Ezetimibe is a potent inhibitor of cholesterol absorption that has been approved for the treatment of hypercholesterolemia, but its molecular target has been elusive. Using a genetic approach, we ...recently identified Niemann-Pick C1-Like 1 (NPC1L1) as a critical mediator of cholesterol absorption and an essential component of the ezetimibe-sensitive pathway. To determine whether NPC1L1 is the direct molecular target of ezetimibe, we have developed a binding assay and shown that labeled ezetimibe glucuronide binds specifically to a single site in brush border membranes and to human embryonic kidney 293 cells expressing NPC1L1. Moreover, the binding affinities of ezetimibe and several key analogs to recombinant NPC1L1 are virtually identical to those observed for native enterocyte membranes. KDvalues of ezetimibe glucuronide for mouse, rat, rhesus monkey, and human NPC1L1 are 12,000, 540, 40, and 220 nM, respectively. Last, ezetimibe no longer binds to membranes from NPC1L1 knockout mice. These results unequivocally establish NPC1L1 as the direct target of ezetimibe and should facilitate efforts to identify the molecular mechanism of cholesterol transport.
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-diazaspiro4.5dec-9-yl-N-(2R)-2'-oxo-1,1',2',3-tetrahydrospiroindene-2,3'-pyrrolo2,3-bpyridin-5-ylacetamide (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 3HMK-3207 was used to study the binding characteristics on the human CGRP receptor. 3HMK-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.
Voltage-gated sodium channels (Nav1) transmit pain signals from peripheral nociceptive neurons, and blockers of these channels have been shown to ameliorate a number of pain conditions. Because these ...drugs can have adverse effects that limit their efficacy, more potent and selective Nav1 inhibitors are being pursued. Recent human genetic data have provided strong evidence for the involvement of the peripheral nerve sodium channel subtype, Nav1.7, in the signaling of nociceptive information, highlighting the importance of identifying selective Nav1.7 blockers for the treatment of chronic pain. Using a high-throughput functional assay, novel Nav1.7 blockers, namely, the 1-benzazepin-2-one series, have recently been identified. Further characterization of these agents indicates that, in addition to high-affinity inhibition of Nav1.7 channels, selectivity against the Nav1.5 and Nav1.8 subtypes can also be achieved within this structural class. The most potent, nonselective member of this class of Nav1.7 blockers has been radiolabeled with tritium. 3HBNZA binds with high affinity to rat brain synaptosomal membranes (K d = 1.5 nM) and to membranes prepared from HEK293 cells stably transfected with hNav1.5 (K d = 0.97 nM). In addition, and for the first time, high-affinity binding of a radioligand to hNav1.7 channels (K d = 1.6 nM) was achieved with 3HBNZA, providing an additional means for identifying selective Nav1.7 channel inhibitors. Taken together, these data suggest that members of the novel 1-benzazepin-2-one structural class of Nav1 blockers can display selectivity toward the peripheral nerve Nav1.7 channel subtype, and with appropriate pharmacokinetic and drug metabolism properties, these compounds could be developed as analgesic agents.
Although zomepirac (ZP) and tolmetin (TM) induce anaphylactic reactions and form reactive acyl glucuronides, a direct link between the two events remains obscure. We report herein that, in addition ...to acyl glucuronidation, both drugs are subject to oxidative bioactivation. Following incubations of ZP with human liver microsomes fortified with NADPH and glutathione (GSH), a metabolite with an MH+ ion at m/z 597 was detected by LC/MS/MS. On the basis of collision-induced dissociation and NMR evidence, the structure of this metabolite was determined to be 5-4'-chlorobenzoyl-1,4-dimethyl-3-glutathionylpyrrole-2-acetic acid (ZP-SG), suggesting that the pyrrole moiety of ZP had undergone oxidation to an epoxide intermediate, followed by addition of GSH and loss of the elements of H2O to yield the observed conjugate. The oxidative bioactivation of ZP most likely is catalyzed by cytochrome P450 (P450) 3A4, since the formation of ZP-SG was reduced to approximately 10% of control values following pretreatment of human liver microsomes with ketoconazole or with an inhibitory anti-P450 3A4 IgG. A similar GSH adduct, namely 5-4'-methylbenzoyl-1-methyl-3-glutathionylpyrrole-2-acetic acid (TM-SG), was identified when TM was incubated with human liver microsomal preparations. The relevance of these in vitro findings to the in vivo situation was established through the detection of the same thiol adducts in rats treated with ZP and TM, respectively. Taken together, these data suggest that, in addition to the formation of acyl glucuronides, oxidative metabolism of ZP and TM affords reactive species that may haptenize proteins and thereby contribute to the drug-mediated anaphylactic reactions.
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.
3HMK-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
3HMK-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
3HMK-0974 in a monophasic fashion. In competitive binding studies with
3HMK-0974 and CGRP, the fraction of high-affinity binding was reduced significantly by incubating the membranes with GTPγS. In kinetic binding experiments, the off-rate of
3HMK-0974 was determined to be 0.51 min
−
1
with a half-life of 1.3 min. In conclusion, the radioligand
3HMK-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.
Novel benzodiazepine-containing γ-secretase inhibitors for potential use in Alzheimer's disease have been designed that incorporate a substituted hydrocinnamide C-3 side chain. A syn combination of ...α-alkyl or aryl and β-hydroxy or hydroxymethyl substituents was shown to give highly potent compounds. In particular, (2S,3R)-3-(3,4-difluorophenyl)-2-(4-fluorophenyl)-4-hydroxy-N-((3S)-2-oxo-5-phenyl-2,3-dihydro-1H-benzoe1,4diazepin-3-yl)butyramide (34) demonstrated excellent in vitro potency (IC50 = 0.06nM). 34 could also be selectively methylated to give 3H-28, which is of use in radioligand binding assays.
MB243 (a 1,3-disubstituted piperazine) is a new, potent, and selective melanocortin receptor subtype-4 agonist with potential application in the treatment of obesity and/or erectile dysfunction. ...MB243 was observed to covalently bind extensively to liver microsomal proteins from rats and humans. In the presence of glutathione, two thioether adducts were detected in liver microsomal incubations by radiochromatography and LC/MS/MS analysis. These adducts were also formed when bile duct-cannulated rats were dosed with MB243. The two adducts were isolated, and their structures were determined by accurate mass MS/MS and NMR analyses. The proposed structures resulted from a novel contraction of the piperazine ring to yield a substituted imidazoline. A mechanism is proposed, which involves an initial six electron oxidation of the piperazine ring to form a reactive intermediate, which is trapped by glutathione. Hydrolysis of the glutamic acid residue followed by internal aminolysis by the cysteine amino group resulted in opening of the piperazine ring, which is followed by ring closure to an imidazoline. The resulting cysteinyl−glycine conjugate underwent subsequent hydrolysis of the glycine residue. Understanding of the mechanism of bioactivation led to the design of MB243 analogues that exhibited reduced covalent protein binding.
Sodium channel blockers are used clinically to treat a number of neuropathic pain conditions, but more potent and selective agents should improve on the therapeutic index of currently used drugs. In ...a high-throughput functional assay, a novel sodium channel (NaV) blocker, N-{2‘-(aminosulfonyl)biphenyl-4-ylmethyl}-N‘-(2,2‘-bithien-5-ylmethyl)succinamide (BPBTS), was discovered. BPBTS is 2 orders of magnitude more potent than anticonvulsant and antiarrhythmic sodium channel blockers currently used to treat neuropathic pain. Resembling block by these agents, block of NaV1.2, NaV1.5, and NaV1.7 by BPBTS was found to be voltage- and use-dependent. BPBTS appeared to bind preferentially to open and inactivated states and caused a dose-dependent hyperpolarizing shift in the steady-state availability curves for all sodium channel subtypes tested. The affinity of BPBTS for the resting and inactivated states of NaV1.2 was 1.2 and 0.14 μM, respectively. BPBTS blocked NaV1.7 and NaV1.2 with similar potency, whereas block of NaV1.5 was slightly more potent. The slow tetrodotoxin-resistant Na+ current in small-diameter DRG neurons was also potently blocked by BPBTS. 3HBPBTS bound with high affinity to a single class of sites present in rat brain synaptosomal membranes (K d = 6.1 nM), and in membranes derived from HEK cells stably expressing NaV1.5 (K d = 0.9 nM). BPBTS dose-dependently attenuated nociceptive behavior in the formalin test, a rat model of tonic pain. On the basis of these findings, BPBTS represents a structurally novel and potent sodium channel blocker that may be used as a template for the development of analgesic agents.
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