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PubMed2002

Function first: a powerful approach to post-genomic drug discovery.

In the post-genomic era, pharmaceutical researchers must evaluate vast numbers of protein sequences and formulate novel, intelligent strategies for identifying valid targets and discovering leads against them. The identification of small molecules that selectively target proteins or protein families will be aided by knowing the function and/or the structure of the target(s). By identifying protein function first, efficiencies are gained that allow subsequent focus of resources on particular protein families of interest. This article reviews current proteomic-scale approaches to identifying function as a way of accelerating lead discovery.

Pharmacology↗

PubMed2002

In need of high-throughput behavioral systems.

One of the current major bottlenecks in drug discovery is in vivo testing of candidate drugs in behavioral paradigms in normal or genetically altered mice. This testing is essential in discovering gene function and predicting potential efficacy of CNS drugs in humans. New efforts in the biotech community aim to alleviate this bottleneck by developing higher-throughput systems of behavioral, neurological and physiological analyses. Together with large pharmacological databases, equipped with state-of-the-art bioinformatic and/or data-mining algorithms, these systems will provide rapid and accurate indices of the therapeutic potential of novel drugs. By providing a substantial increase in the speed of behavioral testing, new high-throughput systems will facilitate current behavioral research with faster, more reliable approaches. Furthermore, screening whole drug-libraries and comparing the profiles of novel compounds to those of known compounds will facilitate the discovery of novel drugs. Target validation will also become more efficient with the fast characterization of novel mutant mice.

Animals↗

PubMed2002

Structure-based virtual screening: an overview.

Enormous advances in genomics have resulted in a large increase in the number of potential therapeutic targets that are available for investigation. This growth in potential targets has increased the demand for reliable target validation, as well as technologies that can identify rapidly several quality lead candidates. Virtual screening, and in particular receptor-based virtual screening, has emerged as a reliable, inexpensive method for identifying leads. Although still an evolving method, advances in computational techniques have enabled virtual screening to have a positive impact on the discovery process. Here, the current strengths and weaknesses of the technology are discussed, and emphasis is placed on aspects of the work-flow of a virtual screening campaign, from preparation through to post-screening analysis.

Animals↗

PubMed2005

Stable isotope methods for high-precision proteomics.

Stable isotope tagging methods provide a useful means of determining the relative expression level of individual proteins between samples in a mass spectrometer with high precision (coefficients of variation less than 10%). Because two or more samples tagged with different numbers of stable isotopes can be mixed before any processing steps, sample-to-sample recovery differences are eliminated. Mass spectrometry also allows post-translational modifications, splice variations and mutations (often unnoticed in immunoassays) to be detected and identified, increasing the clinical relevance of the assay and avoiding the issues of non-specific binding and cross-reactivity observed in immunoassays. Several stable isotope tagging methods are available for use in proteomics research. We discuss the advantages and disadvantages of each technique with respect to biomarker discovery, target validation, efficacy and toxicology screening and clinical diagnostic applications.

Isotope Labeling↗

PubMed2005

Functional genomics in antibacterial drug discovery.

Antibacterial drug discovery has experienced a paradigm shift from phenotypic screening for antibacterial activity to rational inhibition of preselected targets. Functional genomics techniques are implemented at various stages of the early drug discovery process and play a central role in target validation and mode of action determination. The spectrum of methods ranges from genetic manipulations (e.g. knockout studies, mutation analyses and the construction of conditional mutants) to transcriptome and proteome expression profiling. Functional genomics supports antibacterial drug discovery by improving knowledge on gene function, bacterial physiology and virulence and the effects of antibiotics on bacterial metabolism.

Animals↗

PubMed2006

Targeting N-type and T-type calcium channels for the treatment of pain.

Severe chronic pain afflicts a large number of people worldwide but satisfactory relief from such pain is difficult to achieve with drugs that are currently available, and so there is a great need for the development of new, efficacious and safe analgesics. Voltage-gated calcium-permeable ion channels are multi-subunit complexes that regulate neuronal excitability, action-potential firing patterns and neurotransmission in nociceptive pathways. Although multiple subtypes of voltage-gated calcium channels exist, pharmacological and ion-channel gene knockdown approaches in animals have revealed N-type and T-type calcium channels to be particularly attractive molecular targets for the discovery and development of new analgesic drugs. The recent approval of Prialt (Elan Pharmaceuticals) provides the ultimate target validation for N-type calcium channels, namely proof that they are key regulators of nociceptive signaling in humans.

Acute Disease↗

PubMed1999

Reaping the rewards.

Drug discovery is the subject of unprecedented advances in genomics, combinatorial chemistry, ultra-high-throughput screening and information technology. These technologies promise to discover proprietary validated targets and chemical entities faster and more economically than before. Furthermore, the quality of drugs going into development might improve if profiling of drug candidates, performing in vitro substitute adsorption-distribution-metabolism-excretion (ADME)-toxicology and genotyping subjects in clinical studies, is undertaken at an earlier stage.

Journal Article↗

PubMed2000

Using bioinformatics in gene and drug discovery.

Bioinformatics has, out of necessity, become a key aspect of drug discovery in the genomic revolution, contributing to both target discovery and target validation. The author describes the role that bioinformatics has played and will continue to play in response to the waves of genome-wide data sources that have become available to the industry, including expressed sequence tags, microbial genome sequences, model organism sequences, polymorphisms, gene expression data and proteomics. However, these knowledge sources must be intelligently integrated.

Journal Article↗

PubMed2002

Positron emission tomography and drug discovery: contributions to the understanding of pharmacokinetics, mechanism of action and disease state characterization.

As an imaging modality, positron emission tomography (PET) provides unique quantitative in vivo information of value to drug discovery studies. These non-invasive studies span the pharmacokinetic/pharmacodynamic evaluation of potential drug candidates, receptor occupancy as an important determinant of efficacy, the pharmacological characterization of potential mechanisms of action, and the biological characterization of disease with well-characterized PET ligands. PET techniques are also being applied to the assessment of gene-level activities and the longitudinal evaluation of disease progression and therapeutic intervention. As the availability of PET scanners, cyclotrons, and specific PET ligands grows, the techniques highlighted in this review will become central to target validation, drug candidate selection, pharmacokinetic characterization, and clinical evaluation.

Journal Article↗

PubMed2005

Malarial (Plasmodium falciparum) dihydrofolate reductase-thymidylate synthase: structural basis for antifolate resistance and development of effective inhibitors.

Dihydrofolate reductase-thymidylate synthase (DHFR-TS) from Plasmodium falciparum, a validated target for antifolate antimalarials, is a dimeric enzyme with interdomain interactions significantly mediated by the junction region as well as the Plasmodium-specific additional sequences (inserts) in the DHFR domain. The X-ray structures of both the wild-type and mutant enzymes associated with drug resistance, in complex with either a drug which lost, or which still retains, effectiveness for the mutants, reveal features which explain the basis of drug resistance resulting from mutations around the active site. Binding of rigid inhibitors like pyrimethamine and cycloguanil to the enzyme active site is affected by steric conflict with the side-chains of mutated residues 108 and 16, as well as by changes in the main chain configuration. The role of important residues on binding of inhibitors and substrates was further elucidated by site-directed and random mutagenesis studies. Guided by the active site structure and modes of inhibitor binding, new inhibitors with high affinity against both wild-type and mutant enzymes have been designed and synthesized, some of which have very potent anti-malarial activities against drug-resistant P. falciparum bearing the mutant enzymes.

Animals↗

PubMed2005

Identification of potent and selective RNA antagonists of the IFN-gamma-inducible CXCL10 chemokine.

CXCL10 (also known as IP-10 in humans and CRG-2 in mice) is a nonglycosylated chemokine and a member of the non-ELR CXC chemokine subfamily implicated in a variety of inflammatory conditions. The role of CXCL10 in different disease states still requires clarification, and new approaches are necessary to better understand its biological function. We report here the isolation of a series of nuclease-resistant RNA aptamers that act to antagonize human CXCL10 function in a number of in vitro and cell-based assays. The two most potent aptamers identified were highly selective for human CXCL10. A further aptamer was identified that antagonized both the human and the mouse CXCL10. A combination of a molecular-biology-based truncation and solid-phase synthesis enabled the truncation of one of the aptamers from 71 to 34 nucleotides. This was followed by PEGylation, 3' capping, and further stabilization of the RNA aptamer, while its high potency was maintained. These aptamers could be utilized as powerful target validation tools and may also have therapeutic potential. To our knowledge, the CXCL10 aptamers generated are the most potent antagonists of CXCL10/CXCR3 signaling reported to date.

Animals↗

PubMed2006

Long-range cooperative interactions modulate dimerization in SARS 3CLpro.

Severe acute respiratory syndrome (SARS) is an infectious disease caused by the human coronavirus, SARS-CoV. The main viral protease, SARS 3CLpro, is a validated target for the development of antiviral therapies. Since the enzyme is a homodimer and the individual monomers are inactive, two approaches are being used to develop inhibitors: enzyme activity inhibitors that target the active site and dimerization inhibitors. Dimerization inhibitors are usually targeted to the dimerization interface and need to compete with the attractive forces between subunits to be effective. In this paper, we show that the dimerization of SARS 3CLpro is also under allosteric control and that additional and energetically more favorable target sites away from the dimerization interface may also lead to subunit dissociation. We previously identified a cluster of conserved serine residues (Ser139, Ser144, and Ser147) located adjacent to the active site of 3CLpro that could effectively be targeted to inactivate the protease [Bacha, U et al. (2004) Biochemistry 43, 4906-4912]. Mutation of any of these serine residues to alanine had a debilitating effect on the catalytic activity of 3CLpro. In particular, the mutation of Ser147, which does not make any contact with the opposing subunit and is located approximately 9 A away from the dimer interface, totally inhibited dimerization and resulted in a complete loss of enzymatic activity. The finding that residues away from the dimer interface are able to control dimerization defines alternative targets for the design of dimerization inhibitors.

Allosteric Site↗

PubMed2007

Human glycinamide ribonucleotide transformylase: active site mutants as mechanistic probes.

Human glycinamide ribonucleotide transformylase (GART) (EC 2.1.2.2) is a validated target for cancer chemotherapy, but mechanistic studies of this therapeutically important enzyme are limited. Site-directed mutagenesis, initial velocity studies, pH-rate studies, and substrate binding studies have been employed to probe the role of the strictly conserved active site residues, N106, H108, and D144, and the semiconserved K170 in substrate binding and catalysis. Only two conservative substitutions, N106Q and K170R, resulted in catalytically active enzymes, and these active mutant enzymes gave pH-rate profiles and a steady-state kinetic mechanism essentially identical to those of the native enzyme. All inactive mutants were able to bind both substrates, ruling out disrupted formation of the ternary complex as the source of inactivity. Differences between human and Escherichia coli GART, previously used as a model for the human enzyme, were evident.

Amino Acid Sequence↗

PubMed2005

Design and optimization of tricyclic phtalimide analogues as novel inhibitors of HIV-1 integrase.

Human immunodeficiency virus type-1 integrase is an essential enzyme for effective viral replication and hence a valid target for the design of inhibitors. We report here on the design and synthesis of a novel series of phthalimide analogues as integrase inhibitors. The short synthetic pathway enabled us to synthesize a series of analogues with a defined structure diversity. The presence of a single carbonyl-hydroxy-aromatic nitrogen motif was shown to be essential for the enzymatic activity and this was confirmed by molecular docking studies. The enzymatically most active compound from this series is 7-(3,4-dichlorobenzyl)-5,9-dihydroxypyrrolo[3,4-g]quinoxaline-6,8-dione (15l) with an IC(50) value of 112 nM on the HIV-1 integrase enzyme, while ((7-(4-chlorobenzyl)-5,9-dihydroxy-pyrrolo[3,4-g]quinoxaline-6,8-dione (15k)) showed an EC(50) of 270 nM against HIV-1 in a cell-based assay.

Anti-HIV Agents↗

PubMed2006

Identification of novel parasitic cysteine protease inhibitors by use of virtual screening. 2. The available chemical directory.

The incidence of parasitic infections such as malaria, leishmaniasis, and trypanosomiasis has been steadily increasing. Since the existing chemotherapy of these diseases suffers from lack of safe and effective drugs and/or the presence of widespread drug resistance, there is an urgent need for development of potent, mechanism-based antiparasitic agents against these diseases. Cysteine proteases have been established as valid targets for this purpose. The Available Chemical Directory consisting of nearly 355,000 compounds was screened in silico against the homology models of plasmodial cysteine proteases, falcipain-2, and falcipain-3, to identify structurally diverse non-peptide inhibitors. The study led to identification of 22 inhibitors of parasitic cysteine proteases out of which 18 compounds were active against falcipain-2 and falcipain-3. Eight compounds exhibited dual activity against both enzymes. Additionally, four compounds were found to inhibit L. donovani cysteine protease. While one of the cysteine protease inhibitors also exhibited in vitro antiplasmodial activity with an IC50 value of 9.5 microM, others did not show noticeable antiplasmodial activity up to 20 microM. A model identifying important pharmacophoric features common to the structurally diverse falcipain-2 inhibitors has also been developed. Very few potent non-peptide inhibitors of the parasitic cysteine proteases have been reported so far, and identification of these novel and chemically diverse inhibitors should provide leads to be optimized into candidates to treat protozoal infections.

Animals↗

PubMed2006

Novel bifunctional quinolonyl diketo acid derivatives as HIV-1 integrase inhibitors: design, synthesis, biological activities, and mechanism of action.

The virally encoded integrase protein is an essential enzyme in the life cycle of the HIV-1 virus and represents an attractive and validated target in the development of therapeutics against HIV infection. Drugs that selectively inhibit this enzyme, when used in combination with inhibitors of reverse transcriptase and protease, are believed to be highly effective in suppressing the viral replication. Among the HIV-1 integrase inhibitors, the beta-diketo acids (DKAs) represent a major lead for anti-HIV-1 drug development. In this study, novel bifunctional quinolonyl diketo acid derivatives were designed, synthesized, and tested for their inhibitory ability against HIV-1 integrase. The compounds are potent inhibitors of integrase activity. Particularly, derivative 8 is a potent IN inhibitor for both steps of the reaction (3'-processing and strand transfer) and exhibits both high antiviral activity against HIV-1 infected cells and low cytotoxicity. Molecular modeling studies provide a plausible mechanism of action, which is consistent with ligand SARs and enzyme photo-cross-linking experiments.

Catalytic Domain↗

PubMed2006

Structural insights into the ATP binding pocket of the anaplastic lymphoma kinase by site-directed mutagenesis, inhibitor binding analysis, and homology modeling.

Anaplastic lymphoma kinase (ALK) is a valid target for anticancer therapy; however, potent ALK inhibitors suitable for clinical use are lacking. Because the majority of described kinase inhibitors bind in the ATP pocket of the kinase domain, we have characterized this pocket in ALK using site-directed mutagenesis, inhibition studies, and molecular modeling. Mutation of the gatekeeper residue, a key structural determinant influencing inhibitor binding, rendered the fusion protein, NPM/ALK, sensitive to inhibition by SKI-606 in the nanomolar range, while PD173955 inhibited the NPM/ALK mutant at micromolar concentrations. In contrast, both wild type and mutant NPM/ALK were insensitive to imatinib. Computer modeling indicated that docking solutions obtained with a homology model representing the intermediate conformation of the ALK kinase domain reflected closely experimental data. The good agreement between experimental and virtual results indicate that the ALK molecular models described here are useful tools for the rational design of ALK selective inhibitors. In addition, 4-phenylamino-quinoline compounds may have potential as templates for ALK inhibitors.

Adenosine Triphosphate↗

PubMed2006

Design, synthesis, and biological evaluation of phenylamino-substituted 6,11-dihydro-dibenzo[b,e]oxepin-11-ones and dibenzo[a,d]cycloheptan-5-ones: novel p38 MAP kinase inhibitors.

The pathogenesis of chronic inflammatory diseases is promoted by various pro-inflammatory cytokines. p38 MAP kinase seems to be a valid target as it controls proinflammatory cytokine levels on both transcriptional and translational levels. Starting from benzophenone-type inhibitors, a rigidisation strategy lead to 3-amino-6,11-dihydro-dibenzo[b,e]thiepin-11-one, phenylamino-substituted 6,11-dihydro-dibenzo[b,e]oxepin-11-ones, and dibenzo[a,d]cyclohepten-5-ones. Synthesis, p38 inhibition, and CYP-inhibition of selected compounds are described.

Cytochrome P-450 Enzyme Inhibitors↗