High-throughput synthesis and screening of cyclic peptide antibiotics. (1/1231)

Cyclic peptides are a rich source of biologically active compounds and are produced in nature by plants, bacteria, fungi, and lower sea animals. A high-throughput methodology has been developed for the combinatorial synthesis, screening, and identification of cyclic peptide natural product analogues with improved biological activities or useful new activities. The methodology was applied to generate a library of 1716 tyrocidine A analogues, which were screened for antibacterial activity in 96-well plates. The identity of the active peptides was determined by partial Edman degradation/mass spectrometry. This has resulted in the discovery of a series of tyrocidine analogues that have significantly improved therapeutic indices compared to the natural product. The availability of tyrocidine analogues with varying antibacterial activities has provided important insights into the structure-function relationship of tyrocidine A, which should help reveal its mechanism of action.  (+info)

Antibacterial targets in fatty acid biosynthesis. (2/1231)

The fatty acid biosynthesis pathway is an attractive but still largely unexploited target for the development of new antibacterial agents. The extended use of the antituberculosis drug isoniazid and the antiseptic triclosan, which are inhibitors of fatty acid biosynthesis, validates this pathway as a target for antibacterial development. Differences in subcellular organization of the bacterial and eukaryotic multienzyme fatty acid synthase systems offer the prospect of inhibitors with host versus target specificity. Platensimycin, platencin, and phomallenic acids, newly discovered natural product inhibitors of the condensation steps in fatty acid biosynthesis, represent new classes of compounds with antibiotic potential. An almost complete catalog of crystal structures for the enzymes of the type II fatty acid biosynthesis pathway can now be exploited in the rational design of new inhibitors, as well as the recently published crystal structures of type I FAS complexes.  (+info)

The molecular pruning of a phosphoramidate peptidomimetic inhibitor of prostate-specific membrane antigen. (3/1231)

To identify the pharmacophore of a phosphoramidate peptidomimetic inhibitor of prostate-specific membrane antigen (PSMA), a small analog library was designed and screened for inhibitory potency against PSMA. The design of the lead inhibitor was based upon N-acyl derivatives of endogenous substrate folyl-gamma-Glu and incorporates a phosphoramidate group to interact with the PSMA catalytic zinc atoms. The scope of the analog library was designed to test the importance of various functional groups to the inhibitory potency of the lead phosphoramidate. The IC(50) for the lead phosphoramidate inhibitor was 35 nM while the IC(50) values for the analog library presented a range from 0.86 nM to 4.1 microM. Computational docking, utilizing a recently solved X-ray crystal structure of the recombinant protein, along with enzyme inhibition data, was used to propose a pharmacophore model for the PSMA active site.  (+info)

Evolutionary conservation of drug action on lipoprotein metabolism-related targets. (4/1231)

Genetic analysis has shown that the slower than normal rhythmic defecation behavior of the clk-1 mutants of Caenorhabditis elegans is the result of altered lipoprotein metabolism. We show here that this phenotype can be suppressed by drugs that affect lipoprotein metabolism, including drugs that affect HMG-CoA reductase activity, reverse cholesterol transport, or HDL levels. These pharmacological effects are highly specific, as these drugs affect defecation only in clk-1 mutants and not in the wild-type and do not affect other behaviors of the mutants. Furthermore, drugs that affect processes not directly related to lipid metabolism show no or minimal activity. Based on these findings, we carried out a compound screen that identified 190 novel molecules that are active on clk-1 mutants, 15 of which also specifically decrease the secretion of apolipoprotein B (apoB) from HepG2 hepatoma cells. The other 175 compounds are potentially active on lipid-related processes that cannot be targeted in cell culture. One compound, CHGN005, was tested and found to be active at reducing apoB secretion in intestinal Caco-2 cells as well as in HepG2 cells. This compound was also tested in a mouse model of dyslipidemia and found to decrease plasma cholesterol and triglyceride levels. Thus, target processes for pharmacological intervention on lipoprotein synthesis, transport, and metabolism are conserved between nematodes and vertebrates, which allows the use of C. elegans for drug discovery.  (+info)

Efficacy of small-molecule glycogen synthase kinase-3 inhibitors in the postnatal rat model of tau hyperphosphorylation. (5/1231)

BACKGROUND AND PURPOSE: Glycogen synthase kinase-3 (GSK-3) affects neuropathological events associated with Alzheimers disease (AD) such as hyperphosphorylation of the protein, tau. GSK-3beta expression, enzyme activity and tau phosphorylated at AD-relevant epitopes are elevated in juvenile rodent brains. Here, we assess five GSK-3beta inhibitors and lithium in lowering phosphorylated tau (p-tau) and GSK-3beta enzyme activity levels in 12-day old postnatal rats. EXPERIMENTAL APPROACH: Brain levels of inhibitors following treatment in vivo were optimized based on pharmacokinetic data. At optimal doses, p-tau (Ser(396)) levels in brain tissue was measured by immunoblotting and correlated with GSK-3beta enzyme activities in the same tissues. Effects of GSK inhibitors on p-tau, GSK-3beta activities and cell death were measured in a human neuronal cell line (LUHMES). KEY RESULTS: Lithium and CHIR98014 reduced tau phosphorylation (Ser(396)) in the cortex and hippocampus of postnatal rats, while Alsterpaullone and SB216763 were effective only in hippocampus. AR-A014418 and Indirubin-3'-monoxime were ineffective in either brain region. Inhibition of p-tau in brain required several-fold higher levels of GSK inhibitors than the IC(50) values obtained in recombinant or cell-based GSK-3beta enzyme activity assays. The inhibitory effect on GSK-3beta activity ex vivo correlated with protection against cell death and decrease of p-tau- in LUHMES cells, using low microM inhibitor concentrations. CONCLUSIONS AND IMPLICATIONS: Selective small-molecule inhibitors of GSK-3 reduce tau phosphorylation in vivo. These findings corroborate earlier suggestions that GSK-3beta may be an attractive target for disease-modification in AD and related conditions where tau phosphorylation is believed to contribute to disease pathogenesis.  (+info)

High-throughput screening for fatty acid uptake inhibitors in humanized yeast identifies atypical antipsychotic drugs that cause dyslipidemias. (6/1231)

Fatty acids are implicated in the development of dyslipidemias, leading to type 2 diabetes and cardiovascular disease. We used a standardized small compound library to screen humanized yeast to identify compounds that inhibit fatty acid transport protein (FATP)-mediated fatty acid uptake into cells. This screening procedure used live yeast cells expressing human FATP2 to identify small compounds that reduced the import of a fluorescent fatty acid analog, 4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid (C(1)-BODIPY-C(12)). The library used consisted of 2,080 compounds with known biological activities. Of these, approximately 1.8% reduced cell-associated C(1)-BODIPY-C(12) fluorescence and were selected as potential inhibitors of human FATP2-mediated fatty acid uptake. Based on secondary screens, 28 compounds were selected as potential fatty acid uptake inhibitors. Some compounds fell into four groups with similar structural features. The largest group was structurally related to a family of tricyclic, phenothiazine-derived drugs used to treat schizophrenia and related psychiatric disorders, which are also known to cause metabolic side effects, including hypertriglyceridemia. Potential hit compounds were studied for specificity of interaction with human FATP and efficacy in human Caco-2 cells. This study validates this screening system as useful to assess the impact of drugs in preclinical screening for fatty acid uptake.  (+info)

Inhibition of monometalated methionine aminopeptidase: inhibitor discovery and crystallographic analysis. (7/1231)

Two divalent metal ions are commonly seen in the active-site cavity of methionine aminopeptidase, and at least one of the metal ions is directly involved in catalysis. Although ample structural and functional information is available for dimetalated enzyme, methionine aminopeptidase likely functions as a monometalated enzyme under physiological conditions. Information on structure, as well as catalysis and inhibition, of the monometalated enzyme is lacking. By improving conditions of high-throughput screening, we identified a unique inhibitor with specificity toward the monometalated enzyme. Kinetic characterization indicates a mutual exclusivity in binding between the inhibitor and the second metal ion at the active site. This is confirmed by X-ray structure, and this inhibitor coordinates with the first metal ion and occupies the space normally occupied by the second metal ion. Kinetic and structural analyses of the inhibition by this and other inhibitors provide insight in designing effective inhibitors of methionine aminopeptidase.  (+info)

A class of small molecules that inhibit TNFalpha-induced survival and death pathways via prevention of interactions between TNFalphaRI, TRADD, and RIP1. (8/1231)

Small-molecule library screening to find compounds that inhibit TNFalpha-induced, but not interleukin 1beta (IL-1beta)-induced, intercellular adhesion molecule 1 (ICAM-1) expression in lung epithelial cells identified a class of triazoloquinoxalines. These compounds not only inhibited the TNFalpha-induced nuclear factor kappaB (NFkappaB) survival pathway but also blocked death-pathway activation. Such dual activity makes them unique against other known NFkappaB-pathway inhibitors that inhibit only a subset of TNFalpha signals leading to increased TNFalpha-induced cytotoxicity. Interestingly, these compounds inhibited association of TNFalpha receptor (TNFalphaR) I with TNFalphaR-associated death domain protein (TRADD) and receptor interacting protein 1 (RIP1), the initial intracellular signaling event following TNFalpha stimulation. Further study showed that they blocked ligand-dependent internalization of the TNFalpha-TNFalphaR complex, thereby inhibiting most of the TNFalpha-induced cellular responses. Thus, compounds with a triazoloquinoxaline scaffold could be a valuable tool to investigate small molecule-based anti-TNFalpha therapies.  (+info)