Showing papers on "Chemical library published in 2008"

A portable albumin binder from a DNA-encoded chemical library

Abstract: Albumin represents the most abundant protein in human plasma, at a concentration of 45 mgmL . To keep physiological production rates to a minimum, albumin displays a long circulatory half-life in mammals thanks to its size above the renal filtration threshold and its unique ability to interact with the neonatal FcRn receptor. Fusions of biopharmaceuticals to albumin or to albumin-binding peptides have been devised to expose the body to adequate concentrations of the therapeutic agent for a sufficiently long period of time, thus improving efficacy and reducing the number of injections. In principle, small organic albumin-binding molecules could be used as functional analogues of albumin-binding peptides. However, although many small molecules are known to bind to albumin, the success in isolating small molecules as portable albumin-binding moieties has been limited, mainly because most albumin binders (for example, ibuprofen) lose binding affinity upon chemical modification. Myristoylation of insulin has been shown to significantly prolong the circulatory half-life, but this modification is not applicable to a broader set of molecules because of its negative effect on solubility. In another example, a 4,4diphenylcyclohexyl moiety has been connected through a phosphodiester bond to the metal chelator diethylenetriaminepentaacetic acid (DTPA) for magnetic resonance imaging (MRI) applications and to short peptides. These compounds display dissociation constants (Kd) from human serum albumin in the 100 mm range and are susceptible to hydrolysis in vivo. Thus, there is a considerable scientific and biotechnological interest in the identification of small portable binders that display a stable noncovalent interaction with serum albumin. Herein, we report the discovery and characterization of a class of 4-(p-iodophenyl)butyric acid derivatives from a DNA-encoded chemical library, which display a stable noncovalent binding interaction with both mouse serum albumin (MSA) and human serum albumin (HSA). One of these portable albumin-binding moieties was used to improve the performance of the contrast agents fluorescein and GdDTPA. HSA-binding molecules were selected from a DNAencoded chemical library consisting of 619 oligonucleotidecompound conjugates carrying a six-base-pair code for identification. After selection, the DNA sequences of the enriched compounds were amplified by PCR and decoded on oligonucleotide microarrays displaying the complementary sequences (Figure 1a), normalizing the signal intensities after selection against the intensities of compounds selected on empty resin (Figure 1b). Some of the identified binding molecules were excluded from further evaluation based on being promiscuous binders or because of the high standard deviations of the signal intensities on the microarrays (64, 313, 453, 454, 619). Several of the selected molecules (428, 533, 535, 539) displayed striking structural similarities. The basic structure featured a 4-phenylbutanoic acid moiety, with different hydrophobic substituents on the phenyl ring. To obtain further insights into structure–activity relationships, DNA-modified analogues containing propanoyl or pentanoyl skeletons, and/or carrying various substituents on the phenyl ring (Figure 1c; 536, 622–632), were characterized in a radioactivity-based chromatographic albumin-binding assay, which allowed a first classification of the potential binders (Retention: 428> 539> 624> 535> 533> 536> 326> others; see the Supporting Information). The absence of retention of compounds with propanoyl (625) and penta[*] S. Tr ssel, F. Buller, Dr. F. Bootz, Dr. Y. Zhang, L. Mannocci, Dr. J. Scheuermann, Prof. Dr. D. Neri Institut f r Pharmazeutische Wissenschaften Departement f r Chemie und Angewandte Biowissenschaften ETH Z rich Wolfgang-Pauli-Strasse 10, 8093 Z rich (Switzerland) Fax: (+41)44-633-1358 E-mail: neri@pharma.ethz.ch

High-throughput sequencing allows the identification of binding molecules isolated from DNA-encoded chemical libraries

Abstract: DNA encoding facilitates the construction and screening of large chemical libraries. Here, we describe general strategies for the stepwise coupling of coding DNA fragments to nascent organic molecules throughout individual reaction steps as well as the first implementation of high-throughput sequencing for the identification and relative quantification of the library members. The methodology was exemplified in the construction of a DNA-encoded chemical library containing 4,000 compounds and in the discovery of binders to streptavidin, matrix metalloproteinase 3, and polyclonal human IgG.

Estimating Protein−Ligand Binding Affinity Using High-Throughput Screening by NMR

Abstract: Many of today’s drug discovery programs utilize high-throughput screening methods that rely on quick evaluations of protein activity to rank potential chemical leads. By monitoring biologically relevant protein-ligand interactions, NMR can provide a means to validate these discovery leads and to optimize the drug discovery process. NMR-based screens typically use a change in chemical shift or linewidth to detect a protein-ligand interaction. However, the relatively low throughput of current NMR screens and their high demand on sample requirements generally makes it impractical to collect complete binding curves to measure the affinity for each compound in a large and diverse chemical library. As a result, NMR ligand screens are typically limited to identifying candidates that bind to a protein and do not give any estimate of the binding affinity. To address this issue, a methodology has been developed to rank binding affinities for ligands based on NMR-based screens that use 1D 1 H NMR line-broadening experiments. This method was demonstrated by using it to estimate the dissociation equilibrium constants for twelve ligands with the protein human serum albumin (HSA). The results were found to give good agreement with previous affinities that have been reported for these same ligands with HSA.

Structure-based tailoring of compound libraries for high-throughput screening: discovery of novel EphB4 kinase inhibitors.

Abstract: High-throughput docking is a computational tool frequently used to discover small-molecule inhibitors of enzymes or receptors of known three-dimensional structure. Because of the large number of molecules in chemical libraries, automatic procedures to prune multimillion compound collections are useful for high-throughput docking and necessary for in vitro screening. Here, we propose an anchor-based library tailoring approach (termed ALTA) to focus a chemical library by docking and prioritizing molecular fragments according to their binding energy which includes continuum electrostatics solvation. In principle, ALTA does not require prior knowledge of known inhibitors, but receptor-based pharmacophore information (hydrogen bonds with the hinge region) is additionally used here to identify molecules with optimal anchor fragments for the ATP-binding site of the EphB4 receptor tyrosine kinase. The 21,418 molecules of the focused library (from an initial collection of about 730,000) are docked into EphB4 and ranked by force-field-based energy including electrostatic solvation. Among the 43 compounds tested in vitro, eight molecules originating from two different anchors show low-micromolar activity in a fluorescence-based enzymatic assay. Four of them are active in a cell-based assay and are potential anti-angiogenic compounds.

Identification of novel HCV RNA-dependent RNA polymerase inhibitors using pharmacophore-guided virtual screening.

Abstract: We performed pharmacophore-guided virtual screening experiments using FlexX-Pharm to identify novel inhibitors of hepatitis C virus RNA-dependent RNA polymerase. Pharmacophore model generated from our previous analysis of the binding modes as well as structure-based three-dimensional quantitative structure-activity relationship studies of aryl diketoacid analogues was used. In pharmacophore-guided virtual screening study, among 37 447 compounds in LeadQuest chemical library, 40 compounds were selected as novel candidates of hepatitis C virus RNA-dependent RNA polymerase inhibitors, and their biological activities were evaluated. Especially, T29 was chosen for further development.

A novel high-throughput screening system identifies a small molecule repressive for matrix metalloproteinase-9 expression.

Abstract: Aberrant gene expression is one of the driving forces for cancer progression and is considered an ideal target for chemical intervention. Although emerging bioluminescence reporter systems allow high-throughput searches for small molecules regulatory for gene expression, frequent silencing of reporter genes by epigenetic mechanisms hinders wide application of this drug discovery strategy. Here we report a novel system that directs the integration of a promoter-reporter construct to an open chromosomal location by Flp-mediated homologous recombination, thereby overcoming reporter-gene silencing. Using this system, we have screened more than 8000 compounds in the DIVERSet chemical library for repressors of a matrix metalloproteinase-9 (MMP-9) promoter and identified 5-methyl-2-(4-methylphenyl)-1H-benzimidazole (MPBD) inhibitory for MMP-9 gene expression. Consistent with this effect, MPBD inhibits MMP-9-dependent invasion of UMSCC-1 oral cancer cells, preosteoclast migration, and receptor activator of nuclear factor-κB ligand-induced osteoclast activity over concentration ranges that repressed MMP-9 expression. Mechanistic studies indicated that MPBD antagonizes AP-1 function by inhibiting its transactivation activity. We conclude that the Flp-mediated homologous recombination system to direct reporter integration into open chromatin regions represents a novel strategy allowing for the development of high-throughput systems screening for lead compounds targeting aberrant gene expression in cancer.

Virtual screening, identification, and biochemical characterization of novel inhibitors of the reverse transcriptase of human immunodeficiency virus type-1.

Abstract: The reverse transcriptase (RT) of human immunodeficiency virus type-1 (HIV-1) is a leading target in current antiretroviral therapy. Unfortunately, drug-resistant RT mutants evolve under the pressure of these drugs, and therefore, new anti-RT inhibitors are constantly required for HIV-1/AIDS treatment. We virtually screened a large chemical library of compounds against two crystal structures of HIV-1 RT to identify novel inhibitors. Top-scoring compounds were tested experimentally; 71 inhibited the RT-associated DNA polymerase, while several also inhibited HIV-1 pseudovirus infection in a cell-based assay. A combination of substituents from two structurally related inhibitors in a single molecule improved the inhibition efficacy. This compound strongly suppressed the RT-associated activity also protecting human lymphocytes from HIV-1 infection. RT inhibition by this compound was reversible and noncompetitive. This molecule and another structurally unrelated potent compound inhibited a known drug-resistant mutant of HIV-1 RT and affected moderately the HIV-2 RT-associated DNA polymerase. These inhibitors may serve as promising anti-HIV lead compounds.

Expression and purification of human mPGES-1 in E. coli and identification of inhibitory compounds from a drug-library.

Abstract: Human microsomal prostaglandin E synthase-1 (mPGES-1) is a membrane associated protein that catalyzes the conversion of prostaglandin H2 (PGH2) into prostaglandin E2 (PGE2). In this study, the expression of human mPGES-1 in E. coli was significantly enhanced by modifying the utility of specific codons and the recombinant mPGES-1 was efficiently purified to homogeneity. The Km and Vmax of the purified enzyme were determined and the trimeric state characterized by chemical cross-linking with glutaraldehyde. The purified mPGES-1 was used for the screening of a chemical library of bioactive or drug compounds to identify novel inhibitors, and oxacillin and dyphylline were identified as moderately inhibiting mPGES-1 with IC50 values of 100 and 200 μM, respectively. As these compounds competitively inhibited the catalysis of PGH2, their binding sites appeared to be located near the PGH2 binding pocket. [BMB reports 2008; 41(11): 808-813]

New developments in small molecules targeting p53 pathways in anticancer therapy

Abstract: The tumor suppressor p53 is frequently inactivated in a wide variety of cancers and point mutations or deletions of the p53 gene are associated with poor prognosis in cancer. About half of all human tumors carry wildtype p53 but p53 wildtype functions are often suppressed by the overexpression of murine double minute 2 (MDM2), a negative regulator of p53. Restoration of p53 functions in tumor cells, therefore, represents an attractive strategy in combating cancer and has been the focus of intensive anticancer drug discovery. One strategy is to antagonize MDM2 functions and initial success was demonstrated in vitro and in xenograft tumor models using newly discovered small molecule inhibitors and antisense oligonucleotides. The new discovery of a compound targeting SirT1 (a member of the sirtuin family) in a p53-dependent reporter screen highlighted the importance of another negative regulator of p53 and offers an additional avenue for drug discovery and research on p53-activating therapeutics. Here, we discuss the developments of p53-activating small molecules and their potential use in combination therapy with established chemotherapeutics. These small molecules were discovered from chemical library screening using biochemical assays or cellular-based assays, and/or structure-based rational drug design strategies. Drug Dev Res 69:289–296, 2008. © 2008 Wiley-Liss, Inc.

Profiling signalling pathways of the receptor activator of NF-κB ligand-induced osteoclast formation in mouse monocyte cells, RAW264.7

Abstract: Cell-based signal chemical genomics can profile the signalling pathway for certain cellular events by using a target-known chemical library. To ascertain its usefulness, the receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis in mouse monocyte/macrophage cells RAW264.7 was used as an in vitro experimental model. Of 180 target-known inhibitors/activators formatted in a 384-well plate, 8 chemicals were shown to inhibit the osteoclast formation, but 4 chemicals enhanced this process. A variety of references support, or possibly lead one to expect the effects of these 12 chemicals on the cellular process of osteoclastogenesis in RAW264.7 cells, but several signalling pathways were newly found in this study; for example, CA-074 Me inhibiting cathepsin B and nitrendipine blocking the calcium channel could have the potential to inhibit the osteoclast formation as well as bone resorption. This is a simple but very fast and powerful method of profiling the signalling pathway of certain cellular events. Signal chemical genomics could provide invaluable information for the exploration of new target signalling processes and further target-based drug discovery strategies.

Direct Deconvolution Techniques for Pool Libraries of Small Organic Molecules

Abstract: The tremendous development of combinatorial chemistry during the last few years has contributed to increase steadily the options for a chemist to synthesize a chemical library, and many excellent r...