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PubMed2002

Potent, novel in vitro inhibitors of the Pseudomonas aeruginosa deacetylase LpxC.

Deacetylation of uridyldiphospho-3-O-(R-hydroxydecanoyl)-N-acetylglucosamine by LpxC is the first committed step in the Pseudomonas aeruginosa biosynthetic pathway to lipid A; homologous enzymes are found widely among Gram-negative bacteria. As an essential enzyme for which no inhibitors have yet been reported, the P. aeruginosa LpxC represents a highly attractive target for a novel antibacterial drug. We synthesized several focused small-molecule libraries, each composed of a variable aromatic ring, one of four heterocyclic/spacer moieties, and a hydroxamic acid and evaluated the LpxC inhibition of these compounds against purified P. aeruginosa enzyme. To ensure that the in vitro assay would be as physiologically relevant as possible, we synthesized a tritiated form of the specific P. aeruginosa glycolipid substrate and measured directly the enzymatically released acetate. Several of our novel compounds, predominantly those having fluorinated substituents on the aromatic ring and an oxazoline as the heterocyclic moiety, demonstrated in vitro IC(50) values less than 1 microM. We now report the synthesis and in vitro evaluation of these P. aeruginosa LpxC inhibitors.

Amidohydrolases↗
PubMed2006

Automatic and efficient decomposition of two-dimensional structures of small molecules for fragment-based high-throughput docking.

The computer program DAIM (Decomposition and Identification of Molecules) has been developed to automatically break up compounds in small-molecule libraries for fragment-based docking as well as database analysis. Here, DAIM is evaluated on 130 ligands derived from known crystal structures of ligand-protein complexes. The decomposition and a new fingerprint-based identification technique are used to select anchor fragments for docking. The docking results show that the DAIM selection is superior to size-based or random selection of fragments. To evaluate the usefulness for analyzing the fragment composition of a large library, DAIM is applied to a collection of about 1.85 million commercially available compounds. Interestingly, it is found that the set of most frequent cyclic and acyclic fragments originating from the decomposition of the 1.85 million molecules shows a large overlap with the most frequent fragments in a library of 5120 known drugs. DAIM has been successfully used in the in silico screening for inhibitors of beta-secretase and EphB4 kinase by fragment-based high-throughput docking. Possible future applications for de novo ligand design are briefly discussed.

Amyloid Precursor Protein Secretases↗
PubMed1998

Key Intermediates in Combinatorial Chemistry: Access to Various Heterocycles from alpha,beta-Unsaturated Ketones on the Solid Phase.

The value of alpha,beta-unsaturated ketones as key intermediates for the combinatorial assembly of four different templates on the solid phase, namely pyrimidines, dihydropyrimidinones, pyridines, and pyrazoles, was explored with individual syntheses of variably substituted model compounds. Starting from aldehydes grafted on polystyrene support, the Wittig and the Claisen-Schmidt reaction conditions were adapted to efficiently prepare alpha,beta-unsaturated ketones on the solid phase. Further derivatization of the alpha,beta-unsaturated ketones to form pyrimidines succeeded with a number of amidines. In a feasibility study, the potential to obtain, in a modular fashion, other small heterocycles from the same intermediates was assessed. In this solid-phase approach alpha,beta-unsaturated carbonyl intermediates can act as a three-carbon component and a primary enamine is utilized to complement the system for pyridine ring formation. Instead, with N-methylurea a dihydropyrimidinone is obtained. As an alternative, substituted hydrazines are incorporated in one orientation, providing pyrazoles with defined regioisomerism. The study indicates that alpha,beta-unsaturated ketones grafted on the solid phase can take a pivotal role as branching points in a number of synthetic diversity schemes and, therefore, represent versatile intermediates for the efficient preparation of combinatorial small molecule libraries.

Journal Article↗
PubMed2004

TRAM linker: a safety-catch linker for the traceless release of acrylamides.

[reaction: see text] A novel safety-catch linker for the solid-phase synthesis of small-molecule libraries containing electrophilic reactive groups has been developed. Upon cleavage from solid support, the linker generates a Michael acceptor (an acrylamide) on each library member. Utilization of a two-resin system in the final cleavage step provides crude products in high purity, allowing direct use in biological assays following filtration and evaporation.

Acrylamides↗
PubMed2002

A small-molecule inhibitor of skeletal muscle myosin II.

We screened a small-molecule library for inhibitors of rabbit muscle myosin II subfragment 1 (S1) actin-stimulated ATPase activity. The best inhibitor, N-benzyl-p-toluene sulphonamide (BTS), an aryl sulphonamide, inhibited the Ca2+-stimulated S1 ATPase, and reversibly blocked gliding motility. Although BTS does not compete for the nucleotide-binding site of myosin, it weakens myosin's interaction with F-actin. BTS reversibly suppressed force production in skinned skeletal muscle fibres from rabbit and frog skin at micromolar concentrations. BTS suppressed twitch production of intact frog fibres with minimum alteration of Ca2+ metabolism. BTS is remarkably specific, as it was much less effective in suppressing contraction in rat myocardial or rabbit slow-twitch muscle, and did not inhibit platelet myosin II. The isolation of BTS and the recently discovered Eg5 kinesin inhibitor, monastrol, suggests that motor proteins may be potential targets for therapeutic applications.

Adenosine Triphosphatases↗
PubMed2006

Fluorescent cell barcoding in flow cytometry allows high-throughput drug screening and signaling profiling.

Flow cytometry allows high-content, multiparameter analysis of single cells, making it a promising tool for drug discovery and profiling of intracellular signaling. To add high-throughput capacity to flow cytometry, we developed a cell-based multiplexing technique called fluorescent cell barcoding (FCB). In FCB, each sample is labeled with a different signature, or barcode, of fluorescence intensity and emission wavelengths, and mixed with other samples before antibody staining and analysis by flow cytometry. Using three FCB fluorophores, we were able to barcode and combine entire 96-well plates, reducing antibody consumption 100-fold and acquisition time to 5-15 min per plate. Using FCB and phospho-specific flow cytometry, we screened a small-molecule library for inhibitors of T cell-receptor and cytokine signaling, simultaneously determining compound efficacy and selectivity. We also analyzed IFN-gamma signaling in multiple cell types from primary mouse splenocytes, revealing differences in sensitivity and kinetics between B cells, CD4+ and CD4- T cells and CD11b-hi cells.

Animals↗
PubMed2001

Reversible inhibition of calcineurin by the polyphenolic aldehyde gossypol.

The reversible inhibition of calcineurin (CaN), which is the only Ca(2+)/calmodulin-dependent protein Ser/Thr phosphatase, is thought to be a key functional event for most cyclosporin A (CsA)- and tacrolimus (FK506)-mediated biological effects. In addition to CaN inhibition, however, CsA and FK506 have multiple biochemical effects because of their action in a gain-of-function model that requires prior binding to immunophilic proteins. We screened a small molecule library for direct inhibitors of CaN using CaN-mediated dephosphorylation of (33)P-labeled 19-residue phosphopeptide substrate (RII phosphopeptide) as an assay and found the polyphenolic aldehyde gossypol to be a novel CaN inhibitor. Unlike CsA and FK506, gossypol does not require a matchmaker protein for reversible CaN inhibition with an IC(50) value of 15 microm. Gossypolone, a gossypol analog, showed improved inhibition of both RII phosphopeptide and p-nitrophenyl phosphate dephosphorylation with an IC(50) of 9 and 6 microm, respectively. In contrast, apogossypol hexaacetate was inactive. Gossypol acts noncompetitively, interfering with the binding site for the cyclophilin 18.CsA complex in CaN. In contrast to CsA and FK506, gossypol does not inactivate the peptidyl-prolyl-cis/trans-isomerase activity of immunophilins. Similar to CsA and FK506, T cell receptor signaling induced by phorbol 12-myristate 13-acetate/ionomycin is inhibited by gossypol in a dose-dependent manner, demonstrated by the inhibition of nuclear factor of activated T cell (NFAT) c1 translocation from the cytosol into the nucleus and suppression of NFAT-luciferase reporter gene activity.

Amino Acid Sequence↗
PubMed2004

Enzymatic characterization of the pancreatic islet-specific glucose-6-phosphatase-related protein (IGRP).

Glucose is the main physiological stimulus for insulin biosynthesis and secretion by pancreatic beta-cells. Glucose-6-phosphatase (G-6-Pase) catalyzes the dephosphorylation of glucose-6-phosphate to glucose, an opposite process to glucose utilization. G-6-Pase activity in pancreatic islets could therefore be an important factor in the control of glucose metabolism and, consequently, of glucose-dependent insulin secretion. While G-6-Pase activity has been shown to be present in pancreatic islets, the gene responsible for this activity has not been conclusively identified. A homolog of liver glucose-6-phosphatase (LG-6-Pase) specifically expressed in islets was described earlier; however, the authors could not demonstrate enzymatic activity for this protein. Here we present evidence that the previously identified islet-specific glucose-6-phosphatase-related protein (IGRP) is indeed the major islet glucose-6-phosphatase. IGRP overexpressed in insect cells possesses enzymatic activity comparable to the previously described G-6-Pase activity in islets. The K(m) and V(max) values determined using glucose-6-phosphate as the substrate were 0.45 mm and 32 nmol/mg/min by malachite green assay, and 0.29 mm and 77 nmol/mg/min by glucose oxidase/peroxidase coupling assay, respectively. High-throughput screening of a small molecule library led to the identification of an active compound that specifically inhibits IGRP enzymatic activity. Interestingly, this inhibitor did not affect LG-6-Pase activity, while conversely LG-6-Pase inhibitors did not affect IGRP activity. These data demonstrate that IGRP is likely the authentic islet-specific glucose-6-phosphatase catalytic subunit, and selective inhibitors to this molecule can be obtained. IGRP inhibitors may be an attractive new approach for the treatment of insulin secretion defects in type 2 diabetes.

Animals↗
PubMed2006

A simple cell-based HTS assay system to screen for inhibitors of p53-Hdm2 protein-protein interactions.

Green fluorescent protein-assisted readout for interacting proteins (GRIP) is a universal protein interaction discovery system that can be used to generate truly high throughput screening-compatible cellular assays to be used to screen for inhibitors of protein-protein interactions. The technology uses a "bait and prey" principle based on the distinct translocation behavior of the human cyclic AMP phosphodiesterase 4A4. Here we use the p53-Hdm2 Redistribution assay (Fisher BioImage ApS, Søborg, Denmark) as an example to describe the GRIP technology. The p53-Hdm2 Redistribution assay is a high content imaging assay based on the GRIP technology that is designed to measure the interaction between Hdm2 and the tumor suppressor p53. Hdm2 regulates p53 and inhibits its function by modulating its transcriptional activity and stability. Activation of p53 in tumor cells through inhibition of its physical interaction with Hdm2 is therefore a focus of cancer drug discovery. We have performed a pilot screen by screening 3,165 compounds from a diverse small-molecule library for inhibitors of the p53-Hdm2 interaction by using the p53-Hdm2 Redistribution assay. Here we show that by taking advantage of the translocation behavior of nonbound p53, it is possible to identify true inhibitors of the p53-Hdm2 interaction by extracting high content information from the acquired images.

Animals↗
PubMed2004

A three-fluorophore FRET assay for high-throughput screening of small-molecule inhibitors of ribosome assembly.

In one of the first steps of prokaryotic ribosome assembly, the ribosomal protein S15 binds to a three-way junction in the central domain of the 16S rRNA. Binding causes a conformational change that is required for subsequent binding events. Using a novel fluorescence resonance energy transfer assay with three fluorophores, two on the RNA and one on the S15 protein, small-molecule libraries can be screened for potential inhibitors of this initial step in ribosome assembly. The employment of three fluorophores allows both the conformational change of the RNA and the binding of S15 to be monitored in a single assay.

Aminoglycosides↗
PubMed2006

Mimicking damaged DNA with a small molecule inhibitor of human UNG2.

Human nuclear uracil DNA glycosylase (UNG2) is a cellular DNA repair enzyme that is essential for a number of diverse biological phenomena ranging from antibody diversification to B-cell lymphomas and type-1 human immunodeficiency virus infectivity. During each of these processes, UNG2 recognizes uracilated DNA and excises the uracil base by flipping it into the enzyme active site. We have taken advantage of the extrahelical uracil recognition mechanism to build large small-molecule libraries in which uracil is tethered via flexible alkane linkers to a collection of secondary binding elements. This high-throughput synthesis and screening approach produced two novel uracil-tethered inhibitors of UNG2, the best of which was crystallized with the enzyme. Remarkably, this inhibitor mimics the crucial hydrogen bonding and electrostatic interactions previously observed in UNG2 complexes with damaged uracilated DNA. Thus, the environment of the binding site selects for library ligands that share these DNA features. This is a general approach to rapid discovery of inhibitors of enzymes that recognize extrahelical damaged bases.

Binding Sites↗
PubMed2005

Proteomics meets microbiology: technical advances in the global mapping of protein expression and function.

The availability of complete genome sequences for a large number of pathogenic organisms has opened the door for large-scale proteomic studies to dissect both protein expression/regulation and function. This review highlights key proteomic methods including two-dimensional gel electrophoresis, reference mapping, protein expression profiling and recent advances in gel-free separation techniques that have made a significant impact on the resolution of complex proteomes. In addition, we highlight recent developments in the field of chemical proteomics, a branch of proteomics aimed at functionally profiling a proteome. These techniques include the development of activity-based probes and activity-based protein profiling methods as well as the use of synthetic small molecule libraries to screen for pharmacological tools to perturb basic biological processes. This review will focus on the applications of these technologies to the field of microbiology.

Bacterial Proteins↗
PubMed2004

Discovery and characterization of novel small molecule inhibitors of human Cdc25B dual specificity phosphatase.

Cdc25A and Cdc25B dual-specificity phosphatases are key regulators of cell cycle transition and proliferation. They have oncogenic properties and are overexpressed in many human tumors. Because selective Cdc25 phosphatase inhibitors would be valuable biological tools and possible therapeutic agents, we have assayed a small molecule library for in vitro inhibition of Cdc25. We now report the identification of two new structurally distinct classes of Cdc25 inhibitors with cellular activity. The cyclopentaquinoline 3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-4,8-dicarboxylic acid (5661118) and the naphthofurandione 3-benzoyl-naphtho[1,2-b]furan-4,5-dione (5169131) had in vitro IC50 values of 2.5 to 11 microM against recombinant Cdc25 and were less potent inhibitors of other phosphatases. Unlike 5661118, 5169131 caused reversible inhibition of Cdc25B and displayed competitive inhibitor kinetics. No growth inhibitory activity was seen with 5661118, whereas 10 to 30 microM 5169131 caused G1/S and G2/M arrest. We also found that 5169131 inhibited human PC-3 prostate and MDA-MB-435 breast cancer cell proliferation. Concentration-dependent Tyr15 hyperphosphorylation was seen on cyclin-dependent kinase with a 1-h 5169131 treatment, consistent with Cdc25 inhibition. Cells resistant to DNA toposiomerase II inhibitors were as sensitive to 5169131 as parental cells, indicating that this quinone compound does not inhibit topoisomerase II in vivo. Molecular modeling was used to predict a potential interaction site between the inhibitor and Cdc25B and to provide insights as to the molecular origins of the experimental observations. Based on its kinetic profile and cellular activity, we suggest that 5169131 could be an excellent tool for further studies on the cellular roles of Cdc25.

Cell Cycle↗
PubMed2006

Differential targeting of Gbetagamma-subunit signaling with small molecules.

G protein betagamma subunits have potential as a target for therapeutic treatment of a number of diseases. We performed virtual docking of a small-molecule library to a site on Gbetagamma subunits that mediates protein interactions. We hypothesized that differential targeting of this surface could allow for selective modulation of Gbetagamma subunit functions. Several compounds bound to Gbetagamma subunits with affinities from 0.1 to 60 muM and selectively modulated functional Gbetagamma-protein-protein interactions in vitro, chemotactic peptide signaling pathways in HL-60 leukocytes, and opioid receptor-dependent analgesia in vivo. These data demonstrate an approach for modulation of G protein-coupled receptor signaling that may represent an important therapeutic strategy.

Analgesics↗
PubMed2004

Affinity capillary electrophoresis for the screening of novel antimicrobial targets.

The increasing number of multiantibiotic-resistant organisms, including methicillin-resistant Staphylococcus aureus (MRSA), requires the development of novel chemotherapies that are structurally distinct and exempt from current resistance mechanisms. Bioinformatics data mining of microbial genomes has revealed numerous previously unexploited essential open reading frames (ORFs) of unknown biochemical function. The potential of these proteins as screening targets is not readily apparent because most screening technologies rely on knowledge of biological function. To address this problem, the authors employed affinity capillary electrophoresis (ACE) to identify antimicrobial compounds that bound the novel target YihA. Screening a small-molecule library of 44,000 compounds initially identified 115 binders, of which 76% were confirmed. Furthermore, the ACE assay distinguished diverse compounds that possessed drug-like properties and antimicrobial activity against drug-resistant clinical isolates. These data validate ACE as a valuable tool for the fast, efficient detection of specific binding molecules that possess biological activity.

Anti-Bacterial Agents↗
PubMed2000

Polymer-supported reagents and catalysts: recent advances in synthetic applications.

The current surge in parallel array synthesis for the production of small molecule libraries has generated keen interest in the application of solid-supported reagents and catalysts in solution-phase chemistry. The strategy assimilates the advantages of product isolation and purification of solid-phase organic synthesis with the flexible choice of chemistry from the vast repertoire of solution-phase organic reactions. This review summarizes the significant recent advances in the application of polymer-bound reagents and catalysts in solution-phase synthesis of organic molecules. Multi-step reaction sequences employing sequential use of polymer-supported reagents are also discussed. In view of the earlier review publications on this topic, only the recent literature covering 1998 and 1999 is included.

Combinatorial Chemistry Techniques↗
PubMed2005

Cyclic peptides, a chemical genetics tool for biologists.

The genomics era has presented scientists with an overwhelming amount of sequence information that encodes for a continuously expanding array of metabolic pathways and regulatory networks. In order to unravel this elaborate maze, effective approaches need to be pioneered, and cyclic peptides represent an exciting new tool that can aid this challenging task. There are considerable benefits to cyclic peptides including enhanced protein binding affinity and metabolic stability, but perhaps most important is the ability to biologically synthesize them using intein chemistry. This capacity for genetic encoding allows biologists access to diverse, intracellular small-molecule libraries that can be tailored for numerous chemical genetics approaches. An increasing number of molecular strategies are being developed to aid in the implementation of cyclic peptides in forward and reverse genetics experiments, which will provide researchers with a powerful toolbox to complement existing genomic methodologies.

Animals↗
PubMed2004

DNA display II. Genetic manipulation of combinatorial chemistry libraries for small-molecule evolution.

Biological in vitro selection techniques, such as RNA aptamer methods and mRNA display, have proven to be powerful approaches for engineering molecules with novel functions. These techniques are based on iterative amplification of biopolymer libraries, interposed by selection for a desired functional property. Rare, promising compounds are enriched over multiple generations of a constantly replicating molecular population, and subsequently identified. The restriction of such methods to DNA, RNA, and polypeptides precludes their use for small-molecule discovery. To overcome this limitation, we have directed the synthesis of combinatorial chemistry libraries with DNA "genes," making possible iterative amplification of a nonbiological molecular species. By differential hybridization during the course of a traditional split-and-pool combinatorial synthesis, the DNA sequence of each gene is read out and translated into a unique small-molecule structure. This "chemical translation" provides practical access to synthetic compound populations 1 million-fold more complex than state-of-the-art combinatorial libraries. We carried out an in vitro selection experiment (iterated chemical translation, selection, and amplification) on a library of 10(6) nonnatural peptides. The library converged over three generations to a high-affinity protein ligand. The ability to genetically encode diverse classes of synthetic transformations enables the in vitro selection and potential evolution of an essentially limitless collection of compound families, opening new avenues to drug discovery, catalyst design, and the development of a materials science "biology."

Catalysis↗