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PubMed2005

Rational selection of structurally diverse natural product scaffolds with favorable ADME properties for drug discovery.

Natural product analogs are significant sources for therapeutic agents. To capitalize efficiently on the effective features of naturally occurring substances, a natural product-based library production platform has been devised at Aurigene for drug lead discovery. This approach combines the attractive biological and physicochemical properties of natural product scaffolds, provided by eons of natural selection, with the chemical diversity available from parallel synthetic methods. Virtual property analysis, using computational methods described here, guides the selection of a set of natural product scaffolds that are both structurally diverse and likely to have favorable pharmacokinetic properties. The experimental characterization of several in vitro ADME properties of twenty of these scaffolds, and of a small set of designed congeners based upon one scaffold, is also described. These data confirm that most of the scaffolds and the designed library members have properties favorable to their utilization for creating libraries of lead-like molecules.

Alkaloids↗
PubMed2004

Development of small molecules that mimic the binding of omega-conotoxins at the N-type voltage-gated calcium channel.

Cone snails (Conidae) are marine predators with some extraordinary features. Their venom contains a hundred or more peptides that target numerous ion channels and receptors in mammals, including several that are involved in disease. omega-Conotoxins from fish hunting snails are 24-27 residue peptides with a rigid 4-loop cysteine framework that target the N-type voltage-gated calcium channel (VGCC). Two omega-conotoxins, MVIIA and CVID are currently in clinical development for chronic pain management (Ziconotide or Prialt, and AM336, respectively). In an attempt to develop small molecule equivalents of CVID, we defined the Calpha-Cbeta vectors of the residues believed to be important for binding to the N-type VGCC. Using these vectors, we undertook a virtual screening of virtual libraries approach to identify compounds that matched the pharmacophore. Cyclic pentapeptides containing residues of loop 2 of CVID, with one or more being a D-amino acid were designed and synthesised and were found to be active at the N-type VGCC (IC50 approximately 20 microM). Agreeing with the specificity profile of CVID, molecules were inactive at the P/Q-type VGCC.

Animals↗
PubMed2005

Protein "fingerprinting" in complex mixtures with peptoid microarrays.

We report here that microarrays comprised of several thousand peptoids (oligo-N-substituted glycines) are useful tools for the identification of proteins via a "fingerprinting" approach. By using maltose-binding protein, glutathione S-transferase, and ubiquitin, a specific and highly reproducible pattern of binding was observed when fluorescently labeled protein was hybridized to the array. A similar pattern was obtained when binding of an unlabeled protein to the array was visualized by secondary hybridization of a labeled antibody against that protein, showing that native proteins can be identified without the requirement for prior chemical labeling. This work suggests that small-molecule microarrays might be used for more complex fingerprinting assays of potential diagnostic value.

Antibodies↗
PubMed2000

A CDC6 protein-binding peptide selected using a bacterial two-hybrid-like system is a cell cycle inhibitor.

Peptides or small molecules able to modulate protein-protein interactions hold promise as tools with which to probe and manipulate biological pathways. An important issue in this nascent field is to evaluate different methods with which to search libraries for molecules that modulate the function of specific target proteins. One strategy is to screen libraries for molecules that bind specifically to a protein known to be critical in the pathway of interest, with the expectation that the molecules isolated will recognize regions of the target protein important for its function and thereby exhibit biological activity. Here, a peptide library was screened using a two-hybrid-like system for molecules able to bind human CDC6 protein (CDC6p), required for the initiation of DNA replication in eukaryotic cells. From a collection of over a million peptides, a single species that exhibited good affinity and specificity for binding CDC6p was obtained. When expressed in human cells, the peptide inhibited cell cycle progression and exhibited other properties expected of a CDC6p inhibitor. This approach, which does not require detailed knowledge of the mechanism of action of a protein target, may be generally useful for isolating peptides capable of manipulating biological pathways.

Amino Acid Sequence↗
PubMed2004

Cloning single-chain antibody fragments (scFv) from hybridoma cells.

Despite the availability of antibody libraries for the selection of receptor molecules, the large number of established and well-characterized hybridoma lines still represent a useful source for recombinant antibody genes. This protocol describes the PCR amplification, cloning, and a small-scale expression test for the generation of scFv fragments from hybridoma cell lines. Particular emphasis was placed on frequently observed problems and pitfalls of this method.

Animals↗
PubMed2003

Small molecule regulation of phosphatase-dependent cell signaling pathways.

Small molecules provide exceptionally useful tools for probing signaling targets relevant for cancer and stem cell differentiation. In contrast to genetic approaches, the application of small molecules generally offers a graded and reversible disruption of a particular pathway. The vast array of theoretical chemical entities that exist in the chemical universe are now becoming available through the production and distribution of chemical libraries generated by both traditional and combinatorial methods, which are suitable for pharmacological use. Convenient and inexpensive cell-free and cell-based assays can be used to identify chemicals that exhibit desirable antisignaling properties. We illustrate a model of how agents targeted against signaling macromolecules involved in cancer, namely dual specificity protein phosphatases, can be identified.

Antineoplastic Agents↗
PubMed2001

Macromolecular therapeutics: emerging strategies for drug discovery in the postgenome era.

The postgenome era offers a plethora of potential therapeutic targets. Many of these targets will be addressable using small organic molecules as drug candidates. However, certain aspects of cell function, particularly those that rely on protein-protein or protein-nucleic acid interactions, will be difficult to influence using small molecules. Thus, the possibility of using highly specific macromolecules as potential therapeutic agents is an intriguing concept. Recent developments in several areas of research have brought this possibility closer to fruition. Peptide and nucleic acid combinatorial libraries allow the generation of novel molecules having exquisite selectivity. Structural information and molecular modeling also contribute to the design of new macromolecules with therapeutic potential. Perhaps most importantly, approaches for delivering macromolecules into the cell interior have been developed and applied with considerable success. Thus, the therapeutic use of macromolecules, including oligonucleotides, peptides, and proteins, may be an idea whose time has come.

Animals↗
PubMed2003

Simultaneous prediction of binding capacity for multiple molecules of the HLA B44 supertype.

We selected for study a set of B44-supertype molecules collectively represented in >40% of the individuals in all major ethnicities (B*1801, B*4001, B*4002, B*4402, B*4403, and B*4501). The peptide-binding specificity of each molecule was characterized using single amino acid substitution analogues and nonredundant peptide libraries. In all cases, only peptide ligands with glutamic acid in position 2 were preferred. At the C terminus, each allele was associated with a unique but broad pattern of preferences, but all molecules tolerated hydrophobic/aliphatic (leucine, isoleucine, valine, methionine), aromatic (tyrosine, phenylalanine, tryptophan), and small (alanine, glycine, threonine) residues. Secondary anchor motifs were also defined for all molecules. Together, these features were used to define a B44 supermotif and a novel algorithm for calculating degeneracy scores that can be used to predict B44-supertype degenerate binders. Approximately 90% of the peptides with a B44 supermotif degeneracy score of >10 bound at least three of the six B44-supertype molecules studied with high affinity. Finally, a number of peptides derived from hepatitis B and C viruses, HIV, and Plasmodium falciparum have been identified that have degenerate B44 supertype-binding capacity. Taken together, these findings have important implications for epitope-based approaches to vaccination, immunotherapy, and the monitoring of immune responses.

Algorithms↗
PubMed2001

Anti-HLA-DR/anti-DOTA diabody construction in a modular gene design platform: bispecific antibodies for pretargeted radioimmunotherapy.

Recombinant immunoglobulin libraries of single chain molecules (sc) from the variable domains of antibody light and heavy chains (Fv), have great promise for new approaches to radioimmunotherapy (RIT). However, creating and evaluating scFv from diverse sources is time consuming and differences in molecular format can influence in vitro and in vivo characteristics. Furthermore, scFv do not have optimal characteristics for targeting therapy to tumor because of their small size and univalent binding. Diabody molecules at least twice the size of scFv are better for RIT because bivalent and bispecific molecules can be constructed. A polymerase chain reaction (PCR) based primer system was created to easily convert scFv genes into a diabody gene format, once they have been placed into pCANTAB 5E, a readily available vector. The primer system for this diabody gene platform was developed and tested by constructing an anti-lymphoma/anti-chelate, bispecific diabody (anti-HLA-DR/anti-DOTA). Two mouse scFv libraries were screened for reactive clones using recombinant phage display techniques. Selected mouse anti-HLA-DR and anti-DOTA scFv genes were combined, ligated into the pCANTAB 5E vector that co-expressed these self-assembling scFv in E. coli as two mismatched nonlinked pairs (VHA-link-VLB; VHB-link-VLA). The diabody protein that was purified from periplasm had the expected molecular characteristics when analyzed by sequencing, chromatography, electrophoresis and Western blot. This modular gene design platform provides methodology for easy and rapid creation of diabody molecules from diverse scFv libraries. Diabodies from various scFv can easily be produced, thereby facilitating comparative preclinical studies en route to development of new tumor targeting molecules.

Antibodies, Bispecific↗
PubMed1991

The erythropoietin receptor gene: cloning and identification of multiple transcripts in an erythroid cell line OCIM1.

The hormone erythropoietin is necessary for the maintenance, generation, and maturation of erythroid cells. This kidney-derived glycoprotein binds to cell-surface receptors, mediating both the proliferation and differentiation of erythroid cells. We have characterized 16 human erythropoietin receptor cDNA clones from two cDNA libraries prepared from OCIM1 poly(A)-containing mRNA. Of these 16 isolates, many showed evidence of incorrect RNA splicing events; others contained small deletions yielding multiple transcripts that may play a role in their erythroleukemic state. The human erythropoietin receptor, as deduced from the sequence of these clones, encodes a 508-amino-acid molecule, including a 24-residue signal peptide, a 226-amino-acid external domain, a transmembrane spanning region of 22 amino acids, and a cytoplasmic domain of 236 amino acids. There is an 80% overall homology between the human and murine erythropoietin receptor sequences.

Amino Acid Sequence↗
PubMedDate not supplied

T cell epitope characterization in tandemly repetitive Trypanosoma cruzi B13 protein.

Proteins containing tandemly repetitive sequences are present in several immunodominant protein antigens in pathogenic protozoan parasites. The tandemly repetitive Trypanosoma cruzi B13 protein is recognized by IgG antibodies from 98% of Chagas' disease patients. Little is known about the molecular mechanisms that lead to the immunodominance of the repeated sequences, and there is limited information on T cell epitopes in such repetitive antigens. We finely characterized the T cell recognition of the tandemly repetitive, degenerate B13 protein by T cell lines, clones and PBMC from Chagas' disease cardiomyopathy (CCC), asymptomatic T. cruzi infected (ASY) and non-infected individuals (N). PBMC proliferative responses to recombinant B13 protein were restricted to individuals bearing HLA-DQA1*0501(DQ7), -DR1, and -DR2; B13 peptides bound to the same HLA molecules in binding assays. The HLA-DQ7-restricted minimal T cell epitope [FGQAAAG(D/E)KP] was identified with an overlapping combinatorial peptide library including all B13 sequence variants in T. cruzi Y strain B13 protein; the underlined small residues GQA were the major HLA contact residues. Among natural B13 15-mer variant peptides, molecular modeling showed that several variant positions were solvent (TCR)-exposed, and substitutions at exposed positions abolished recognition. While natural B13 variant peptide S15.9 seems to be the immunodominant epitope for Chagas' disease patients, S15.4 was preferentially recognized by CCC rather than ASY patients, which may be pathogenically relevant. This is the first thorough characterization of T cell epitopes of a tandemly repetitive protozoan antigen and may suggest a role for T cell help in the immunodominance of protozoan repetitive antigens.

Amino Acid Sequence↗
PubMed2004

Building predictive models for protein tyrosine phosphatase 1B inhibitors based on discriminating structural features by reassembling medicinal chemistry building blocks.

A new approach to predicting the biological activity of small molecule pharmaceutics is demonstrated. Structural features of medicinal chemistry building blocks are used as 2-D molecular descriptors. These descriptors include predefined structural features and macrostructures obtained from a supervised process in which features in the core library are reassembled to provide larger features that strongly differentiate the desired biological response variable. Chemical features derived in this manner can serve as predictor variables for diverse modeling algorithms, and application using partial least squares techniques is demonstrated here. Models are presented for inhibition by benzofuran and benzothiophene biphenyl analogues of protein tyrosine phosphatase 1B (PTP1B), a target for insulin-resistant disease states. Results are compared to models for PTP1B inhibitors available in the literature based on CoMFA-related techniques and 3-D molecular descriptors.

Models, Molecular↗
PubMed2005

Discovery of positive allosteric modulators of metabotropic glutamate receptor subtype 5 (mGluR5).

This review provides an overview of the drug discovery process used to identify, develop and characterize the first positive allosteric modulators of the metabotropic glutamate receptor (mGluR) subtype 5 (mGluR5). Discovery and optimization of three series of positive allosteric modulators are described, each using different approaches. The symmetric benzaldazine series was discovered and optimized from samples already existing in our sample collection without an active synthetic program to further elucidate SAR. This series yielded a family of highly selective pharmacological tools that produced positive, negative and neutral allosteric modulation of mGluR5 activity. The original compound in the benzamide series was discovered from screening and this series was optimized using an iterative library synthesis approach to explore SAR in each of three regions of the molecule. This series produced more potent positive allosteric mGluR5 modulators than the benzaldazine series which could be evaluated for their effect on mGluR5 in brain slice electrophysiological studies. The pyrazole series used a fragment library approach based on small structural motives from the benzamide series to discover lead compounds and establish SAR. This series produced still more potent positive allosteric mGluR5 modulators with improved pharmacokinetic and physical properties. These modulators showed efficacy in animal behavioral models in which other antipsychotic drugs were active. Evaluation of assay data in mathematical models of allosterism to constrain possible mechanisms of action is briefly discussed. Other reviews of this emerging field with different emphases have been published recently [1-3].

Allosteric Regulation↗
PubMed2000

Chemical approaches to control gene expression.

A current goal in molecular medicine is the development of new strategies to interfere with gene expression in living cells in the hope that novel therapies for human disease will result from these efforts. This review focuses on small-molecule or chemical approaches to manipulate gene expression by modulating either transcription of messenger RNA-coding genes or protein translation. The molecules under study include natural products, designed ligands, and compounds identified through functional screens of combinatorial libraries. The cellular targets for these molecules include DNA, messenger RNA, and the protein components of the transcription, RNA processing, and translational machinery. Studies with model systems have shown promise in the inhibition of both cellular and viral gene transcription and mRNA utilization. Moreover, strategies for both repression and activation of gene transcription have been described. These studies offer promise for treatment of diseases of pathogenic (viral, bacterial, etc.) and cellular origin (cancer, genetic diseases, etc.).

Animals↗
PubMed1999

Parallel solution synthesis of a "Carbohybrid" library designed to inhibit galactose-binding proteins.

Parallel solution S-alkylations of a 1-thio-beta-D-galactopyranoside derivative with Michael acceptors and alpha-chloroketones, followed by ketone reductions, reductive aminations, and acylations were developed to yield a library of 1-thio-beta-D-galactopyranosides carrying small and diverse polar-neutral, hydrophobic, aromatic, cationic, or anionic non-carbohydrate aglycon structures. Screening of the library against a panel of galactose recognizing plant lectins revealed microM inhibitors of toxin A of A. precatorius superior to the reference ligands lactose and N-acetyl lactosamine. Such small, monosaccharide based inhibitors are attractive lead-molecules for therapeutic development, since they are low-molecular, hydrolytically stable and more hydrophobic than natural oligosaccharides.

Calcium-Binding Proteins↗
PubMed1998

Ion transporters and receptors in cDNA libraries from lens and cornea epithelia.

PURPOSE: To construct tissue-specific cDNA libraries containing copies of genes of ion transporting and receptor proteins in lens and cornea epithelia. To screen the libraries for the cDNA of these molecules and to deduce the protein sequence from the cDNA sequence. METHOD: Lens and corneal cDNA libraries are not commercially available and are difficult to prepare from microdissected single animal eyes or cadaver human eyes due to the small amount of tissue. To overcome these problems, we refined an approach for preparing high efficiency, non-PCR amplified plasmid cDNA libraries, where only nanogram quantities of poly (A) RNA are required. Plasmids were electrotransformed into DH10B bacteria and routinely yielded >10(6) independent colonies with typically >90% recombinants. Randomly selected colonies were subjected to colony PCR analysis to determine the libraries average insert size (typically approximately 1-kb). Primers to known genes were used in PCR amplification to check for representation of the genes in the cDNA libraries. RESULTS: Using libraries from rabbit cornea epithelium and endothelium, cultured human lens epithelium, and alphaTN4 cells, we have found the libraries to contain the cDNA for 3 common housekeeping proteins expected to be at high copy numbers in all cells. In addition, we identified 22 rare proteins and for many we determined the complete coding regions. These include K+ channels, Cl- channels, Ca++ channels, Na+ channels, three exchangers, the Na+-HCO3- cotransporter, and three kinds of receptors. CONCLUSION: Cornea and lens libraries have been constructed from single cornea or lens preparations. The libraries often contain the cDNA sequences for ion transporting and receptor proteins which are expected to be relatively rare in these cells. Many of these cDNA's have now been sequenced and the encoded proteins identified.

Animals↗
PubMed1987

A library of trimethylguanosine-capped small RNAs in Physarum polycephalum.

We have constructed a cDNA library for the trimethylguanosine-capped small RNAs (sRNAs) in the acellular slime mold Physarum polycephalum. Capped sRNAs were purified from total cellular RNA of vegetative microplasmodia by preparative immunoprecipitation with anti-trimethylguanosine antibody. The purified RNA was analyzed by polyacrylamide gel electrophoresis. Approx. eleven different capped sRNAs were observed with a size range of 70-204 nucleotides (nt). Based on their approximate sizes, the presence of trimethylguanosine cap, and the presence of a lupus type-Sm antigen, molecules U1-U7 (excluding U3) were identified. Further confirmation of the identity of molecule U1a was established by Northern hybridization, U4a by colony hybridization, and U6 and U7a by direct chemical sequence analysis. Purified capped sRNAs were tailed with oligo(A), and inserted into oligo(dT)-tailed plasmid pCDV1. The cDNAs were used to transform Escherichia coli strain HB101. Approx. 1.9 X 10(5) ampicillin-resistant (ApR) transformants were obtained per microgram of tailed sRNA. Dot-blot hybridization, using Physarum RNA precipitated with anti-cap antibody as a probe, indicated that approx. 94% of the ApR colonies contained recombinant DNAs. The library was screened by colony hybridization using heterologous sRNA probes. Clones hybridizing with heterologous sRNAs U1, U2, U4 and U7 were each represented in the library in approximately the same frequency as their relative abundance in the Physarum sRNA population they were derived from. The insert of one Physarum U4 clone was sequenced and was found to have 57.1% homology with nt 1-91 of the published sequence for rat U4 RNA. A 12-nt 'functional' subdomain of the rat U4 molecule was 83.3% conserved in Physarum U4.

Autoantigens↗
PubMed1998

Catalyst discovery through combinatorial chemistry.

Combinatorial chemistry and high-throughput strategies can be used to identify effective small-molecule chiral catalysts. Infrared thermography has been used recently as the means to identify the most active catalyst.

Catalysis↗