G protein coupled receptors -in silico drug discovery and design.

In silico drug discovery is a complex process requiring flexibility and ingenuity in method selection and a careful validation of work protocols. GPCR in silico drug discovery poses additional challenges due to the paucity of crystallographic data. This paper starts by reviewing selected GPCR in silico screening programs reported in the literature, including both structure-based and ligand-based approaches. Particular emphasis is given to library design, binding mode selection, process validation and compound selection for biological testing. Following literature review, we provide insights into in silico methodologies and process workflows used at EPIX to drive over 20 highly successful screening and lead optimization programs performed since 2001. Applications of the various methodologies discussed are demonstrated by examples from recent programs that have not yet been published.

Proton-collecting properties of bovine heart cytochrome C oxidase: kinetic and electrostatic analysis.

Proton-transfer reactions on the surface of bovine heart cytochrome c oxidase were investigated by combining a laser-induced proton-pulse technique with molecular modeling. The experimental approach simultaneously monitors the state of pyranine protonation in the bulk phase and that of a fluorescein indicator specifically attached to the native Cys(III-115) residue of subunit III of cytochrome oxidase. The reversible dynamics of the acid-base equilibration between the surface and the bulk phase were measured with submicrosecond time resolution and analyzed by numerical integration of coupled nonlinear differential rate equations. Kinetic analysis shows that carboxylates on the surface of the protein act as a proton-collecting antenna, which is able to rapidly transfer protons to nearby histidines that function as a local proton reservoir. These properties enable cytochrome oxidase to carry out its redox-linked proton translocation. Molecular modeling of the fluorescein-binding site indicates that, in addition to the covalent bond, the dye is anchored through a hydrogen bond to the hydroxyl moiety of Tyr(VII-50). The protonation of the dye is mediated through three residues that shuttle protons between the bulk and the dye. A correlation between the measured kinetic properties of the bound fluorescein and the different configurations of the dye allows us to predict the identity of the proton-binding sites in the fluorescein-binding domain.

Modeling the 3D structure of GPCRs from sequence.

G-protein-coupled receptors (GPCRs) are a large and functionally diverse protein superfamily, which form a seven transmembrane (TM) helices bundle with alternating extra-cellular and intracellular loops. GPCRs are considered to be one of the most important groups of drug targets because they are involved in a broad range of body functions and processes and are related to major diseases. In this paper we present a new technology, named PREDICT, for modeling the 3D structure of any GPCR from its amino acid sequence. This approach takes into account both internal protein properties (i.e., the amino acid sequence) and the properties of the membrane environment. Unlike competing approaches, the new technology does not rely on the single known structure of rhodopsin, and is thus capable of predicting novel GPCR conformations. We demonstrate the capabilities of PREDICT in reproducing the known experimental structure of rhodopsin. In principle, PREDICT-generated models offer new opportunities for structure-based drug discovery towards GPCR targets.

Dynamics of the proton transfer reaction on the cytoplasmic surface of bacteriorhodopsin.

The cytoplasmic surface of bacteriorhodopsin is characterized by a group of carboxylates that function as a proton attractive domain [Checover, S., Nachliel, E., Dencher, N. A., and Gutman, M. (1997) Biochemistry 36, 13919-13928]. To identify these carboxylates, we selectively mutated them into cysteine residues and monitored the effects of the dynamics of proton transfer between the bulk and the surface of the protein. The measurements were carried out without attachment of a pH-sensor to the cysteine residue, thus avoiding any structural perturbation and change in the surface charge caused by the attachment of a reporter group, and the protein was in its BR state. The purple membranes were suspended in an unbuffered solution of pyranine (8-hydroxypyrene-1,3,6-trisulfonate) and exposed to a train of 1000 laser pulses (2.1 mJ/pulse, lambda = 355 nm, at 10 Hz). The excitation of the dye ejected the hydroxyl's proton, and a few nanoseconds later, a pair of free protons and ground-state pyranine anion was formed. The experimental observation was the dynamics of the relaxation of the system to the prepulse state. The observed signals were reconstructed by a numeric method that replicates the chemical reactions proceeding in the perturbed space. The detailed reconstruction of the measured signal assigned the various proton-binding sites with rate constants for proton binding and proton exchange and the pK values. Comparison of the results obtained by the various mutants indicates that the dominant proton-binding cluster of the wild-type protein consists of D104, E161, and E234. The replacement of D104 or E161 with cysteine lowered the proton binding capacity of the cluster to approximately 60% of that of the native protein. The replacement of E234 with cysteine disrupted the structure of the cluster, causing the two remaining carboxylates to function as isolated residues that do not interact with each other. The possibility of proton transfer between monomers is discussed.

Biophysical aspects of intra-protein proton transfer.

The passage of proton trough proteins is common to all membranal energy conserving enzymes. While the routes differ among the various proteins, the mechanism of proton propagation is based on the same chemical-physical principles. The proton progresses through a sequence of dissociation association steps where the protein and water molecules function as a solvent that lowers the energy penalty associated with the generation of ions in the protein. The propagation of the proton in the protein is a random walk, between the temporary proton binding sites that make the conducting path, that is biased by the intra-protein electrostatic potential. Kinetic measurements of proton transfer reactions, in the sub-ns up to micros time frame, allow to monitor the dynamics of the partial reactions of an overall proton transfer through a protein.

The proton collecting function of the inner surface of cytochrome c oxidase from Rhodobacter sphaeroides.

The experiments presented in this study address the problem of how the cytoplasmic surface (proton-input side) of cytochrome c oxidase interacts with protons in the bulk. For this purpose, the cytoplasmic surface of the enzyme was labeled with a fluorescein (Flu) molecule covalently bound to Cys223 of subunit III. Using the Flu as a proton-sensitive marker on the surface and phiOH as a soluble excited-state proton emitter, the dynamics of the acid-base equilibration between the surface and the bulk was measured in the time-resolved domain. The results were analyzed by using a rigorous kinetic analysis that is based on numeric integration of coupled nonliner differential rate equations in which the rate constants are used as adjustable parameters. The analysis of 11 independent measurements, carried out under various initial conditions, indicated that the protonation of the Flu proceeds through multiple pathways involving diffusion-controlled reactions and proton exchange among surface groups. The surface of the protein carries an efficient system made of carboxylate and histidine moieties that are sufficiently close to each other as to form a proton-collecting antenna. It is the passage of protons among these sites that endows cytochrome c oxidase with the capacity to pick up protons from the buffered cytoplasmic matrix within a time frame compatible with the physiological turnover of the enzyme.

Involvement of protein phosphatases in gonadotropin releasing hormone regulated gonadotropin secretion.

The role of persistent protein phosphorylation upon gonadotropin releasing hormone (GnRH) stimulated luteinizing hormone (LH) release was investigated by the use of the selective inhibitors of protein phosphatase type 1 (PP1) and 2A (PP2A), okadaic acid (OA) and calyculin A. Pre-incubation of cultured rat pituitary cells with OA (24 h) or calyculin A (30 min) resulted in inhibition of GnRH-stimulated LH release with significant inhibition being detected at 10 nM and 30 nM for OA and calyculin A, respectively. The inactive OA analog norokadone and the protein tyrosine phosphatase inhibitor vanadyl hydroperoxide had no significant effect on GnRH-induced LH release. The stimulatory effects of the protein kinase C (PKC) activator 12-O-tetradecanoylphorbol 13-acetate (TPA, 50 ng/ml) or the Ca2+ ionophore, ionomycin (1 micron), upon LH release were also abolished by pretreatment with OA (10-20 nM) or calyculin A (30 nM). Stimulation of LH release by high K+ (28 mM) or residual LH release stimulated by GnRH in Ca(2+)-free medium were also blocked by OA. These observations indicate that protein dephosphorylation is involved positively in GnRH-stimulated LH release. The site of action of the protein phosphatases PP1 and PP2A is most likely downstream to Ca2+ elevation and PKC activation by GnRH.

Developmental expression of protein kinase C subspecies in rat brain-pituitary axis.

We have examined the neonatal developmental expression of protein kinase C subspecies (PKCs) in rat brain, pituitary glands and cells by enzymatic activity assays, immunohistochemistry and Western blot analysis with type-specific antibodies. A very large increase (455%) was noticed in brain PKC activity during the first week of life with the particulate fraction (22% of total enzyme activity on day 1) increasing dramatically (900%) during the first week to 50% of enzyme activity. In contrast, the pituitary gland showed high activity on day 1 that decreased progressively to reach the lowest levels at 1 year of age. Paradoxically, the number of pituitary cells immunolabeled for PKC increases as a function of age. Western blot analysis showed only small changes in PKC alpha, PKC beta and PKC epsilon when brains from 6-day-old and 3-month-old female rats were compared, whereas PKC tau and PKC delta increased markedly during this period. On the other hand, brain PKC zeta decreased between 6 days and 3 months of age. Western blot analysis showed no major changes in pituitary PKC alpha, PKC beta and PKC zeta when 6-day-old and 3-month-old female rats were compared, while PKC tau was not detected. The major band of pituitary PKC delta (76 kDa) decreased markedly between 6 days and 3 months of age whereas the minor band (68 kDa) did not change.(ABSTRACT TRUNCATED AT 250 WORDS)