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At least 199 records · Page 11Linked to original sources
PubMed2000

Statistical relationships among docking scores for different protein binding sites.

This report describes the existence of statistical relationships among scores computed with the DOCK program for a library of small molecules and a panel of protein binding sites. Multivariate relationships are observed in docking scores computed for a constant set of ligands in different binding sites of proteins that are dissimilar in structure and function. The structural basis for the correlations found among scores is analyzed in terms of size, shape and charge characteristics of the binding sites considered. Interestingly, these results parallel a growing body of evidence demonstrating the promiscuous behavior of small molecules in their interactions with macromolecules that could have an impact in future efforts in drug design.

Binding Sites↗
PubMed2005

Metabolomics in human nutrition: opportunities and challenges.

Metabolomics has been widely adopted in pharmacology and toxicology but is relatively new in human nutrition. The ultimate goal, to understand the effects of exogenous compounds on human metabolic regulation, is similar in all 3 fields. However, the application of metabolomics to nutritional research will be met with unique challenges. Little is known of the extent to which changes in the nutrient content of the human diet elicit changes in metabolic profiles. Moreover, the metabolomic signal from nutrients absorbed from the diet must compete with the myriad of nonnutrient signals that are absorbed, metabolized, and secreted in both urine and saliva. The large-bowel microflora also produces significant metabolic signals that can contribute to and alter the metabolome of biofluids in human nutrition. Notwithstanding these possible confounding effects, every reason exists to be optimistic about the potential of metabolomics for the assessment of various biofluids in nutrition research. This potential lies both in metabolic profiling through the use of pattern-recognition statistics on assigned and unassigned metabolite signals and in the collection of comprehensive data sets of identified metabolites; both objectives have the potential to distinguish between different dietary treatments, which would not have been targeted with conventional techniques. The latter objective sets out a well-recognized challenge to modern biology: the development of libraries of small molecules to aid in metabolite identification. The purpose of the present review was to highlight some early challenges that need to be addressed if metabolomics is to realize its great potential in human nutrition.

Diet↗
PubMed2004

Identification and characterization of a novel and specific inhibitor of the ataxia-telangiectasia mutated kinase ATM.

The serine/threonine protein kinase ATM signals to cell cycle and DNA repair components by phosphorylating downstream targets such as p53, CHK2, NBS1, and BRCA1. Mutation of ATM occurs in the human autosomal recessive disorder ataxia-telangiectasia, which is characterized by hypersensitivity to ionizing radiation and a failure of cells to arrest the cell cycle after the induction of DNA double-strand breaks. It has thus been proposed that ATM inhibition would cause cellular radio- and chemosensitization. Through screening a small molecule compound library developed for the phosphatidylinositol 3'-kinase-like kinase family, we identified an ATP-competitive inhibitor, 2-morpholin-4-yl-6-thianthren-1-yl-pyran-4-one (KU-55933), that inhibits ATM with an IC(50) of 13 nmol/L and a Ki of 2.2 nmol/L. KU-55933 shows specificity with respect to inhibition of other phosphatidylinositol 3'-kinase-like kinases. Cellular inhibition of ATM by KU-55933 was demonstrated by the ablation of ionizing radiation-dependent phosphorylation of a range of ATM targets, including p53, gammaH2AX, NBS1, and SMC1. KU-55933 did not show inhibition of UV light DNA damage induced cellular phosphorylation events. Exposure of cells to KU-55933 resulted in a significant sensitization to the cytotoxic effects of ionizing radiation and to the DNA double-strand break-inducing chemotherapeutic agents, etoposide, doxorubicin, and camptothecin. Inhibition of ATM by KU-55933 also caused a loss of ionizing radiation-induced cell cycle arrest. By contrast, KU-55933 did not potentiate the cytotoxic effects of ionizing radiation on ataxia-telangiectasia cells, nor did it affect their cell cycle profile after DNA damage. We conclude that KU-55933 is a novel, specific, and potent inhibitor of the ATM kinase.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2003

Fluorescence polarization competition assay: the range of resolvable inhibitor potency is limited by the affinity of the fluorescent ligand.

For the development of fluorescence polarization (FP) competition assays, there is a widespread belief that tight-binding fluorescent ligands should be avoided to identify inhibitors of low or intermediate potency in the screening of small-molecule compound libraries. It is demonstrated herein that this statement is a misconception; in fact, the higher the affinity of the fluorescent ligand, the wider the range of inhibitor potency that can be resolved. An approximate estimate for the low end of inhibitor K(i) values that can be resolved is the K(d) value of the fluorescent ligand. Because FP competition assays are typically conducted under nonstoichiometric titration conditions, it is suggested that a fluorescent ligand of highest affinity that also has an adequate quantum yield to satisfy such conditions be selected.

Fluorescence Polarization↗
PubMed2006

Screening for caspase-3 inhibitors: effect of a reducing agent on identified hit chemotypes.

When studying cysteinyl proteases in general and caspases in particular, it is generally accepted that a reaction buffer must contain a reducing agent to prevent essential cysteinyl groups from spontaneous oxidation. Dithiothreitol (DTT) and beta-mercaptoethanol (beta-MCE) are 2 of the most broadly used reducing agents. While screening a library of small molecules against caspase-3, the authors have found that the nature of the reducing agent used, DTT or beta-MCE, dramatically affects screening results and leads to identification of nonoverlapping hits. Screening in DTT-containing buffer revealed few novel classes of small molecules that selectively and reversibly inhibit caspase-3 but failed to identify isatin sulfonamides recently found to be potent and selective caspase-3 inhibitors (false negatives). On the other hand, screening in the presence of beta-MCE failed to identify a series of hit compounds, 1,3-dioxo-2,3-dichloro-1H-pyrrolo[3,4-c]quinolines, discovered with DTT, whereas isatin sulphonamides in these conditions exhibited strong caspase-3 inhibition. In this work, the authors show that thiol-containing reducing agents can affect catalytic activity of caspase-3 and modify its thermostability in a redox-potential-independent manner. The authors speculate that the differential structural modifications of caspase-3 seen with different reducing agents represent structurally different caspase-3 conformations and are responsible for its differential sensitivity to small molecules of different chemotypes. Hence, selection of the reducing agent may dramatically affect the quality of high-throughput screening campaigns.

Caspase 3↗
PubMed2006

Screening the receptorome.

The term 'receptorome' is now being used to describe receptors, ion channels and transporters in the human genome that are potential drug targets. These proteins comprise a considerable fraction of the human genome, and include the G protein-coupled receptors, which are the targets for many medications. In this review, we summarize recent advances in the field, including the concept that the ultimate goal of drug discovery may not be the development of highly selective single-target drugs, the idea that potential side-effects can also be the goal of multi-target drug screening, and a discussion of the application of computational screening and public domain databases available to interested investigators.

Animals↗
PubMed2004

Identification of vasoactive nonpeptidic positive and negative modulators of adrenomedullin using a neutralizing antibody-based screening strategy.

Adrenomedullin (AM) is a peptide hormone implicated in blood pressure regulation and in the pathophysiology of important diseases, such as hypertension, cancer, and diabetes. However, nonpeptidic modulators of this peptide that could be used to clinically regulate its actions are not available. We present here an efficient new method to screen a large library of small molecules. This technology was applied to the identification of positive and negative modulators of AM function. A two-tier screening strategy was developed in which the first screening entails disruption of the interaction between the peptide and a neutralizing monoclonal antibody. Selected compounds were further characterized by their ability to modulate second messengers in cells containing specific AM receptors. A parallel screen against gastrin-releasing peptide selected a different subset of molecules, confirming the specificity of the screening method. Identified AM-positive regulators reduced blood pressure in vivo, whereas AM-negative regulators mediated vasoconstriction, as predicted by the vasodilatory activity of AM. Binding of the small molecules to immobilized AM was demonstrated by surface plasmon resonance assays, with K(d) values ranging from 7.76 x 10(-9) to 4.14 x 10(-6) m. Preclinical development of AM modulators may result in useful drugs for the prevention and treatment of hypertension, cancer, and diabetes.

Adrenomedullin↗
PubMedDate not supplied

High-throughput identification of phage-derived imaging agents.

The use of phage-displayed peptide libraries is a powerful method for selecting peptides with desired binding properties. However, the validation and prioritization of "hits" obtained from this screening approach remains challenging. Here, we describe the development and testing of a new analysis method to identify and display hits from phage-display experiments and high-throughput enzyme-linked immunosorbent assay screens. We test the method using a phage screen against activated macrophages to develop imaging agents with higher specificity for active disease processes. The new methodology should be useful in identifying phage hits and is extendable to other library screening methods such as small-molecule and nanoparticle libraries.

Affinity Labels↗
PubMed2005

Library versus library recognition and inhibition of the HIV-1 Nef allelome.

Rapid evolution of drug-resistant viruses renders essentially all small-molecule antiviral treatments ineffective. We demonstrate an in vitro library versus library approach to identify small molecules targeting a broad spectrum of HIV-1 Nef protein variants. The technique could provide more effective antiviral therapies. First, a library of clinically derived Nef allelic variants, termed an allelome, was selected for function by binding to Nef ligands p53, actin, or p56lck. Next, a library of small-molecule inhibitors challenged the Nef allelome in competition assays. In contrast to single-variant inhibition, structurally simpler molecules could better inhibit the Nef allelome. Additionally, Nef sequences selected for binding to p53 resembled sequences from patients with a rapid progression to AIDS phenotype. Thus, the allelome versus small-molecule library approach offers a route for improving antiviral drug discovery and elucidating fundamental mechanisms of viral pathogenesis and resistance.

Binding Sites↗
PubMed2006

Self-renewal of embryonic stem cells by a small molecule.

A cell-based screen of chemical libraries was carried out to identify small molecules that control the self-renewal of ES cells. A previously uncharacterized heterocycle, SC1, was discovered that allows one to propagate murine ES cells in an undifferentiated, pluripotent state under chemically defined conditions in the absence of feeder cells, serum, and leukemia inhibitory factor. Long-term SC1-expanded murine ES cells can be differentiated into cells of the three primary germ layers in vitro and also can generate chimeric mice and contribute to the germ line in vivo. Biochemical and cellular experiments suggest that SC1 works through dual inhibition of RasGAP and ERK1. Molecules of this kind may not only facilitate practical applications of stem cells in research and therapy, but also provide previously undescribed insights into the complex biology of stem cells.

Animals↗
PubMed2003

Biological mechanism profiling using an annotated compound library.

We present a method for testing many biological mechanisms in cellular assays using an annotated library of 2036 small organic molecules. This annotated compound library represents a large-scale collection of compounds with diverse, experimentally confirmed biological mechanisms and effects. We found that this chemical library is (1) more structurally diverse than conventional, commercially available libraries, (2) enriched in active compounds in a tumor cell viability assay, and (3) capable of generating hypotheses regarding biological mechanisms underlying cellular processes. We elucidated biological mechanisms relevant to the antiproliferative activity of 85 compounds from this library that were selected using a high-throughput cell viability screen. We developed a novel automated scoring system for identifying statistically enriched mechanisms among such a subset of compounds. This scoring system can identify both previously known and potentially novel antiproliferative mechanisms.

Cell Line, Tumor↗
PubMed2002

An automated LC method for the small-scale purification of organic molecules derived from combinatorial libraries.

This paper presented the development of an automated HPLC small-scale purification method for single bead compounds derived from combinatorial libraries. The method was found to produce higher and more consistent recoveries of purified compounds as compared to conventional manual HPLC purification. Using the manual method, the average percentage recovery of one synthetic compound was determined to be 24% and the coefficients of variation (C.V.%) of recovery were found to be greater than 38%. Using the automated system, the average percentages recovery of a standard compound at 600 and 1000 micromol l(-1) were determined to be 72.63+/-10.17% and 81.34+/-4.39%, respectively. This represented an approximate 3-folds increase in percentage recovery compared to that of the manual small-scale purification process. It was also found that the C.V.% of recovery were less than 15% at both concentration levels. The development of this automated method was found to be straightforward. The importance and implications of this study were discussed.

Chromatography, High Pressure Liquid↗
PubMed1998

Encoded chemical synthesis coupled to screening: "Pot Assay".

A variety of screening methodologies is available to identify lead compounds. Screening methods that would permit the direct use of libraries made via the Radiofrequency Encoded Combinatorial chemistry paradigm (each individual small molecule in the library is presented separately on an individual encoded support) have the potential to diminish burdensome steps in this process. Here we report on our studies leading to such a direct method, which we have termed a Pot Assay. Pot Assay is a multiplex assay, which simultaneously measures specific binding of a number of ligands to at least one target. Pot Assay uses specific radiofrequency signals to decode compounds that are high affinity binders. We validated this approach by evaluating the interaction of biotin and its analogs with labeled streptavidin. This report introduces Pot Assay as a rapid, simple, sensitive and accurate format for identifying active members of libraries synthesized on solid supports. The success of this study demonstrates the power of coupling Radiofrequency Encoded Combinatorial chemistry and screening. This assay format may be applied to a wide range of screens that are based on binding events: ligand/receptor, inhibitor/enzyme, antigen/antibody, protein/protein, DNA/protein, and RNA/DNA.

Alkaline Phosphatase↗
PubMed1995

Search strategies for applied molecular evolution.

A new approach to drug discovery is based on the generation of high diversity libraries of DNA, RNA, peptides or small molecules. Search of such libraries for useful molecules is an optimization problem on high-dimensional molecular fitness landscapes. We utilize a spin-glass-like model, the NK model, to analyze search strategies based on pooling, mutation, recombination and selective hill-climbing. Our results suggest that pooling followed by recombination and/or hill-climbing finds better candidate molecules than pooling alone on most molecular landscapes. Our results point to new experiments to assess the structure of molecular fitness landscapes and improve current models.

Drug Design↗
PubMed1996

Isotope or mass encoding of combinatorial libraries.

BACKGROUND: Combinatorial chemistry using solid-phase synthesis is a rapidly developing technology that can result in a significant reduction in the time required to find and optimize lead compounds. The application of this approach to traditional medicinal chemistry has led to the construction of libraries of small organic molecules on resin beads. A major difficulty in developing large combinatorial libraries is the lack of a facile encoding and decoding methodology to identify active compounds. RESULTS: Several encoding schemes are described which use the ability of mass spectrometry to ascertain isotopic distributions. Molecular tags are attached to resin beads in parallel or on the linker used for chemical library synthesis. The tags are encoded via a controlled ratio of a number of stable isotopes on the tagging molecules, and range from a single to a complex isotopic distribution. CONCLUSIONS: A novel coding scheme is described that is useful for the generation of large encoded combinatorial libraries. The code can be cleaved after assay and analyzed by mass spectrometry in an automated fashion. An important element of the combinatorial discovery process is the ability to extract the structure-activity relationship (SAR) information made available by library screening. The speed and sensitivity of the mass-encoding scheme has the potential to determine the full SAR for a given library.

Chemistry, Organic↗
PubMedDate not supplied

Selection of streptavidin binders from a DNA-encoded chemical library.

DNA-encoded libraries of small organic molecules facilitate the construction of large, encoded self-assembling chemical libraries for the identification of high-affinity binders to protein targets. We have constructed a library of 477 chemical compounds, coupled to 48mer-oligonucleotides, each containing a unique six-base sequence serving as "bar-code" for the identification of the chemical moiety. The functionality of the library was confirmed by selection and amplification of both high- and low-affinity binding molecules specific to streptavidin.

Animals↗
PubMed2005

Dissection of melanogenesis with small molecules identifies prohibitin as a regulator.

Bioactive compounds can be used to selectively modulate gene function. We utilized a chemical genetic approach to dissect the mammalian pigmentation pathway and identify protein regulators. We screened a tagged library of 1170 small molecules in a cell-based assay and discovered a class of pigment-enhancing chemicals. From this class we characterized the small molecule melanogenin. Using melanogenin bound to an affinity matrix and amino acid sequencing, we identified the mitochondrial protein, prohibitin, as an intracellular binding target. Studies employing siRNA demonstrate that prohibitin is required for melanogenin to exert its propigmentary effects and reveal an unsuspected functional role for this protein in melanin induction. This represents a mechanism by which propigmentary signals are transduced and ultimately provides a potential target for the treatment of pigmentary disorders.

Amino Acid Sequence↗