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Pradipsinh K Rathod

Publications and source records attributed to Pradipsinh K Rathod.

11 recordsLinked to original sources
PubMed2006

Resistance mutations at the lipid substrate binding site of Plasmodium falciparum protein farnesyltransferase.

The post-translational farnesylation of proteins serves to anchor a subset of intracellular proteins to membranes in eukaryotic organisms and also promotes protein-protein interactions. This enzymatic reaction is carried out by protein farnesyltransferase (PFT), which catalyzes the transfer of a 15-carbon isoprenoid lipid unit, a farnesyl group, from farnesyl pyrophosphate to the C-termini of proteins containing a CaaX motif. Inhibition of PFT is lethal to the pathogenic protozoa Plasmodium falciparum. Previously, we have shown that parasites resistant to a tetrahydroquinoline (THQ)-based PFT inhibitor BMS-388891 have mutations leading to amino acid substitutions in PFT that map to the peptide substrate binding domain. We now report the selection of parasites resistant to another THQ PFT inhibitor BMS-339941. In whole cell assays sensitivity to BMS-339941 was reduced by 33-fold in a resistant clone, and biochemical analysis demonstrated a corresponding 33-fold increase in the BMS-339941 K(i) for the mutant PFT enzyme. More detailed kinetic analysis revealed that the mutant enzyme required higher concentration of peptide and farnesyl pyrophosphate substrates for optimum catalysis. Unlike previously characterized parasites resistant to BMS-388891, the resistant parasites have a mutation which is predicted to be in a distinct location of the enzymatic pocket, near the farnesyl pyrophosphate binding pocket. This is the first description of a mutation from any species affecting the farnesyl pyrophosphate binding pocket with reduced efficacy of PFT inhibitors. These data provide further support that PFT is the target of THQ inhibitors in P. falciparum and suggest that PFT inhibitors should be combined with other antimalarial agents to minimize the development of resistant parasites.

Alkyl and Aryl Transferases↗
PubMed2005

High-throughput screening for potent and selective inhibitors of Plasmodium falciparum dihydroorotate dehydrogenase.

Plasmodium falciparum is the causative agent of the most serious and fatal malarial infections, and it has developed resistance to commonly employed chemotherapeutics. The de novo pyrimidine biosynthesis enzymes offer potential as targets for drug design, because, unlike the host, the parasite does not have pyrimidine salvage pathways. Dihydroorotate dehydrogenase (DHODH) is a flavin-dependent mitochondrial enzyme that catalyzes the fourth reaction in this essential pathway. Coenzyme Q (CoQ) is utilized as the oxidant. Potent and species-selective inhibitors of malarial DHODH were identified by high-throughput screening of a chemical library, which contained 220,000 drug-like molecules. These novel inhibitors represent a diverse range of chemical scaffolds, including a series of halogenated phenyl benzamide/naphthamides and urea-based compounds containing napthyl or quinolinyl substituents. Inhibitors in these classes with IC(50) values below 600 nm were purified by high pressure liquid chromatography, characterized by mass spectroscopy, and subjected to kinetic analysis against the parasite and human enzymes. The most active compound is a competitive inhibitor of CoQ with an IC(50) against malarial DHODH of 16 nm, and it is 12,500-fold less active against the human enzyme. Site-directed mutagenesis of residues in the CoQ-binding site significantly reduced inhibitor potency. The structural basis for the species selective enzyme inhibition is explained by the variable amino acid sequence in this binding site, making DHODH a particularly strong candidate for the development of new anti-malarial compounds.

Animals↗
PubMed2005

Resistance to a protein farnesyltransferase inhibitor in Plasmodium falciparum.

The post-translational farnesylation of proteins serves to anchor a subset of intracellular proteins to membranes in eukaryotic organisms and also promotes protein-protein interactions. Inhibition of protein farnesyltransferase (PFT) is lethal to the pathogenic protozoa Plasmodium falciparum. Parasites were isolated that were resistant to BMS-388891, a tetrahydroquinoline (THQ) PFT inhibitor. Resistance was associated with a 12-fold decrease in drug susceptibility. Genotypic analysis revealed a single point mutation in the beta subunit in resistant parasites. The resultant tyrosine 837 to cysteine alteration in the beta subunit corresponded to the binding site for the THQ and peptide substrate. Biochemical analysis of Y837C-PFT demonstrated a 13-fold increase in BMS-388891 concentration necessary for inhibiting 50% of the enzyme activity. These data are consistent with PFT as the target of BMS-388891 in P. falciparum and suggest that PFT inhibitors should be combined with other antimalarial agents for effective therapy.

Alkyl and Aryl Transferases↗
PubMed2003

A microfluidic model for single-cell capillary obstruction by Plasmodium falciparum-infected erythrocytes.

Severe malaria by Plasmodium falciparum is a potentially fatal disease, frequently unresponsive to even the most aggressive treatments. Host organ failure is associated with acquired rigidity of infected red blood cells and capillary blockage. In vitro techniques have played an important role in modeling cell deformability. Although, historically they have either been applied to bulk cell populations or to measure single physical parameters of individual cells. In this article, we demonstrate the unique abilities and benefits of elastomeric microchannels to characterize complex behaviors of single cells, under flow, in multicellular capillary blockages. Channels of 8-, 6-, 4-, and 2-microm widths were readily traversed by the 8 microm-wide, highly elastic, uninfected red blood cells, as well as by infected cells in the early ring stages. Trophozoite stages failed to freely traverse 2- to 4-microm channels; some that passed through the 4-microm channels emerged from constricted space with deformations whose shape-recovery could be observed in real time. In 2-microm channels, trophozoites mimicked "pitting," a normal process in the body where spleen beds remove parasites without destroying the red cell. Schizont forms failed to traverse even 6-microm channels and rapidly formed a capillary blockage. Interestingly, individual uninfected red blood cells readily squeezed through the blockages formed by immobile schizonts in a 6-microm capillary. The last observation can explain the high parasitemia in a growing capillary blockage and the well known benefits of early blood transfusion in severe malaria.

Animals↗
PubMed2002

Stochastic versus stable transcriptional differences on Plasmodium falciparum DNA microarrays.

The recent availability of the Plasmodium falciparum genome sequence has opened up convenient, large-scale analysis of transcriptional products in malaria. Protocols for cDNA labelling, cDNA hybridisation, and fluorescent signal detection developed for other organisms can be applied directly to malaria. However, P. falciparum offers unique challenges in data analysis due to stochastic variability in expression of some gene products, such as variable erythrocyte surface proteins. Careful comparison of global transcriptional patterns in two well-studied clones of P. falciparum (Dd2 and HB3) indicates that reliable, stable transcriptional alterations in malaria can be readily distinguished from stochastic processes. To do this, we utilised a complex experimental design which involves a combination of self-hybridisations and cross-hybridisations between two independently grown parasite populations for each clone being examined (for short, we call this a '2x2 CombiScan'). While even a simple 2x2 CombiScan required 12 microarray hybridisations, the effort generated output that was highly interpretable. Reliable RNA transcriptional differences between Dd2 and HB3 could be readily visualised using public algorithms for data normalisation and clustering.

Animals↗
PubMed2002

Malarial dihydroorotate dehydrogenase. Substrate and inhibitor specificity.

The malarial parasite relies on de novo pyrimidine biosynthesis to maintain its pyrimidine pools, and unlike the human host cell it is unable to scavenge preformed pyrimidines. Dihydroorotate dehydrogenase (DHODH) catalyzes the oxidation of dihydroorotate (DHO) to produce orotate, a key step in pyrimidine biosynthesis. The enzyme is located in the outer membrane of the mitochondria of the malarial parasite. To characterize the biochemical properties of the malarial enzyme, an N-terminally truncated version of P. falciparum DHODH has been expressed as a soluble, active enzyme in E. coli. The recombinant enzyme binds 0.9 molar equivalents of the cofactor FMN and it has a pH maximum of 8.0 (k(cat) 8 s(-1), K(m)(app) DHO (40-80 microm)). The substrate specificity of the ubiquinone cofactor (CoQ(n)) that is required for the oxidation of FMN in the second step of the reaction was also determined. The isoprenoid (n) length of CoQ(n) was a determinant of reaction efficiency; CoQ(4), CoQ(6) and decylubiquinone (CoQ(D)) were efficiently utilized in the reaction, however cofactors lacking an isoprenoid tail (CoQ(0) and vitamin K(3)) showed decreased catalytic efficiency resulting from a 4 to 7-fold increase in K(m)(app). Five potent inhibitors of mammalian DHODH, Redoxal, dichloroallyl lawsone (DCL), and three analogs of A77 1726 were tested as inhibitors of the malarial enzyme. All five compounds were poor inhibitors of the malarial enzyme, with IC(50)'s ranging from 0.1-1.0 mm. The IC(50) values for inhibition of the malarial enzyme are 10(2)-10(4)-fold higher than the values reported for the mammalian enzyme, demonstrating that inhibitor binding to DHODH is species specific. These studies provide direct evidence that the malarial DHODH active site is different from the host enzyme, and that it is an attractive target for the development of new anti-malarial agents.

Amino Acid Sequence↗
PubMed2002

Divergent regulation of dihydrofolate reductase between malaria parasite and human host.

For half a century, successful antifolate therapy against Plasmodium falciparum malaria has been attributed to host-parasite differences in drug binding to dihydrofolate reductase-thymidylate synthase (DHFR-TS). Selectivity may also arise through previously unappreciated differences in regulation of this drug target. The DHFR-TS of Plasmodium binds its cognate messenger RNA (mRNA) and inhibits its own translation. However, unlike translational regulation of DHFR or TS in humans, DHFR-TS mRNA binding is not coupled to enzyme active sites. Thus, antifolate treatment does not relieve translational inhibition and parasites cannot replenish dead enzyme.

Animals↗
PubMed2002

DNA microarrays for malaria.

DNA microarrays are a powerful tool for the analysis of RNA and DNA composition on a whole-genome scale. The first applications of this technology in parasitology are in place. This review examines the various approaches to Plasmodium transcript-profiling that are being adopted using DNA microarray analysis and discusses additional strategies for obtaining and collating information relevant to the search for drug and vaccine candidates in malaria.

Animals↗