Identification Of Potential Drug Targets Research Articles

Rapid and sensitive detection of DNA polymerase fidelity by singly labeled smart fluorescent probes

We report here a novel approach to monitor the DNA polymerase fidelity in detailed steps, including mispair extension, mispair formation and 3′ → 5′ proofreading. The method is based on the photo-induced electron transfer between the natural base guanine and the labeled fluorophore. The G:T mispair extension catalyzed by the exonuclease-deficient Klenow fragment DNA polymerase (KF exo −) was easily detected and the effect of the nearest neighbor base pair on the mispair extension rate was clearly observed. More importantly, kinetics of the G:T, G:A and G:G mispair formation and extension under single turnover conditions were measured by continuous fluorescence-based assay for the first time. The probes also showed their applicability to discriminate the 3′ → 5′ proofreading activity of different exonuclease-proficient DNA polymerases. The presented method may greatly simplify the screening and characterization procedures of the increasing number of polymerases that are thought to be potential targets for drug design and cancer treatment. It will also provide important information for deep understanding of the polymerase fidelity mechanism.

New Pharmacotherapy for Airway Mucus Hypersecretion in Asthma and COPD: Targeting Intracellular Signaling Pathways

Airway mucus hypersecretion is a pathophysiological feature of asthma and chronic obstructive pulmonary disease (COPD). The hypersecretion is associated with phenotypic changes in the airways, notably, increases in the number of surface epithelial goblet cells (hyperplasia) and in the size of the submucosal glands (hypertrophy). The hyperplasia and hypertrophy are associated with increased production of mucin, the gel-forming component of mucus. The excess mucus production contributes to morbidity and mortality in many patients, particularly in those with more severe disease. Although current pharmacotherapy is effective in clinical management of patients with stable asthma, severe asthma is poorly treated and there is no current drug treatment for COPD. In neither disease is there specific, effective pharmacotherapy for the hypersecretion. Consequently, identification of potential drug targets for treatment of hypersecretion in asthma and COPD is warranted. The inflammatory mediators and the associated intracellular signaling pathways underlying upregulation of mucin synthesis and development of goblet cell hyperplasia are gradually being elucidated. These include Th2 cytokines (predominantly IL-9 and IL-13), and IL-1 beta, tumor necrosis factor-alpha (TNF-alpha) and cyclooxygenase (COX)-2. IL-9 may act predominantly via calcium-activated chloride channels (CLCA), IL-13 via STAT-6 and FOXA2, TNF-alpha via NF-kappaB, and IL-1 beta via COX-2. Epidermal growth factor receptor (EGF-R) signaling and FOXA2 appear to be convergent intracellular pathways for a number of inflammatory mediators, with EGF-R upregulated in the airways of asthmatic and COPD patients. Thus, preclinical studies have clearly identified a number of intracellular signaling pathways as possible targets for pharmacotherapy of airway mucus hypersecretion in asthma and COPD. Of these, the EGF-R and Th2 cytokine pathways may have the greatest potential for inhibition of excessive mucus production. However, because these targets are so often intimately involved with different aspects of airway (and systemic) homeostasis, there is potential for development of unwanted side effects with drug intervention. Thus, translation of the promising preclinical studies to the clinic will depend on development of drug moieties with low off-target activity. This may be accomplished by maximizing airway selectivity, which may be facilitated by appropriate delivery device design.

Identification of Distinguishing Motifs

Motif identification for DNA sequences has many important applications in biological studies, including diagnostic probe design, locating binding sites and regulatory signals, and potential drug target identification. There are two versions—the Single Group and Two Groups. Here, the occurrences of the motif in the given sequences have errors. Currently, most of existing programs can only handle the case of single group. However, most of the programs do not allow indels (insertions and deletions) in the occurrences of the motif. In this paper, the authors propose a randomized algorithm for the one group problem that can handle indels in the occurrences of the motif. Finally, an algorithm for the two groups’ problem is given along with extensive simulations evaluating algorithms.

A Novel Entamoeba histolytica Cysteine Proteinase, EhCP4, Is Key for Invasive Amebiasis and a Therapeutic Target

Entamoeba histolytica cysteine proteinases (EhCPs) play a key role in disrupting the colonic epithelial barrier and the innate host immune response during invasion of E. histolytica, the protozoan cause of human amebiasis. EhCPs are encoded by 50 genes, of which ehcp4 (ehcp-a4) is the most up-regulated during invasion and colonization in a mouse cecal model of amebiasis. Up-regulation of ehcp4 in vivo correlated with our finding that co-culture of E. histolytica trophozoites with mucin-producing T84 cells increased ehcp4 expression up to 6-fold. We have expressed recombinant EhCP4, which was autocatalytically activated at acidic pH but had highest proteolytic activity at neutral pH. In contrast to the other amebic cysteine proteinases characterized so far, which have a preference for arginine in the P2 position, EhCP4 displayed a unique preference for valine and isoleucine at P2. This preference was confirmed by homology modeling, which revealed a shallow, hydrophobic S2 pocket. Endogenous EhCP4 localized to cytoplasmic vesicles, the nuclear region, and perinuclear endoplasmic reticulum (ER). Following co-culture with colonic cells, EhCP4 appeared in acidic vesicles and was released extracellularly. A specific vinyl sulfone inhibitor, WRR605, synthesized based on the substrate specificity of EhCP4, inhibited the recombinant enzyme in vitro and significantly reduced parasite burden and inflammation in the mouse cecal model. The unique expression pattern, localization, and biochemical properties of EhCP4 could be exploited as a potential target for drug design.

Baculovirus Gene Delivery: A Flexible Assay Development Tool

Modern drug discovery programs utilize a wide variety of technologies to aid in identification of potential drug targets, and progress them through the often long and winding path of finding novel drug-like molecules. Recombinant cell-based assays are an important tool in the drug discovery process for investigating the biological mechanisms of potential drug targets and conducting screening campaigns in the hunt for biologically active molecules. Historically, stable cell lines expressing the target protein(s) of interest have been used for these assays. Although such cell lines can be useful, their development can be laborious and the resulting cell line affords little experimental flexibility. Transient gene expression approaches provide an alternative to the often tedious task of developing and maintaining numerous stable cell lines. Recently the unique properties of modified baculoviruses, containing mammalian expression cassettes and referred to as BacMam viruses, have been exploited to facilitate rapid and reproducible transient cell-based assay development. This review will focus on the many features of BacMam virus gene delivery that make it a powerful system for cell-based assay development and screening.

Novel insertion and deletion mutants of RpoB that render Mycobacterium smegmatis RNA polymerase resistant to rifampicin-mediated inhibition of transcription

The startling increase in the occurrence of rifampicin (Rif) resistance in the clinical isolates of Mycobacterium tuberculosis worldwide is posing a serious concern to tuberculosis management. The majority of Rif resistance in bacteria arises from mutations in the RpoB subunit of the RNA polymerase. We isolated M. smegmatis strains harbouring either an insertion (6 aa) or a deletion (10 aa) in their RpoB proteins. Although these strains showed a compromised fitness for growth in 7H9 Middlebrook medium, their resistance to Rif was remarkably high. The attenuated growth of the strains correlated with decreased specific activities of the RNA polymerases from the mutants. While the RNA polymerases from the parent or a mutant strain (harbouring a frequently occurring mutation, H442Y, in RpoB) were susceptible to Rif-mediated inhibition of transcription from calf thymus DNA, those from the insertion and deletion mutants were essentially refractory to such inhibition. Three-dimensional structure modelling revealed that the RpoB amino acids that interact with Rif are either deleted or unable to interact with Rif due to their unsuitable spatial positioning in these mutants. We discuss possible uses of the RpoB mutants in studying transcriptional regulation in mycobacteria and as potential targets for drug design.

Mining the Proteome of Haemophilus ducreyi for Identification of Potential Drug Targets

Chancroid is an extremely infectious sexually transmitted disease (STD) caused by the bacterium Haemophilus ducreyi, prevalent in Africa, United States and in some parts of South Asia. Chancroid has been recognized as a cofactor for human immunodeficiency virus (HIV) transmission. So, there is a requirement to develop an efficient drug to combat chancroid, which can also diminish the HIV prevalence in those populations where chancroid is a prime source for HIV infection. The availability of the complete proteome information of H. ducreyi help enabled in silico analysis for identification of potential vaccine candidates and drug targets. Our study revealed 1226 proteins in H. ducreyi to be nonhomologous with human proteome. Screening these proteins using the Database of Essential Genes (DEG) resulted in the identification of 451 essential proteins. Analysis of the identified essential proteins, using the KEGG Automated Annotation Server (KAAS), revealed 40 proteins of H. ducreyi as potential drug targets as they are involved in pathogen specific metabolic pathways. Subcellular localization prediction of these 451 essential proteins revealed that 11 proteins lie on the outer membrane of the pathogen which could be potential vaccine candidates. Functional family prediction for the 50 putative uncharacterized essential proteins of H. ducreyi by SVM-Prot web server revealed that out of 50, 3 proteins as transmembrane proteins, which may be potential drug targets. Identification of potential inhibitors against these targets through virtual screening may consequence in detection of novel lead compounds effective against H. ducreyi to combat chancroid.

Identification of Potential Drug Targets of Leishmania infantum by In-silico Genome Analysis

Identification of Potential Drug Targets of Leishmania infantum by In-silico Genome Analysis

Protein interaction network analysis—Approach for potential drug target identification in Mycobacterium tuberculosis

In host–parasite diseases like tuberculosis, non-homologous proteins (enzymes) as drug target are first preference. Most potent drug target can be identified among large number of non-homologous protein through protein interaction network analysis. In this study, the entire promising dimension has been explored for identification of potential drug target. A comparative metabolic pathway analysis of the host Homo sapiens and the pathogen M. tuberculosis H37Rv has been performed with three level of analysis. In first level, the unique metabolic pathways of M. tuberculosis have been identified through its comparative study with H. sapiens and identification of non-homologous proteins has been done through BLAST similarity search. In second level, choke-point analysis has been performed with identified non-homologous proteins of metabolic pathways. In third level, two type of analysis have been performed through protein interaction network. First analysis has been done to find out the most potential metabolic functional associations among all identified choke point proteins whereas second analysis has been performed to find out the functional association of high metabolic interacting proteins to pathogenesis causing proteins. Most interactive metabolic proteins which have highest number of functional association with pathogenesis causing proteins have been considered as potential drug target. A list of 18 potential drug targets has been proposed which are various stages of progress at the TBSGC and proposed drug targets are also studied for other pathogenic strains. As a case study, we have built a homology model of identified drug targets histidinol-phosphate aminotransferase (HisC1) using MODELLER software and various information have been generated through molecular dynamics which will be useful in wetlab structure determination. The generated model could be further explored for insilico docking studies with suitable inhibitors.

Genomes2Drugs: Identifies Target Proteins and Lead Drugs from Proteome Data

BackgroundGenome sequencing and bioinformatics have provided the full hypothetical proteome of many pathogenic organisms. Advances in microarray and mass spectrometry have also yielded large output datasets of possible target proteins/genes. However, the challenge remains to identify new targets for drug discovery from this wealth of information. Further analysis includes bioinformatics and/or molecular biology tools to validate the findings. This is time consuming and expensive, and could fail to yield novel drugs if protein purification and crystallography is impossible. To pre-empt this, a researcher may want to rapidly filter the output datasets for proteins that show good homology to proteins that have already been structurally characterised or proteins that are already targets for known drugs. Critically, those researchers developing novel antibiotics need to select out the proteins that show close homology to any human proteins, as future inhibitors are likely to cross-react with the host protein, causing off-target toxicity effects later in clinical trials.Methodology/Principal FindingsTo solve many of these issues, we have developed a free online resource called Genomes2Drugs which ranks sequences to identify proteins that are (i) homologous to previously crystallized proteins or (ii) targets of known drugs, but are (iii) not homologous to human proteins. When tested using the Plasmodium falciparum malarial genome the program correctly enriched the ranked list of proteins with known drug target proteins.Conclusions/SignificanceGenomes2Drugs rapidly identifies proteins that are likely to succeed in drug discovery pipelines. This free online resource helps in the identification of potential drug targets. Importantly, the program further highlights proteins that are likely to be inhibited by FDA-approved drugs. These drugs can then be rapidly moved into Phase IV clinical studies under ‘change-of-application’ patents.

Structures of dihydrofolate reductase-thymidylate synthase ofTrypanosoma cruziin the folate-free state and in complex with two antifolate drugs, trimetrexate and methotrexate

The flagellate protozoan parasite Trypanosoma cruzi is the pathogenic agent of Chagas disease (also called American trypanosomiasis), which causes approximately 50,000 deaths annually. The disease is endemic in South and Central America. The parasite is usually transmitted by a blood-feeding insect vector, but can also be transmitted via blood transfusion. In the chronic form, Chagas disease causes severe damage to the heart and other organs. There is no satisfactory treatment for chronic Chagas disease and no vaccine is available. There is an urgent need for the development of chemotherapeutic agents for the treatment of T. cruzi infection and therefore for the identification of potential drug targets. The dihydrofolate reductase activity of T. cruzi, which is expressed as part of a bifunctional enzyme, dihydrofolate reductase-thymidylate synthase (DHFR-TS), is a potential target for drug development. In order to gain a detailed understanding of the structure-function relationship of T. cruzi DHFR, the three-dimensional structure of this protein in complex with various ligands is being studied. Here, the crystal structures of T. cruzi DHFR-TS with three different compositions of the DHFR domain are reported: the folate-free state, the complex with the lipophilic antifolate trimetrexate (TMQ) and the complex with the classical antifolate methotrexate (MTX). These structures reveal that the enzyme is a homodimer with substantial interactions between the two TS domains of neighboring subunits. In contrast to the enzymes from Cryptosporidium hominis and Plasmodium falciparum, the DHFR and TS active sites of T. cruzi lie on the same side of the monomer. As in other parasitic DHFR-TS proteins, the N-terminal extension of the T. cruzi enzyme is involved in extensive interactions between the two domains. The DHFR active site of the T. cruzi enzyme shows subtle differences compared with its human counterpart. These differences may be exploited for the development of antifolate-based therapeutic agents for the treatment of T. cruzi infection.

Unfolding Individual Als5p Adhesion Proteins on Live Cells

Elucidating the molecular mechanisms behind the strength and mechanics of cell adhesion proteins is of central importance in cell biology and offers exciting avenues for the identification of potential drug targets. Here we use single-molecule force spectroscopy to investigate the adhesive and mechanical properties of the widely expressed Als5p cell adhesion protein from the opportunistic pathogen Candida albicans . We show that the forces required to unfold individual tandem repeats of the protein are in the 150-250 pN range, both on isolated molecules and on live cells. We also find that the unfolding probability increases with the number of tandem repeats and correlates with the level of cell adherence. We suggest that the modular and flexible nature of Als5p conveys both strength and toughness to the protein, making it ideally suited for cell adhesion. The single-molecule measurements presented here open new avenues for understanding the mechanical properties of adhesion molecules from mammalian and microbial cells and may help us to elucidate their potential implications in diseases such as inflammation, cancer, and infection.

Structure-function analysis of gC1qR/p33 using deletion mutants show additional binding sites for C1q. (134.69)

Abstract Surface expressed or soluble gC1qR has been shown to activate complement and to play a role in various inflammatory processes. By peptide mapping and antibody inhibition studies, we had previously identified a binding site for C1q in a gC1qR domain contained within residues 76-93. However, although mAbs 60.11, which recognizes residues 76-93, can inhibit C1q binding to gC1qR, the inhibition is not complete. Similarly, binding of C1q to a truncated form of gC1qR lacking residues 76-93 is reduced by only 40-50% when compared to the full-length gC1qR. These findings therefore prompted us to look for additional binding sites elsewhere in the molecule. To this end, we generated several recombinant gC1qR proteins by deletion of domains of very high negative charge— and which, based on their location in the crystal structure—were predicted to interact with solution-phase ligands. The capabilities of these mutants to bind to C1q were therefore examined by comparing to the binding of the full-length gC1qR. Analyses of the data reveal the presence of two additional binding sites for C1q—one contained within residues 190-192 and another within residues 144-162. These results not only demonstrate well the value of crystal-based structure-function predictions, but also allow the identification of potential targets for drug design to alleviate complement-mediated inflammation. [Supported by NIH-NIAID Grant R01 AI-060866]

A Multiplexed Quantitative Strategy for Membrane Proteomics: Opportunities for Mining Therapeutic Targets for Autosomal Dominant Polycystic Kidney Disease

Toward multiplexed, comprehensive, and robust quantitation of the membrane proteome, we report a strategy combining gel-assisted digestion, iTRAQ (isobaric tags for relative and absolute quantitation) labeling, and LC-MS/MS. Quantitation of four independently purified membrane fractions from HeLa cells gave high accuracy (<8% error) and precision (<12% relative S.D.), demonstrating a high degree of consistency and reproducibility of this quantitation platform. Under stringent identification criteria (false discovery rate = 0%), the strategy efficiently quantified membrane proteins; as many as 520 proteins (91%) were membrane proteins, each quantified based on an average of 14.1 peptides per integral membrane protein. In addition to significant improvements in signal intensity for most quantified proteins, most remarkably, topological analysis revealed that the biggest improvement was achieved in detection of transmembrane peptides from integral membrane proteins with up to 19 transmembrane helices. To the best of our knowledge, this level of coverage exceeds that achieved previously using MS and provides superior quantitation accuracy compared with other methods. We applied this approach to the first proteomics delineation of phenotypic expression in a mouse model of autosomal dominant polycystic kidney disease (ADPKD). By characterizing kidney cell plasma membrane from wild-type versus PKD1 knock-out mice, 791 proteins were quantified, and 67 and 37 proteins showed > or =2-fold up-regulation and down-regulation, respectively. Some of these differentially expressed membrane proteins are involved in the mechanisms underlying major abnormalities in ADPKD, including epithelial cell proliferation and apoptosis, cell-cell and cell-matrix interactions, ion and fluid secretion, and membrane protein polarity. Among these proteins, targeting therapeutics to certain transporters/receptors, such as epidermal growth factor receptor, has proven effective in preclinical studies of ADPKD; others are known drug targets in various diseases. Our method demonstrates how comparative membrane proteomics can provide insight into the molecular mechanisms underlying ADPKD and the identification of potential drug targets, which may lead to new therapeutic opportunities to prevent or retard the disease.

Structure of the C-terminal domain of the arginine repressor protein from Mycobacterium tuberculosis.

The Mycobacterium tuberculosis (Mtb) gene product encoded by open reading frame Rv1657 is an arginine repressor (ArgR). All genes involved in the L-arginine (hereafter arginine) biosynthetic pathway are essential for optimal growth of the Mtb pathogen, thus making MtbArgR a potential target for drug design. The C-terminal domains of arginine repressors (CArgR) participate in oligomerization and arginine binding. Several crystal forms of CArgR from Mtb (MtbCArgR) have been obtained. The X-ray crystal structures of MtbCArgR were determined at 1.85 A resolution with bound arginine and at 2.15 A resolution in the unliganded form. These structures show that six molecules of MtbCArgR are arranged into a hexamer having approximate 32 point symmetry that is formed from two trimers. The trimers rotate relative to each other by about 11 degrees upon binding arginine. All residues in MtbCArgR deemed to be important for hexamer formation and for arginine binding have been identified from the experimentally determined structures presented. The hexamer contains six regular sites in which the arginine molecules have one common binding mode and three sites in which the arginine molecules have two overlapping binding modes. The latter sites only bind the ligand at high (200 mM) arginine concentrations.

Construction of a cancer-perturbed protein-protein interaction network for discovery of apoptosis drug targets

BackgroundCancer is caused by genetic abnormalities, such as mutations of oncogenes or tumor suppressor genes, which alter downstream signal transduction pathways and protein-protein interactions. Comparisons of the interactions of proteins in cancerous and normal cells can shed light on the mechanisms of carcinogenesis.ResultsWe constructed initial networks of protein-protein interactions involved in the apoptosis of cancerous and normal cells by use of two human yeast two-hybrid data sets and four online databases. Next, we applied a nonlinear stochastic model, maximum likelihood parameter estimation, and Akaike Information Criteria (AIC) to eliminate false-positive protein-protein interactions in our initial protein interaction networks by use of microarray data. Comparisons of the networks of apoptosis in HeLa (human cervical carcinoma) cells and in normal primary lung fibroblasts provided insight into the mechanism of apoptosis and allowed identification of potential drug targets. The potential targets include BCL2, caspase-3 and TP53. Our comparison of cancerous and normal cells also allowed derivation of several party hubs and date hubs in the human protein-protein interaction networks involved in caspase activation.ConclusionOur method allows identification of cancer-perturbed protein-protein interactions involved in apoptosis and identification of potential molecular targets for development of anti-cancer drugs.

Biological characteristics of dengue virus and potential targets for drug design

Dengue infection is a major cause of morbidity in tropical and subtropical regions, bringing nearly 40% of the world population at risk and causing more than 20,000 deaths per year. But there is neither a vaccine for dengue disease nor antiviral drugs to treat the infection. In recent years, dengue infection has been particularly prevalent in India, Southeast Asia, Brazil, and Guangdong Province, China. In this article, we present a brief summary of the biological characteristics of dengue virus and associated flaviviruses, and outline the progress on studies of vaccines and drugs based on potential targets of the dengue virus.

The biology and future prospects of antivirulence therapies

The emergence and increasing prevalence of bacterial strains that are resistant to available antibiotics demand the discovery of new therapeutic approaches. Targeting bacterial virulence is an alternative approach to antimicrobial therapy that offers promising opportunities to inhibit pathogenesis and its consequences without placing immediate life-or-death pressure on the target bacterium. Certain virulence factors have been shown to be potential targets for drug design and therapeutic intervention, whereas new insights are crucial for exploiting others. Targeting virulence represents a new paradigm to empower the clinician to prevent and treat infectious diseases.

Linking Gene Expression and Functional Network Data in Human Heart Failure

BackgroundGene expression profiling and the analysis of protein-protein interaction (PPI) networks may support the identification of disease bio-markers and potential drug targets. Thus, a step forward in the development of systems approaches to medicine is the integrative analysis of these data sources in specific pathological conditions. We report such an integrative bioinformatics analysis in human heart failure (HF). A global PPI network in HF was assembled, which by itself represents a useful compendium of the current status of human HF-relevant interactions. This provided the basis for the analysis of interaction connectivity patterns in relation to a HF gene expression data set.ResultsRelationships between the significance of the differentiation of gene expression and connectivity degrees in the PPI network were established. In addition, relationships between gene co-expression and PPI network connectivity were analysed. Highly-connected proteins are not necessarily encoded by genes significantly differentially expressed. Genes that are not significantly differentially expressed may encode proteins that exhibit diverse network connectivity patterns. Furthermore, genes that were not defined as significantly differentially expressed may encode proteins with many interacting partners. Genes encoding network hubs may exhibit weak co-expression with the genes encoding their interacting protein partners. We also found that hubs and superhubs display a significant diversity of co-expression patterns in comparison to peripheral nodes. Gene Ontology (GO) analysis established that highly-connected proteins are likely to be engaged in higher level GO biological process terms, while low-connectivity proteins tend to be engaged in more specific disease-related processes.ConclusionThis investigation supports the hypothesis that the integrative analysis of differential gene expression and PPI network analysis may facilitate a better understanding of functional roles and the identification of potential drug targets in human heart failure.

Yeast Genomics and Drug Target Identification

The budding yeast Saccharomyces cerevisiae is well recognized as a preferred eukaryote for the development of genomic technologies and approaches. Accordingly, a sizeable complement of genomic resources has been developed in yeast, and this genomic foundation is now informing a wide variety of disciplines. In particular, yeast genomic methodologies are gaining an expanding foothold in drug development studies, most notably as a preliminary tool towards drug target identification. In this review, we highlight many applications of yeast genomics in the identification of targeted genes and pathways of small molecules or therapeutic drugs. The applicability of genome-wide resources of yeast disruption and deletion mutants for drug-sensitivity/resistance screening is presented here, along with a summary of microarray technologies for drug-based transcriptional profiling and synthetic interaction mapping. Applications of protein-interaction traps for potential drug target identification are also considered. Collectively, this overview of yeast genomics emphasizes the growing intersection between high-throughput model organism biology and medicinal chemistry an intersection promising tangible advances for both academic and pharmaceutical fields alike.