors and is currently in Phase III clinical studies for the acute treatment of migraine. The pharmacology of MK-0974 has been studied extensively, but there has not been a thorough characterization of its binding properties. Here, we characterize the binding of a tritiated analog of MK-0974 on human neuroblastoma (SK-N-MC) membranes and rhesus cerebellum. [(3)H]MK-0974 displayed reversible and saturable binding to both SK-N-MC membranes and rhesus cerebellum with a K(D) of 1.9 nM and 1.3 nM, respectively. Agonists and antagonists of the CGRP receptor displaced [(3)H]MK-0974 in a concentration-dependent manner in competition binding experiments. Both CGRP and adrenomedullin demonstrated biphasic competition while MK-0974 and the peptide antagonist CGRP(8-37) displaced [(3)H]MK-0974 in a monophasic fashion. In competitive binding studies with [(3)H]MK-0974 and CGRP, the fraction of high-affinity binding was reduced significantly by incubating the membranes with GTPgammaS. In kinetic binding experiments, the off-rate of [(3)H]MK-0974 was determined to be 0.51 min(-1) with a half-life of 1.3 min. In conclusion, the radioligand [(3)H]MK-0974 has proven to be a useful tool for studying the binding characteristics of MK-0974 and has broadened our understanding of this promising molecule.

HC-030031, a TRPA1 selective antagonist, attenuates inflammatory- and neuropathy-induced mechanical hypersensitivity.

BACKGROUND: Safe and effective treatment for chronic inflammatory and neuropathic pain remains a key unmet medical need for many patients. The recent discovery and description of the transient receptor potential family of receptors including TRPV1 and TRPA1 has provided a number of potential new therapeutic targets for treating chronic pain. Recent reports have suggested that TRPA1 may play an important role in acute formalin and CFA induced pain. The current study was designed to further explore the therapeutic potential of pharmacological TRPA1 antagonism to treat inflammatory and neuropathic pain. RESULTS: The in vitro potencies of HC-030031 versus cinnamaldehyde or allyl isothiocyanate (AITC or Mustard oil)-induced TRPA1 activation were 4.9 +/- 0.1 and 7.5 +/- 0.2 microM respectively (IC50). These findings were similar to the previously reported IC50 of 6.2 microM against AITC activation of TRPA1 1. In the rat, oral administration of HC-030031 reduced AITC-induced nocifensive behaviors at a dose of 100 mg/kg. Moreover, oral HC-030031 (100 mg/kg) significantly reversed mechanical hypersensitivity in the more chronic models of Complete Freunds Adjuvant (CFA)-induced inflammatory pain and the spinal nerve ligation model of neuropathic pain. CONCLUSION: Using oral administration of the selective TRPA1 antagonist HC-030031, our results demonstrated that TRPA1 plays an important role in the mechanisms responsible for mechanical hypersensitivity observed in inflammatory and neuropathic pain models. These findings suggested that TRPA1 antagonism may be a suitable new approach for the development of a potent and selective therapeutic agent to treat both inflammatory and neuropathic pain.

Pharmacological characterization of MK-0974 [N-[(3R,6S)-6-(2,3-difluorophenyl)-2-oxo-1-(2,2,2-trifluoroethyl)azepan-3-yl]-4-(2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-1-yl)piperidine-1-carboxamide], a potent and orally active calcitonin gene-related peptide receptor antagonist for the treatment of migraine.

Calcitonin gene-related peptide (CGRP) is a potent neuropeptide that plays a key role in the pathophysiology of migraine headache. CGRP levels in the cranial circulation are increased during a migraine attack, and CGRP itself has been shown to trigger migraine-like headache. The correlation between CGRP release and migraine headache points to the potential utility of CGRP receptor antagonists as novel therapeutics in the treatment of migraine. Indeed, clinical proof-of-concept in the acute treatment of migraine was demonstrated with an intravenous formulation of the CGRP receptor antagonist BIBN4096BS (olcegepant). Here we report on the pharmacological characterization of the first orally bioavailable CGRP receptor antagonist in clinical development, MK-0974 [N-[(3R,6S)-6-(2,3-difluorophenyl)-2-oxo-1-(2,2,2-trifluoroethyl)azepan-3-yl]-4-(2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-1-yl)piperidine-1-carboxamide]. In vitro, MK-0974 is a potent antagonist of the human (K(i) = 0.77 nM) and rhesus (K(i) = 1.2 nM) CGRP receptors but displays >1500-fold lower affinity for the canine and rat receptors as determined via (125)I-human CGRP competition binding assays. A rhesus pharmacodynamic assay measuring capsaicin-induced changes in forearm dermal blood flow via laser Doppler imaging was utilized to determine the in vivo activity of CGRP receptor antagonism. MK-0974 produced a concentration-dependent inhibition of dermal vasodilation, generated by capsaicin-induced release of endogenous CGRP, with plasma concentrations of 127 and 994 nM required to block 50 and 90% of the blood flow increase, respectively. In conclusion, MK-0974 is a highly potent, selective, and orally bioavailable CGRP receptor antagonist, which may be valuable in the acute treatment of migraine.

Identification of novel, orally bioavailable spirohydantoin CGRP receptor antagonists.

A rapid analogue approach to identification of spirohydantoin-based CGRP antagonists provided novel, low molecular weight leads. Modification of these leads afforded a series of nanomolar benzimidazolinone-based CGRP receptor antagonists. The oral bioavailability of these antagonists was inversely correlated with polar surface area, suggesting that membrane permeability was a key limitation to absorption. Optimization provided compound 12, a potent CGRP receptor antagonist (K(i)=21nM) with good oral bioavailability in three species.