Identification of potent inhibitors for Leishmania donovani homoserine kinase: an integrated in silico and kinetic study.

Leishmaniasis is caused by ∼20 species of Leishmania that affects millions in endemic areas. Available therapies are not sufficient to effectively control the disease, cause severe side effects and eventually lead to drug resistance, making the discovery of novel therapeutic molecules an immediate need. Molecular target-based drug discovery, where the target is a defined molecular gene, protein or a mechanism, is a rationale driven approach for novel therapeutics. Humans obtain the essential amino acid such as threonine from dietary sources, while Leishmania synthesize it de-novo. Enzymes of the threonine biosynthesis pathway, including the rate limiting Homoserine kinase (HSK) which converts L-homoserine into ortho-phospho homoserine are thus attractive targets for rationale driven therapy. The absence of HSK in humans and its presence in Leishmania donovani enhances the opportunity to exploit HSK as a molecular target for anti-leishmanials therapeutic development. In this study, we utilize structure-based high throughput drug discovery (SBDD), followed by biochemical validation and identified two potential inhibitors (RH00038 and S02587) from Maybridge chemical library that targets L. donovani HSK. These two inhibitors effectively induced the mortality of Leishmania donovani in both amastigote and promastigote stages, with one of them being specific to parasite and twice as effective as the standard therapeutic molecule.

Crystal structure and characterization of nucleoside diphosphate kinase from Vibrio cholerae.

Nucleoside diphosphate kinases (NDK) are ubiquitous enzymes that catalyse the transfer of the γ phosphate from nucleoside triphosphates (NTPs) to nucleoside diphosphate (NDPs), to maintain appropriate NTP levels in cells. NDKs are associated with signal transduction, cell development, proliferation, differentiation, tumor metastasis, apoptosis and motility. The critical role of NDK in bacterial virulence renders it a potential drug target. The present manuscript reports crystal structure and functional characterization of Vibrio cholerae NDK (VNDK). The 16 kDa VNDK was crystallized in a solution containing 30% PEG 4000, 100 mM Tris-HCl pH 8.5 and 200 mM sodium acetate in orthorhombic space group P212121 with unit cell parameters a = 48.37, b = 71.21, c = 89.14 Å, α = ß = Î³ = 90° with 2 molecules in asymmetric unit. The crystal structure was solved by molecular replacement and refined to crystallographic Rfactor and Rfree values of 22.8% and 25.8% respectively. VNDK exists as both dimer and tetramer in solution as confirmed by size exclusion chromatography, glutaraldehyde crosslinking and small angle X-ray scattering while the crystal structure appears to be a dimer. The biophysical characterization states that VNDK has kinase and DNase activity with maximum stability at pH 8-9 and temperature up to 40 °C. VNDK shows elevated thermolability as compared to other NDK and shows preferential binding with GTP rationalized using computational studies.

PfKsgA1 functions as a transcription initiation factor and interacts with the N-terminal region of the mitochondrial RNA polymerase of Plasmodium falciparum.

The small mitochondrial genome (mtDNA) of the malaria parasite is known to transcribe its genes polycistonically, although promoter element(s) have not yet been identified. An unusually large Plasmodium falciparum candidate mitochondrial phage-like RNA polymerase (PfmtRNAP) with an extended N-terminal region is encoded by the parasite nuclear genome. Using specific antibodies against the enzyme, we established that PfmtRNAP was targeted exclusively to the mitochondrion and interacted with mtDNA. Phylogenetic analysis showed that it is part of a separate apicomplexan clade. A search for PfmtRNAP-associated transcription initiation factors using sequence homology and in silico protein-protein interaction network analysis identified PfKsgA1. PfKsgA1 is a dual cytosol- and mitochondrion-targeted protein that functions as a small subunit rRNA dimethyltransferase in ribosome biogenesis. Chromatin immunoprecipitation showed that PfKsgA1 interacts with mtDNA, and in vivo crosslinking and pull-down experiments confirmed PfmtRNAP-PfKsgA1 interaction. The ability of PfKsgA1 to serve as a transcription initiation factor was demonstrated by complementation of yeast mitochondrial transcription factor Mtf1 function in Rpo41-driven in vitro transcription. Pull-down experiments using PfKsgA1 and PfmtRNAP domains indicated that the N-terminal region of PfmtRNAP interacts primarily with the PfKsgA1 C-terminal domain with some contacts being made with the linker and N-terminal domain of PfKsgA1. In the absence of full-length recombinant PfmtRNAP, solution structures of yeast mitochondrial RNA polymerase Rpo41 complexes with Mtf1 or PfKsgA1 were determined by small-angle X-ray scattering. Protein interaction interfaces thus identified matched with those reported earlier for Rpo41-Mtf1 interaction and overlaid with the PfmtRNAP-interfacing region identified experimentally for PfKsgA1. Our results indicate that in addition to a role in mitochondrial ribosome biogenesis, PfKsgA1 has an independent function as a transcription initiation factor for PfmtRNAP.

The coiled-coil domain of glycosomal membrane-associated Leishmania donovani PEX14: cloning, overexpression, purification and preliminary crystallographic analysis.

The glycosomal membrane-associated Leishmania donovani protein PEX14, which plays a crucial role in protein import from the cytosol to the glycosomal matrix, consists of three domains: an N-terminal domain where the signalling molecule binds, a transmembrane domain and an 84-residue coiled-coil domain (CC) that is responsible for oligomerization. CCs are versatile domains that participate in a variety of functions including supramolecular assembly, cellular signalling and transport. Recombinant PEX14 CC was cloned, overexpressed, affinity-purified with in-column thrombin cleavage and further purified by size-exclusion chromatography. Crystals that diffracted to 1.98 Šresolution were obtained from a condition consisting of 1.4 M sodium citrate tribasic dihydrate, 0.1 M HEPES buffer pH 7.5. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 143.98, b = 32.62, c = 95.62 Å, ß = 94.68°. Structure determination and characterization are in progress.

Biochemical and biophysical characterization of Leishmania donovani gamma-glutamylcysteine synthetase.

γ-glutamylcysteine synthetase (Gcs) is a vital enzyme catalyzing the first and rate limiting step in the trypanothione biosynthesis pathway, the ATP-dependent ligation of L-Glutamate and L-Cysteine to form gamma-glutamylcysteine. The Trypanothione biosynthesis pathway is unique metabolic pathway essential for trypanosomatid survival rendering Gcs as a potential drug target. Here we report the cloning, expression, purification and characterization of L. donovani Gcs. Three other constructs of Gcs (GcsN, GcsC and GcsT) were designed on the basis of S. cerevisiae and E. coli Gcs crystal structures. The study shows Gcs possesses ATPase activity even in the absence of substrates L-glutamate and L-Cysteine. Divalent ions however plays an indispensable role in LdGcs ATPase activity. Isothermal titration calorimetry and fluorescence studies illustrates that L. donovani Gcs binds substrate in order ATP >L-glutamate>L-cysteine with Glu 92 and Arg 498 involved in ATP hydrolysis and Glu 92, Glu 55 and Arg 498 involved in glutamate binding. Homology modeling and molecular dynamic simulation studies provided the structural rationale of LdGcs catalytic activity and emphasized on the possibility of involvement of three Mg2+ ions along with Glutamates 52, 55, 92, 99, Met 322, Gln 328, Tyr 397, Lys 483, Arg 494 and Arg 498 in the catalytic function of L. donovani Gcs.