A large superfamily of receptors containing seven transmembrane (TM) helices transmits hormonal and sensory signals across the plasma membrane to heterotrimeric G proteins at the cytoplasmic face of the membrane. To investigate how G-protein-coupled receptors work at the molecular level, we have engineered metal-ion-binding sites between TM helices to restrain activation-induced conformational change in specific locations. In rhodopsin, the photoreceptor of retinal rod cells, we substituted histidine residues for natural amino acids at the cytoplasmic ends of the TM helices C and F. The resulting mutant proteins were able to activate the visual G protein transducin in the absence but not in the presence of metal ions. These results indicate that the TM helices C and F are in close proximity and suggest that movements of these helices relative to one another are required for transducin activation. Thus a change in the orientations of TM helices C and F is likely to be a key element in the mechanism for coupling binding of ligands (or isomerization of retinal) to the activation of G-protein-coupled receptors.

A method was developed to measure Fouriertransform infrared (FTIR) difference spectra of detergentsolubilized rhodopsin expressed in COS cells. Experiments were performed on native bovine rhodopsin, rhodopsin expressed in COS cells, and three expressed rhodopsin mutants with amino acid replacements of membrane-embedded carboxylic acid groups: Asp-83 -* Asn (D83N), Gln (E122Q), and the double mutant D83N/E122Q. Each of the mutant opsins bound 11-cis-retinal to yield a visible light-absorbing pigment. Upon illumination, each of the mutant pigments formed a metarhodopsin fl-like species with maximal absorption at 380 nm that was able to activate guanine nucleotide exchange by btanducin. Rhodopsin versus metarhodopsin iH-like photoproduct FTIR-difference spectra were recorded for each sample. The COS-ceil rhodopsin and mutant difference spectra showed close correspondence to that of rhodopsin from disc membranes.Difference bands (rhodopsin/metarhodopsin II) at 1767/1750 cm'i and at 1734/1745 cm-' were absent from the spectra of mutants D83N and E122Q, respectively. Both bands were absent from the spectrum of the double mutant D83N/E122Q. These results show that Asp-83 and Glu-122 are protonated both in rhodopsin and in metarhodopsin H, in agreement with the isotope effects observed in spectra measured in 2H20. A photoproduct band at 1712 cm-' was not affected by either single or double replacements at positions 83 and 122. We deduce that the 1712 cm-' band arises from the protonation of Glu-113 in metarhodopsin II. Rhodopsin is a member of the superfamily of seventransmembrane-helix, G protein-coupled receptors. The rhodopsin chromophore 11-cis-retinal is covalently bound to the protein via a protonated Schiff base linkage (1) to a lysine residue (Lys-296 in bovine rhodopsin) (2, 3). After photoisomerization of the chromophore, thermal relaxation leads to an active conformation, R*, which binds the G protein transducin and thereby couples photon absorption to the visual signal transduction cascade. It has been shown by chemical modifications of Lys-296 in bovine rhodopsin that the deprotonation of the Schiff base is a prerequisite for R* formation (4, 5). Spectroscopically, this state is designated metarhodopsin II (MII) and characterized by a visible absorption maximum (Am.) at 380 nm, indicative of the unprotonated Schiff base of all-trans-retinal. Biochemical studies (6-10) and resonance Raman spectroscopy (11) of recombinant rhodopsins have shown that the positive charge at the Schiff base nitrogen in rhodopsin is stabilized by Glu-113, which acts as a Schiff base counterion in the transmembrane domain of the opsin.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.To investigate the protonation states and possible protonation changes of membrane-embedded carboxyl groups in rhodopsin and its MII photoproduct, we have performed Fourier-transform i...

Rhodopsin is a seven-transmembrane helix receptor that binds and catalytically activates the heterotrimeric G protein transducin (G t ). This interaction involves the cytoplasmic surface of rhodopsin, which comprises four putative loops and the carboxyl-terminal tail. The fourth loop connects the carboxyl end of transmembrane helix 7 with Cys 322 and Cys 323 , which are both modified by membrane-inserted palmitoyl groups. Published data on the roles of the fourth loop in the binding and activation of G t are contradictory. Here, we attempt to reconcile these conflicts and define a role for the fourth loop in rhodopsin-G t interactions. Fluorescence experiments demonstrated that a synthetic peptide corresponding to the fourth loop of rhodopsin inhibited the activation of G t by rhodopsin and interacted directly with the ␣ subunit of G t . A series of rhodopsin mutants was prepared in which portions of the fourth loop were replaced with analogous sequences from the ␤ 2 -adrenergic receptor or the m1 muscarinic receptor. Chimeric receptors in which residues 310 -312 were replaced could not efficiently activate G t . The defect in G t interaction in the fourth loop mutants was not affected by preventing palmitoylation of Cys 322 and Cys 323 . We suggest that the amino terminus of the fourth loop interacts directly with G t , particularly with G␣ t , and with other regions of the intracellular surface of rhodopsin to support G t binding.Rhodopsin, the dim-light photoreceptor of the rod cell, is a prototypical member of the superfamily of G protein-coupled receptors (GPCRs) 1 (1, 2). Following exposure to light, rhodopsin assumes an active signaling conformation, metarhodopsin II (MII). MII can bind and catalytically activate the retinal heterotrimeric G protein, transducin (G t ). G t is composed of a guanine-nucleotide binding ␣ subunit (G␣ t ), and a functional heterodimer of ␤ and ␥ subunits (G␤␥ t ). Interaction of the trimer with MII promotes the release of GDP from G␣ t , leading to the formation of a stable MII-G␣ t (empty pocket) complex. The subsequent binding of GTP activates G␣ t , leading to its dissociation from the receptor and from G␤␥ t . The activated G␣ t binds and activates its effector, cyclic GMP phosphodiesterase.The molecular structure of the complex between rhodopsin and G t , and the mechanism by which rhodopsin catalyzes nucleotide exchange, are not understood in detail. Numerous studies have localized the G t -binding site to the cytoplasmic surface of rhodopsin. The cytoplasmic surface is composed of four loops (Fig. 1) and a carboxyl-terminal tail. The first (C1), second (C2), and third (C3) cytoplasmic loops connect adjacent transmembrane (TM) helices. The fourth cytoplasmic loop (C4) is unique in that it is bounded by a helix only at its amino terminus; its carboxyl terminus is formed by the insertion of two palmitoyl groups into the membrane bilayer (3). The palmitoyl groups are attached to Cys 322 and Cys 323 via thioester linkages (4, 5). The carboxyl-terminal tail is the region dist...

The discovery of asunaprevir (BMS-650032, 24) is described. This tripeptidic acylsulfonamide inhibitor of the NS3/4A enzyme is currently in phase III clinical trials for the treatment of hepatitis C virus infection. The discovery of 24 was enabled by employing an isolated rabbit heart model to screen for the cardiovascular (CV) liabilities (changes to HR and SNRT) that were responsible for the discontinuation of an earlier lead from this chemical series, BMS-605339 (1), from clinical trials. The structure-activity relationships (SARs) developed with respect to CV effects established that small structural changes to the P2* subsite of the molecule had a significant impact on the CV profile of a given compound. The antiviral activity, preclincial PK profile, and toxicology studies in rat and dog supported clinical development of BMS-650032 (24).

A mutation in the gene for the rod photoreceptor molecule rhodopsin causes congenital night blindness. The mutation results in a replacement of Gly90 by an aspartic acid residue. Two molecular mechanisms have been proposed to explain the physiology of affected rod cells. One involves constitutive activity of the G90D mutant opsin [Rao, V. R., Cohen, G. B., & Oprian, D. D. (1994) Nature 367, 639-642]. A second involves increased photoreceptor noise caused by thermal isomerization of the G90D pigment chromophore [Sieving, P. A., Richards, J. E., Naarendorp F., Bingham, E. L., Scott, K., & Alpern, M. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 880-884]. Based on existing models of rhodopsin and in vitro biochemical studies of site-directed mutants, it appears likely that Gly90 is in the immediate proximity of the Schiff base chromophore linkage. We have studied in detail the mutant pigments G90D and G90D/E113A using biochemical and Fourier-transform infrared (FTIR) spectroscopic methods. The photoproduct of mutant pigment G90D, which absorbs maximally at 468 nm and contains a protonated Schiff base linkage, can activate transducin. However, the active photoproduct decays rapidly to opsin and free all-trans-retinal. FTIR studies of mutant G90D show that the dark state of the pigment has several structural features of metarhodopsin II, the active form of rhodopsin. These include a protonated carboxylic acid group at position Glu113 and increased hydrogen-bond strength of Asp83. Additional results, which relate to the structure of the active G90D photoproduct, are also reported. Taken together, these results may be relevant to understanding the molecular mechanism of congenital night blindness caused by the G90D mutation in human rhodopsin.

The discovery of BMS-605339 (35), a tripeptidic inhibitor of the NS3/4A enzyme, is described. This compound incorporates a cyclopropylacylsulfonamide moiety that was designed to improve the potency of carboxylic acid prototypes through the introduction of favorable nonbonding interactions within the S1' site of the protease. The identification of 35 was enabled through the optimization and balance of critical properties including potency and pharmacokinetics (PK). This was achieved through modulation of the P2* subsite of the inhibitor which identified the isoquinoline ring system as a key template for improving PK properties with further optimization achieved through functionalization. A methoxy moiety at the C6 position of this isoquinoline ring system proved to be optimal with respect to potency and PK, thus providing the clinical compound 35 which demonstrated antiviral activity in HCV-infected patients.

An automated process is described for the detailed assessment of the in vitro metabolic stability properties of drug candidates in support of pharmaceutical property profiling. Compounds are incubated with liver microsomes using a robotic liquid handler. Aliquots are taken at various time points, and the resulting samples are quantitatively analyzed by liquid chromatography-mass spectrometry utilizing ion trap mass spectrometers to determine the amount of compound remaining. From these data metabolism rates can be calculated. A high degree of automation is achieved through custom software, which is employed for instrument setup, data processing, and results reporting. The assay setup is highly configurable, allowing for any combination of up to six user-selected time points, variable substrate concentration, and microsomes or other biologically active media. The data, based on relative substrate depletion, affords an estimate of metabolic stability through the calculation of half-life (t(1/2)) and intrinsic clearance, which are used to differentiate and rank order drug leads. In general, t(1/2) is the time necessary for the metabolism, following first-order kinetics, of 50% of the initial compound. Intrinsic clearance is the proportionality constant between rate of metabolism of a compound and its concentration at the enzyme site. Described here is the setup of the assay, and data from assay test compounds are presented.

ABSTRACT:Six proton pump inhibitors (PPIs), omeprazole, lansoprazole, esomeprazole, dexlansoprazole, pantoprazole, and rabeprazole, were shown to be weak inhibitors of cytochromes P450 (CYP3A4, -