Modeling G protein-coupled receptors for structure-based drug discovery using low-frequency normal modes for refinement of homology models: application to H3 antagonists.

G Protein-Coupled Receptors (GPCRs) are integral membrane proteins that play important role in regulating key physiological functions, and are targets of about 50% of all recently launched drugs. High-resolution experimental structures are available only for very few GPCRs. As a result, structure-based drug design efforts for GPCRs continue to rely on in silico modeling, which is considered to be an extremely difficult task especially for these receptors. Here, we describe Gmodel, a novel approach for building 3D atomic models of GPCRs using a normal mode-based refinement of homology models. Gmodel uses a small set of relevant low-frequency vibrational modes derived from Random Elastic Network model to efficiently sample the large-scale receptor conformation changes and generate an ensemble of alternative models. These are used to assemble receptor-ligand complexes by docking a known active into each of the alternative models. Each of these is next filtered using restraints derived from known mutation and binding affinity data and is refined in the presence of the active ligand. In this study, Gmodel was applied to generate models of the antagonist form of histamine 3 (H3) receptor. The validity of this novel modeling approach is demonstrated by performing virtual screening (using the refined models) that consistently produces highly enriched hit lists. The models are further validated by analyzing the available SAR related to classical H3 antagonists, and are found to be in good agreement with the available experimental data, thus providing novel insights into the receptor-ligand interactions.

Use of a murine cell line for identification of human nitric oxide synthase inhibitors.

INTRODUCTION: Nitric oxide (NO) has been implicated in a wide range of physiological and pathological processes. Low concentrations of this mediator play homeostatic roles, whereas many acute and chronic responses are associated with excessive production of NO. This upregulation is due in part to the induction of inducible nitric oxide synthase (iNOS) by proinflammatory cytokines in several different cell types, including macrophages and their CNS derivative, microglia. METHODS: The crystal structures of the oxygenase domains of mouse and human iNOS were superimposed using the "align by homology" feature in Sybyl (SYBYL 7.0, Tripos Inc.). NOS isoform expression was assessed by TaqMan, Western blotting, and activity assays. RESULTS: We demonstrate that there is a high degree of three-dimensional overlap between the mouse and human iNOS active centers and propose that the murine isoform can serve as a suitable substitute for the human in assays. We also demonstrate that LPS stimulation of the mouse macrophage cell line RAW 264.7 induces the expression of iNOS, but not nNOS or eNOS, at the levels of mRNA transcription and protein expression. Furthermore, the pharmacology and calcium dependency of the NO formation support the finding that it is due to iNOS alone. Also reported is the demonstration of LPS-induced RAW 264.7 macrophages in simple cell-based and cell-free screening assays for iNOS inhibitors. Both assays were reproducible, as demonstrated by Z' factors of 0.69 and 0.71, and had high signal to noise ratios of 11- and 6-fold for the cell-based and cell-free assay, respectively. DISCUSSION: Our computational analyses indicate that there is a high degree of three-dimensional overlap between the oxygenase domains of human and murine iNOS. This observation together with the selective induction of murine iNOS in RAW 264.7 macrophages demonstrates the potential utility of the mouse iNOS assay to identify inhibitors of the human enzyme.

Pyrazolidine-3,5-diones and 5-hydroxy-1H-pyrazol-3(2H)-ones, inhibitors of UDP-N-acetylenolpyruvyl glucosamine reductase.

A series of pyrazolidine-3,5-dione and 5-hydroxy-1H-pyrazol-3(2H)-one inhibitors of Escherichia coli UDP-N-acetylenolpyruvyl glucosamine reductase (MurB) has been prepared. The 5-hydroxy-1H-pyrazol-3(2H)-ones show low micromolar IC(50) values versus E. coli MurB and submicromolar minimal inhibitory concentrations (MIC) against Staphylococcus aureus GC 1131, Enterococcus faecalis GC 2242, Streptococcus pneumoniae GC 1894, and E. coli GC 4560 imp, a strain with increased outer membrane permeability. None of these compounds show antimicrobial activity against Candida albicans, a marker of eukaryotic toxicity. Moreover, these compounds inhibit peptidoglycan biosynthesis, as assessed by measuring the amount of soluble peptidoglycan produced by Streptococcus epidermidis upon incubation with compounds. A partial least squares projection to latent structures analysis shows that improving MurB potency and MIC values correlate with increasing lipophilicity of the C-4 substituent of the 5-hydroxy-1H-pyrazol-3(2H)-one core. Docking studies using FLO and PharmDock produced several binding orientations for these molecules in the MurB active site.

Identification and characterization of small molecule modulators of KChIP/Kv4 function.

Potassium channels and their associated subunits are important contributors to electrical excitability in many cell types. In this study, a yeast two-hybrid assay was used to identify inhibitors such as a diaryl-urea compound (CL-888) that binds to and modulates the formation of the Kv4/KChIP complex. CL-888 altered the apparent affinity of KChIP1 to Kv4.3-N in a Biacore assay, but did not dissociate the two proteins in size-exclusion chromatography experiments. Kv4.2/KChIP1 current amplitude and kinetics were altered with compound exposure, supporting the hypothesis of a compound-induced conformational change in the protein complex. Fluorescence spectroscopy of a unique tryptophan residue in KChIP1 was consistent with compound binding to the protein. Molecular modeling using the KChIP1 crystal structure indicates that compound binding may occur in a small tryptophan-containing binding pocket located on the hydrophilic side of the protein.

4-Alkyl and 4,4'-dialkyl 1,2-bis(4-chlorophenyl)pyrazolidine-3,5-dione derivatives as new inhibitors of bacterial cell wall biosynthesis.

Over 195 4-alkyl and 4,4-dialkyl 1,2-bis(4-chlorophenyl)pyrazolidine-3,5-dione derivatives were synthesized, utilizing microwave accelerated synthesis, for evaluation as new inhibitors of bacterial cell wall biosynthesis. Many of them demonstrated good activity against MurB in vitro and low MIC values against gram-positive bacteria, particularly penicillin-resistant Streptococcus pneumoniae (PRSP). Derivative 7l demonstrated antibacterial activity against both gram-positive and gram-negative bacteria. Derivatives 7f and 10a also demonstrated potent nanomolar Kd values in their binding to MurB.

Two N-terminal domains of Kv4 K(+) channels regulate binding to and modulation by KChIP1.

The family of calcium binding proteins called KChIPs associates with Kv4 family K(+) channels and modulates their biophysical properties. Here, using mutagenesis and X-ray crystallography, we explore the interaction between Kv4 subunits and KChIP1. Two regions in the Kv4.2 N terminus, residues 7-11 and 71-90, are necessary for KChIP1 modulation and interaction with Kv4.2. When inserted into the Kv1.2 N terminus, residues 71-90 of Kv4.2 are also sufficient to confer association with KChIP1. To provide a structural framework for these data, we solved the crystal structures of Kv4.3N and KChIP1 individually. Taken together with the mutagenesis data, the individual structures suggest that that the Kv4 N terminus is required for stable association with KChIP1, perhaps through a hydrophobic surface interaction, and that residues 71-90 in Kv4 subunits form a contact loop that mediates the specific association of KChIPs with Kv4 subunits.

Phenyl thiazolyl urea and carbamate derivatives as new inhibitors of bacterial cell-wall biosynthesis.

Over 50 phenyl thiazolyl urea and carbamate derivatives were synthesized for evaluation as new inhibitors of bacterial cell-wall biosynthesis. Many of them demonstrated good activity against MurA and MurB and gram-positive bacteria including MRSA, VRE and PRSP. 3,4-Difluorophenyl 5-cyanothiazolylurea (3p) with clog P of 2.64 demonstrated antibacterial activity against both gram-positive and gram-negative bacteria.

Structure-based design approaches to cell wall biosynthesis inhibitors.

This review summarizes some of the published attempts to incorporate protein and NMR structures in the design of new antibiotics that specifically target Cell Wall biosynthesis. Most of the steps involved in peptidglycan synthesis have been investigated as potential strategies against cell wall inhibition. Structural information has been most useful in the design of molecules in the Mur enzyme pathway, penicillin binding proteins and lactamases, as well as proteins that are part of the final steps of transglycosylation - in particular, d-Ala-d-Ala ligase. Several unique issues exist in the design of effective antibacterials, such as the significant differences in protein structure between organisms, such as the case of MurB in which a large amino acid loop that occupies the active site of the E. Coli is gone in the Staph aureus enzyme. Additionally, bacterial resistance is an important issue, and in some cases, structural information can be used to understand the source of this resistance. For example, mutations within the d-Ala-d-Ala ligases lead to the inability of Vancomycin antibiotics to bind.