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Potential Drug Target (potential + drug_target)
Selected AbstractsSequence of the Mitochondrial Genome of Pneumocystis carinii: Implications for Biological Function and Identification of Potential Drug TargetsTHE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 2006THOMAS M. SESTERHENN [source] Viral proteinases: targets of opportunityDRUG DEVELOPMENT RESEARCH, Issue 6 2006Chelsea M. Byrd Abstract During antiviral drug development, any essential stage of the viral life cycle can serve as a potential drug target. Since most viruses encode specific proteases whose cleavage activity is required for viral replication, and whose structure and activity are unique to the virus and not the host cell, these enzymes make excellent targets for drug development. Success using this approach has been demonstrated with the plethora of protease inhibitors approved for use against HIV. This discussion is designed to review the field of antiviral drug development, focusing on the search for protease inhibitors, while highlighting some of the challenges encountered along the way. Protease inhibitor drug discovery efforts highlighting progress made with HIV, HCV, HRV, and vaccinia virus as a model system are included. Drug Dev. Res. 67:501,510, 2006. © 2006 Wiley-Liss, Inc. [source] Characterization of a Leishmania stage-specific mitochondrial membrane protein that enhances the activity of cytochrome c oxidase and its role in virulenceMOLECULAR MICROBIOLOGY, Issue 2 2010Ranadhir Dey Summary Leishmaniasis is caused by the dimorphic protozoan parasite Leishmania. Differentiation of the insect form, promastigotes, to the vertebrate form, amastigotes, and survival inside the vertebrate host accompanies a drastic metabolic shift. We describe a gene first identified in amastigotes that is essential for survival inside the host. Gene expression analysis identified a 27 kDa protein-encoding gene (Ldp27) that was more abundantly expressed in amastigotes and metacyclic promastigotes than in procyclic promastigotes. Immunofluorescence and biochemical analysis revealed that Ldp27 is a mitochondrial membrane protein. Co-immunoprecipitation using antibodies to the cytochrome c oxidase (COX) complex, present in the inner mitochondrial membrane, placed the p27 protein in the COX complex. Ldp27 gene-deleted parasites (Ldp27,/,) showed significantly less COX activity and ATP synthesis than wild type in intracellular amastigotes. Moreover, the Ldp27,/, parasites were less virulent both in human macrophages and in BALB/c mice. These results demonstrate that Ldp27 is an important component of an active COX complex enhancing oxidative phosphorylation specifically in infectious metacyclics and amastigotes and promoting parasite survival in the host. Thus, Ldp27 can be explored as a potential drug target and parasites devoid of the p27 gene could be considered as a live attenuated vaccine candidate against visceral leishmaniasis. [source] The 1.25,Å resolution structure of phosphoribosyl-ATP pyrophosphohydrolase from Mycobacterium tuberculosisACTA CRYSTALLOGRAPHICA SECTION D, Issue 6 2008Farah Javid-Majd Phosphoribosyl-ATP pyrophosphohydrolase is the second enzyme in the histidine-biosynthetic pathway, irreversibly hydrolyzing phosphoribosyl-ATP to phosphoribosyl-AMP and pyrophosphate. It is encoded by the hisE gene, which is present as a separate gene in many bacteria and archaea but is fused to hisI in other bacteria, fungi and plants. Because of its essentiality for growth in vitro, HisE is a potential drug target for tuberculosis. The crystal structures of two native (uncomplexed) forms of HisE from Mycobacterium tuberculosis have been determined to resolutions of 1.25 and 1.79,Å. The structure of the apoenzyme reveals that the protein is composed of five ,-helices with connecting loops and is a member of the ,-helical nucleoside-triphosphate pyrophosphatase superfamily. The biological unit of the protein is a homodimer, with an active site on each subunit composed of residues exclusively from that subunit. A comparison with the Campylobacter jejuni dUTPase active site allowed the identification of putative metal- and substrate-binding sites in HisE, including four conserved glutamate and glutamine residues in the sequence that are consistent with a motif for pyrophosphohydrolase activity. However, significant differences between family members are observed in the loop region between ,-helices H1 and H3. The crystal structure of M. tuberculosis HisE provides insights into possible mechanisms of substrate binding and the diversity of the nucleoside-triphosphate pyrophosphatase superfamily. [source] The 1.9,Å resolution structure of Mycobacterium tuberculosis 1-deoxy- d -xylulose 5-phosphate reductoisomerase, a potential drug targetACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2006Lena M. Henriksson 1-Deoxy- d -xylulose 5-phosphate reductoisomerase catalyzes the NADPH-dependent rearrangement and reduction of 1-deoxy- d -xylulose 5-phosphate to form 2- C -methyl- d -erythritol 4-phosphate, as the second step of the deoxyxylulose 5-phosphate/methylerythritol 4-phosphate pathway found in many bacteria and plants. The end product, isopentenyl diphosphate, is the precursor of various isoprenoids vital to all living organisms. The pathway is not found in humans; the mevalonate pathway is instead used for the formation of isopentenyl diphosphate. This difference, combined with its essentiality, makes the reductoisomerase an excellent drug target in a number of pathogenic organisms. The structure of 1-deoxy- d -xylulose 5-phosphate reductoisomerase from Mycobacterium tuberculosis (Rv2870c) was solved by molecular replacement and refined to a resolution of 1.9,Å. The enzyme exhibited an estimated kcat of 5.3,s,1 and Km and kcat/Km values of 7.2,µM and 7.4 × 105,M,1,s,1 for NADPH and 340,µM and 1.6 × 104,M,1,s,1 for 1-deoxy- d -xylulose 5-phosphate. In the structure, a sulfate is bound at the expected site of the phosphate moiety of the sugar substrate. The M. tuberculosis enzyme displays a similar fold to the previously published structures from Escherichia coli and Zymomonas mobilis. Comparisons offer suggestions for the design of specific drugs. Furthermore, the new structure represents an intermediate conformation between the open apo form and the closed holo form observed previously, giving insights into the conformational changes associated with catalysis. [source] Preliminary X-ray crystallographic analysis of a nitric oxide-inducible lactate dehydrogenase from Staphylococcus aureusACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 10 2009Jia-Bin Dong Recent studies have indicated that Staphylococcus aureus can survive the nitrosative stress (caused by the radical nitric oxide; NO·) mounted by the immune system of the infected host. It does this by expressing a nitric oxide-inducible l -lactate dehydrogenase (Sa-LDH-1). Therefore, if efficient inhibitors of Sa-LDH-1 can be designed then Sa-LDH-1 could be a potential drug target against the pathogen S. aureus. For this purpose, the nitric acid-inducible LDH-1 from S. aureus COL strain has been cloned into the expression vector pET-28a(+) and the protein has been expressed, purified and crystallized. The Sa-LDH-1 crystal diffracted to 2.4,Å resolution at a home X-ray source and belonged to space group C2, with unit-cell parameters a = 131.4, b = 74.4, c = 103.2,Å, , = 133.4°. [source] Geobacillus stearothermophilus 6-phosphogluconate dehydrogenase complexed with 6-phosphogluconateACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2009Scott Cameron Two crystal structures of recombinant Geobacillus stearothermophilus 6-phosphogluconate dehydrogenase (Gs6PDH) in complex with the substrate 6-phosphogluconate have been determined at medium resolution. Gs6PDH shares significant sequence identity and structural similarity with the enzymes from Lactococcus lactis, sheep liver and the protozoan parasite Trypanosoma brucei, for which a range of structures have previously been reported. Comparisons indicate that amino-acid sequence conservation is more pronounced in the two domains that contribute to the architecture of the active site, namely the N-terminal and C-terminal domains, compared with the central domain, which is primarily involved in the subunit,subunit associations required to form a stable dimer. The active-site residues are highly conserved, as are the interactions with the 6-phosphogluconate. There is interest in 6PDH as a potential drug target for the protozoan parasite T. brucei, the pathogen responsible for African sleeping sickness. The recombinant T. brucei enzyme has proven to be recalcitrant to enzyme,ligand studies and a surrogate protein might offer new opportunities to investigate and characterize 6PDH inhibitors. The high degree of structural similarity, efficient level of expression and straightforward crystallization conditions mean that Gs6PDH may prove to be an appropriate model system for structure-based inhibitor design targeting the enzyme from Trypanosoma species. [source] Expression, purification, crystallization and preliminary X-ray diffraction analysis of ,-11 giardin from Giardia lambliaACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 11 2006Puja Pathuri ,-11 Giardin, a protein from the annexin superfamily, is a 35.0,kDa protein from the intestinal protozoan parasite Giardia lamblia which triggers a form of diarrhea called giardiasis. Here, the cloning, expression, purification and the crystallization of ,-11 giardin under two different conditions and in two different space groups is reported. Crystals from the first condition diffracted to 1.1,Å and belong to a primitive orthorhombic space group, while crystals from the second condition, which included calcium in the crystallization solution, diffracted to 2.93,Å and belong to a primitive monoclinic space group. Determination of the detailed atomic structure of ,-11 giardin will provide a better insight into its biological function and might establish whether this class of proteins is a potential drug target against giardiasis. [source] Development and Biological Evaluation of a Novel Aurora A Kinase InhibitorCHEMBIOCHEM, Issue 3 2009Teresa Sardon Dr. Abstract Stop dividing: In the quest for antitumorigenic compounds, aurora A kinase has recently emerged as a potential drug target. In this paper three novel aurora inhibitors (shown in the illustration) have been tested for their biological activity in cultured cells. One of them (TC-28) appears to be a promising specific aurora A inhibitor in vivo. The aurora kinase family groups several serine/threonine kinases with key regulatory functions during cell division. The three mammalian members, aurora A, B and C, are frequently over-expressed in human tumors and the aurora A gene is located in a genomic region frequently amplified in breast and colon cancer. All these data have fuelled the idea that aurora kinases are promising targets for anticancer therapy. Indeed some inhibitory compounds are currently being evaluated in clinical trials. However, it was recently shown that mutations in the targeted kinase can confer resistance to a broad range of inhibitors and render patients resistant to treatments. Moreover, aurora A over-expression results in increased resistance to antimitotic agents. The development of new compounds targeting aurora A is therefore highly relevant. We describe here the synthesis of three novel aurora kinase inhibitors, TC-28, TC-34 and TC-107. We report their properties as aurora inhibitors in vitro and their effect on human tissue culture cell lines. Interestingly, our results show that TC-28 has properties compatible with the specific inhibition of aurora A, in vivo. [source] Biosynthesis of New Indigoid Inhibitors of Protein Kinases Using Recombinant Cytochrome P450 2A6CHEMISTRY & BIODIVERSITY, Issue 1 2005Zhongliu-Liu Wu Glycogen synthase kinase-3 (GSK-3) is a potential drug target for a number of human diseases. Some indigoids have been found to be potent inhibitors of GSK-3, and individual compounds with better activity, specificity, and solubility are desired. In this work, a new disubstituted indigoid generation system was developed with a tryptophanase-deficient Escherichia coli strain as a host to express the human cytochrome P450 2A6 mutant L240C/N297Q, which catalyzes the oxidation of indole to isatin and indoxyl, which in turn react to generate indigoids. Forty-five substituted 1H -indoles from commercial sources were used as substrates in the system, and indigoid mixtures were tested as potential inhibitors of GSK-3. After preliminary screening, cell extracts with high inhibitory activity towards GSK-3,/, were fractionated, and the IC50 values of twelve individual indigoids were measured for GSK-3,/, as well as the protein kinases CDK1/cyclinB and CDK5/p25. Several indigoids, including an indigo, showed stronger inhibition than found in previous work. The most potent towards GSK-3,/,, dimethyl indirubin 5,5,-dicarboxylate (IC50 of 51,nM), was modified by chemical reactions. One product, indirubin 5,5,-dicarboxylic acid 5-methyl ester, inhibited GSK-3,/, with an IC50 of 14,nM and selectivity nearly 40-fold over CDK1 and CDK5. Indirubin-5-5,-dicarbonitrile was also modified to the corresponding 3,-oxime, which had low specificity but showed very high inhibition of all three kinases with IC50 values of 5, 13, and 10,nM towards GSK-3,/,, CDK1, and CDK5, respectively. Thus, this system has the potential to generate new indigoids with therapeutic potential. [source] Synthesis and Evaluation of 1-(1-(Benzo[b]thiophen-2-yl)cyclohexyl)piperidine (BTCP) Analogues as Inhibitors of Trypanothione ReductaseCHEMMEDCHEM, Issue 8 2009Stephen Patterson Dr. Abstract Thirty two analogues of phencyclidine were synthesised and tested as inhibitors of trypanothione reductase (TryR), a potential drug target in trypanosome and leishmania parasites. The lead compound BTCP (1, 1-(1-benzo[b]thiophen-2-yl-cyclohexyl) piperidine) was found to be a competitive inhibitor of the enzyme (Ki=1,,M) and biologically active against bloodstream T.,brucei (EC50=10,,M), but with poor selectivity against mammalian MRC5 cells (EC50=29,,M). Analogues with improved enzymatic and biological activity were obtained. The structure,activity relationships of this novel series are discussed. [source] Molecular recognition: Identifying compounds and their targetsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue S37 2001Prabhavathi B. Fernandes Ph.D. Abstract As a result of gene sequencing and proteomic efforts, thousands of new genes and proteins are now available as potential drug targets. The milieu of these proteins is complex and interactive; thousands of proteins activate, inhibit, and control each other's actions. The effect of blocking or activating a protein in a cell is far-reaching, and can affect whole, as well as adjacent pathways. This network of pathways is dynamic and a cellular response can change depending on the stimulus. In this section, the identification and role of individual proteins within the context of networked pathways, and the regulation of the activity of these proteins is discussed. Diverse chemical libraries, combinatorial libraries, natural products, as well as unnatural natural products that are derived from combinatorial biology (Chiu [2001] Proc. Natl. Acad. Sci. USA. 98:8548,8553), provide the chemical diversity in the search for new drugs to block new targets. Identifying new compounds that can become drugs is a long, expensive, and arduous task and potential targets must be carefully defined so as not to waste valuable resources. Equally important is the selection of compounds to be future drug candidates. Target selectivity in no way guarantees clinical efficacy, as the compound must meet pharmaceutical requirements, such as solubility, absorption, tissue distribution, and lack of toxicity. Thus matching biological diversity with chemical diversity involves something more than tight interactions, it involves interactions of the compounds with a variety host factors that can modulate its activity. J. Cell. Biochem. Suppl. 37: 1,6, 2001. © 2002 Wiley-Liss, Inc. [source] Microsomal prostaglandin E synthase-1 and 5-lipoxygenase: potential drug targets in cancerJOURNAL OF INTERNAL MEDICINE, Issue 1 2010O. Rådmark Abstract., Rådmark O, Samuelsson B (Karolinska Institutet, Stockholm, Sweden). Microsomal prostaglandin Esynthase-1 and 5-lipoxygenase: potential drug targets in cancer (Review). J Intern Med 2010; 268:5,14. There is strong evidence for a role of prostaglandin (PG)E2 in cancer cell proliferation and tumour development. In PGE2 biosynthesis, cyclooxygenases (COX-1/2) convert arachidonic acid to PGH2, which can be isomerized to PGE2 by PGE synthases, including microsomal PGE synthase-1 (MPGES-1). Data describing genetic deletions of MPGES-1 are reviewed. The results suggest that MPGES-1 is an alternative therapeutic target for cancer cells and tumours that express this enzyme. Several compounds that target COX-2 or MPGES-1 also inhibit 5-lipoxygenase. This may be advantageous for treatment of some forms of cancer. [source] Intra-articular depot formulation principles: Role in the management of postoperative pain and arthritic disordersJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 11 2008Claus Larsen Abstract The joint cavity constitutes a discrete anatomical compartment that allows for local drug action after intra-articular injection. Drug delivery systems providing local prolonged drug action are warranted in the management of postoperative pain and not least arthritic disorders such as osteoarthritis. The present review surveys various themes related to the accomplishment of the correct timing of the events leading to optimal drug action in the joint space over a desired time period. This includes a brief account on (patho)physiological conditions and novel potential drug targets (and their location within the synovial space). Particular emphasis is paid to (i) the potential feasibility of various depot formulation principles for the intra-articular route of administration including their manufacture, drug release characteristics and in vivo fate, and (ii) how release, mass transfer and equilibrium processes may affect the intra-articular residence time and concentration of the active species at the ultimate receptor site. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:4622,4654, 2008 [source] Proteome analysis of apoptosis signaling by S -trityl- L -cysteine, a potent reversible inhibitor of human mitotic kinesin Eg5PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 2 2008Frank Kozielski Abstract Mitotic kinesins represent potential drug targets for anticancer chemotherapy. Inhibitors of different chemical classes have been identified that target human Eg5, a kinesin responsible for the establishment of the bipolar spindle. One potent Eg5 inhibitor is S -trityl- L -cysteine (STLC), which arrests cells in mitosis and exhibits tumor growth inhibition activity. However, the underlying mechanism of STLC action on the molecular level is unknown. Here, cells treated with STLC were blocked in mitosis through activation of the spindle assembly checkpoint as shown by the phosphorylated state of BubR1 and the accumulation of mitosis specific phosphorylation on histone H3 and aurora A kinase. Using live cell imaging, we observed prolonged mitotic arrest and subsequent cell death after incubation of GFP-,-tubulin HeLa cells with STLC. Activated caspase-9 occurred before cleavage of caspase-8 leading to the accumulation of the activated executioner caspase-3 suggesting that STLC induces apoptosis through the intrinsic apoptotic pathway. Proteome analysis following STLC treatment revealed 33 differentially regulated proteins of various cellular processes, 31 of which can be linked to apoptotic cell death. Interestingly, four identified proteins, chromobox protein homolog, RNA-binding Src associated in mitosis 68,kDa protein, stathmin, and translationally controlled tumor protein can be linked to mitotic and apoptotic processes. [source] Structures of dihydrofolate reductase-thymidylate synthase of Trypanosoma cruzi in the folate-free state and in complex with two antifolate drugs, trimetrexate and methotrexateACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2009Olga Senkovich 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. [source] Lysosomal cysteine proteases (cathepsins): promising drug targetsACTA CRYSTALLOGRAPHICA SECTION D, Issue 2 2003an Turk Papain-like lysosomal cysteine proteases are processive and digestive enzymes expressed in organisms from bacteria to humans. Their ubiquity alone makes them potential drug targets, with the assumption that appropriate specificities may be achieved. These enzymes have rather short active-site clefts, comprising three well defined substrate-binding subsites (S2, S1 and S1,) and additionally have comparatively broad binding areas (S4, S3, S2,, S3,). This geometry distinguishes them from other protease classes, such as serine and aspartic proteases, with six and eight substrate-binding sites, respectively. Exopeptidases (cathepsins B, C, H and X), in contrast to endopeptidases (such as cathepsins L, S, V and F), possess structural features that facilitate binding of N- and C-terminal groups of substrates in the active-site cleft. Other than a clear preference for free chain termini in the case of exopeptidases, the substrate-binding sites exhibit no strict specificities. Instead, their subsite preferences arise more from specific exclusions of substrate type. This presents a challenge for the design of inhibitors to target a specific cathepsin: only the cumulative effect of an assembly of inhibitor fragments can produce the desired result. The small number of papain-like lysosomal cysteine proteases (11 human enzymes are known) and the small number of substrate-binding sites calls for a innovative and empirical approach. [source] A triclinic crystal form of Escherichia coli 4-diphosphocytidyl-2C -methyl- d -erythritol kinase and reassessment of the quaternary structureACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2010Justyna Kalinowska-T 4-Diphosphocytidyl-2C -methyl- d -erythritol kinase (IspE; EC 2.7.1.148) contributes to the 1-deoxy- d -xylulose 5-phosphate or mevalonate-independent biosynthetic pathway that produces the isomers isopentenyl diphosphate and dimethylallyl diphosphate. These five-carbon compounds are the fundamental building blocks for the biosynthesis of isoprenoids. The mevalonate-independent pathway does not occur in humans, but is present and has been shown to be essential in many dangerous pathogens, i.e. Plasmodium species, which cause malaria, and Gram-negative bacteria. Thus, the enzymes involved in this pathway have attracted attention as potential drug targets. IspE produces 4-diphosphosphocytidyl-2C -methyl- d -erythritol 2-phosphate by ATP-dependent phosphorylation of 4-diphosphocytidyl-2C -methyl- d -erythritol. A triclinic crystal structure of the Escherichia coli IspE,ADP complex with two molecules in the asymmetric unit was determined at 2,Å resolution and compared with a monoclinic crystal form of a ternary complex of E. coli IspE also with two molecules in the asymmetric unit. The molecular packing is different in the two forms. In the asymmetric unit of the triclinic crystal form the substrate-binding sites of IspE are occluded by structural elements of the partner, suggesting that the `triclinic dimer' is an artefact of the crystal lattice. The surface area of interaction in the triclinic form is almost double that observed in the monoclinic form, implying that the dimeric assembly in the monoclinic form may also be an artifact of crystallization. [source] Automated classification of Plasmodium sporozoite movement patterns reveals a shift towards productive motility during salivary gland infectionBIOTECHNOLOGY JOURNAL, Issue 6 2009Stephan Hegge Abstract The invasive stages of malaria and other apicomplexan parasites use a unique motility machinery based on actin, myosin and a number of parasite-specific proteins to invade host cells and tissues. The crucial importance of this motility machinery at several stages of the life cycle of these parasites makes the individual components potential drug targets. The different stages of the malaria parasite exhibit strikingly diverse movement patterns, likely reflecting the varied needs to achieve successful invasion. Here, we describe a Tool for Automated Sporozoite Tracking (ToAST) that allows the rapid simultaneous analysis of several hundred motile Plasmodium sporozoites, the stage of the malaria parasite transmitted by the mosquito. ToAST reliably categorizes different modes of sporozoite movement and can be used for both tracking changes in movement patterns and comparing overall movement parameters, such as average speed or the persistence of sporozoites undergoing a certain type of movement. This allows the comparison of potentially small differences between distinct parasite populations and will enable screening of drug libraries to find inhibitors of sporozoite motility. Using ToAST, we find that isolated sporozoites change their movement patterns towards productive motility during the first week after infection of mosquito salivary glands. [source] Proteome mapping of overexpressed membrane-enriched and cytosolic proteins in sodium antimony gluconate (SAG) resistant clinical isolate of Leishmania donovaniBRITISH JOURNAL OF CLINICAL PHARMACOLOGY, Issue 4 2010Awanish Kumar WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT Over 60% of patients with visceral leishmaniasis (VL) in India and Sudan have become unresponsive to treatment with pentavalent antimonials, the first line of drugs for over 60 years. The drug resistance mechanism, studied so far in in vitro selected laboratory strains, has been attributed to various biochemical parameters. The resistance to Sb (V) in Leishmania field isolates is still unexplored. WHAT THIS STUDY ADDS In order to elucidate for the first time the mechanism of drug resistance in field isolates, this study was done in those clinically relevant field isolates which were either responsive or non responsive to SAG. A comparison of proteome profiles of membrane-enriched as well as cytosolic protein fractions of these isolates has pinpointed the multiple overexpressed proteins in resistant isolates. This study has indicated their possible essential role in antimony resistance of the parasite and provides a vast field to be exploited to find much needed novel treatment strategies against VL. AIMS This study aimed to identify differentially overexpressed membrane-enriched as well as cytosolic proteins in SAG sensitive and resistant clinical strains of L. donovani isolated from VL patients which are involved in the drug resistance mechanism. METHODS The proteins in the membrane-enriched as well as cytosolic fractions of drug-sensitive as well as drug-resistant clinical isolates were separated using two-dimensional gel electrophoresis and overexpressed identified protein spots of interest were excised and analysed using MALDI-TOF/TOF. RESULTS Six out of 12 overexpressed proteins were identified in the membrane-enriched fraction of the SAG resistant strain of L. donovani whereas 14 out of 18 spots were identified in the cytosolic fraction as compared with the SAG sensitive strain. The major proteins in the membrane-enriched fraction were ABC transporter, HSP-83, GPI protein transamidase, cysteine,leucine rich protein and 60S ribosomal protein L23a whereas in the cytosolic fraction proliferative cell nuclear antigen (PCNA), proteasome alpha 5 subunit, carboxypeptidase, HSP-70, enolase, fructose-1,6-bisphosphate aldolase, tubulin-beta chain have been identified. Most of these proteins have been reported as potential drug targets, except 60S ribosomal protein L23a and PCNA which have not been reported to date for their possible involvement in drug resistance against VL. CONCLUSION This study for the first time provided a cumulative proteomic analysis of proteins overexpressed in drug resistant clinical isolates of L. donovani indicating their possible role in antimony resistance of the parasite. Identified proteins provide a vast field to be exploited for novel treatment strategies against VL such as cloning and overexpression of these targets to produce recombinant therapeutic/prophylactic proteins. [source] | ||||||||||||||||||||||||||||