Cloning, Expression and Characterization of Spore Wall Protein 5 (SWP5) of Indian Isolate NIK-1S of Nosema bombycis.

SWPs are the major virulence component of microsporidian spores. In microsporidia, SWPs can be found either in exospore or endospore to serve as a putative virulence factor for host cell invasion. SWP5 is a vital protein that involves in exospore localization and supports the structural integrity of the spore wall and this action potentially modulates the course of infection in N. bombycis. Here we report recombinant SWP5 purification using Ni-NTA IMAC and SEC. GFC analysis reveals SWP5 to be a monomer which correlates with the predicted theoretical weight and overlaps with ovalbumin peak in the chromatogram. The raised polyclonal anti-SWP5 antibodies was confirmed using blotting and enterokinase cleavage experiments. The resultant fusion SWP5 and SWP5 in infected silkworm samples positively reacts to anti-SWP5 antibodies is shown in ELISA. Immunoassays and Bioinformatic analysis reveal SWP5 is found to be localized on exospore and this action could indicate the probable role of SWP5 in host pathogen interactions during spore germination and its contribution to microsporidian pathogenesis. This study will support development of a field-based diagnostic kit for the detection N. bombycis NIK-1S infecting silkworms. The analysis will also be useful for the formulation of drugs against microsporidia and pebrine disease.

Profilin is involved in G1 to S phase progression and mitotic spindle orientation during Leishmania donovani cell division cycle.

Profilin is a multi-ligand binding protein, which is a key regulator of actin dynamics and involved in regulating several cellular functions. It is present in all eukaryotes, including trypanosomatids such as Leishmania. However, not much is known about its functions in these organisms. Our earlier studies have shown that Leishmania parasites express a single homologue of profilin (LdPfn) that binds actin, phosphoinositides and poly- L- proline motives, and depletion of its intracellular pool to 50%of normal levels affects the cell growth and intracellular trafficking. Here, we show, employing affinity pull-down and mass spectroscopy, that LdPfn interacted with a large number of proteins, including those involved in mRNA processing and protein translation initiation, such as eIF4A1. Further, we reveal, using mRNA Seq analysis, that depletion of LdPfn in Leishmania cells (LdPfn+/-) resulted in significantly reduced expression of genes which encode proteins involved in cell cycle regulation, mRNA translation initiation, nucleosides and amino acids transport. In addition, we show that in LdPfn+/- cells, cellular levels of eIF4A1 protein were significantly decreased, and during their cell division cycle, G1-to-S phase progression was delayed and orientation of mitotic spindle altered. These changes were, however, reversed to normal by episomal expression of GFP-LdPfn in LdPfn+/- cells. Taken together, our results indicate that profilin is involved in regulation of G1-to-S phase progression and mitotic spindle orientation in Leishmania cell cycle, perhaps through its interaction with elF4A1 protein.

Discovery of MAP855, an Efficacious and Selective MEK1/2 Inhibitor with an ATP-Competitive Mode of Action.

Mutations in MEK1/2 have been described as a resistance mechanism to BRAF/MEK inhibitor treatment. We report the discovery of a novel ATP-competitive MEK1/2 inhibitor with efficacy in wildtype (WT) and mutant MEK12 models. Starting from a HTS hit, we obtained selective, cellularly active compounds that showed equipotent inhibition of WT MEK1/2 and a panel of MEK1/2 mutant cell lines. Using a structure-based approach, the optimization addressed the liabilities by systematic analysis of molecular matched pairs (MMPs) and ligand conformation. Addition of only three heavy atoms to early tool compound 6 removed Cyp3A4 liabilities and increased the cellular potency by 100-fold, while reducing log P by 5 units. Profiling of MAP855, compound 30, in pharmacokinetic-pharmacodynamic and efficacy studies in BRAF-mutant models showed comparable efficacy to clinical MEK1/2 inhibitors. Compound 30 is a novel highly potent and selective MEK1/2 kinase inhibitor with equipotent inhibition of WT and mutant MEK1/2, whose drug-like properties allow further investigation in the mutant MEK setting upon BRAF/MEK therapy.

Draft Genome Sequence of the Community-Associated Staphylococcus aureus Sequence Type 88 Strain LVP-7, Isolated from an Ocular Infection.

We report a de novo-assembled draft genome sequence of the Indian Staphylococcus aureus sequence type 88 (ST88) strain LVP-7, isolated from an ocular infection. The genome harbors a Panton-Valentine leukocidin phage, a type V staphylococcal cassette chromosome mec element, the delta-hemolysin-converting Newman phage ΦNM3, and the pathogenicity island SaPI3, encoding the superantigen enterotoxin B.

Discovery of CA-4948, an Orally Bioavailable IRAK4 Inhibitor for Treatment of Hematologic Malignancies.

Small molecule potent IRAK4 inhibitors from a novel bicyclic heterocycle class were designed and synthesized based on hits identified from Aurigene's compound library. The advanced lead compound, CA-4948, demonstrated good cellular activity in ABC DLBCL and AML cell lines. Inhibition of TLR signaling leading to decreased IL-6 levels was also observed in whole blood assays. CA-4948 demonstrated moderate to high selectivity in a panel of 329 kinases as well as exhibited desirable ADME and PK profiles including good oral bioavailability in mice, rat, and dog and showed >90% tumor growth inhibition in relevant tumor models with excellent correlation with in vivo PD modulation. CA-4948 was well tolerated in toxicity studies in both mouse and dog at efficacious exposure. The overall profile of CA-4948 prompted us to select it as a clinical candidate for evaluation in patients with relapsed or refractory hematologic malignancies including non-Hodgkin lymphoma and acute myeloid leukemia.

Network pharmacology study of Curcuma longa L.: potential target proteins and their functional enrichment analysis.

OBJECTIVE: This study's primary goal is unraveling the mechanism of action of bioactives of Curcuma longa L. at the molecular level using protein-protein interaction network. RESULTS: We used target proteins to create protein-protein interaction network (PPIN) and identified significant node and edge attributes of PPIN. We identified the cluster of proteins in the PPIN, which were used to identify enriched pathways. We identified closeness centrality and jaccard score as most important node and edge attribute of the PPIN respectively. The enriched pathways of various clusters were overlapped suggesting synergistic mechanism of action. The three pathways found to be common among three clusters were Gonadotropin-releasing hormone receptor pathway, Endothelin signaling pathway, and Inflammation mediated by chemokine and cytokine signaling pathway.

Actin sequestering protein, profilin, regulates intracellular vesicle transport in Leishmania.

Profilins are the key regulators of actin dynamics in all eukaryotic cells. However, little information is available on their biochemical properties and functions in kinetoplastids, such as Trypanosoma and Leishmania. We show here that Leishmania parasites express only one homolog of profilin (LdPfn), which catalyzes nucleotide exchange on G-actin and promotes actin polymerization at its low concentrations. However, at high concentrations, it strongly inhibits the polymerization process by sequestering actin monomers. We further demonstrate that LdPfn binds to actin in Leishmania promastigotes, by both immunofluorescence microscopy and IgG affinity chromatography. Further, we reveal that this protein besides binding to poly-null-proline motifs, also binds more efficiently to PI(3,5)P2, which is found on early or late endosomes or lysosomes, than to PI(4,5)P2 and PI(3,4,5)P3. Additionally, we show that heterozygous mutants of profilin display significantly slower growth and intracellular vesicle trafficking activity, which is reversed on episomal gene complementation. Together, these findings suggest that profilin regulates intracellular vesicle trafficking in Leishmania perhaps through its binding to polyphosphoinositides.

Discovery of O-(3-carbamimidoylphenyl)-l-serine amides as matriptase inhibitors using a fragment-linking approach.

Matriptase is a cell-surface trypsin-like serine protease of epithelial origin, which cleaves and activates proteins including hepatocyte growth factor/scatter factor and proteases such as uPA, which are involved in the progression of various cancers. Here we report a fragment-linking approach, which led to the discovery of O-(3-carbamimidoylphenyl)-l-serine amides as potent matriptase inhibitors. The co-crystal structure of one of the potent inhibitors, 6 in complex with matriptase catalytic domain validated the working hypothesis guiding the development of this congeneric series and revealed the structural basis for matriptase inhibition. Replacement of a naphthyl group in 6 with 2,4,6-tri-isopropyl phenyl resulted in 10 with improved matriptase inhibition, which exhibited significant primary tumor growth inhibition in a mouse model of prostate cancer. Compounds such as 10, identified using a fragment-linking approach, can be explored further to understand the role of matriptase as a drug target in cancer and inflammation.

Structure-guided discovery of 1,3,5 tri-substituted benzenes as potent and selective matriptase inhibitors exhibiting in vivo antitumor efficacy.

Matriptase is a serine protease implicated in cancer invasion and metastasis. Expression of matriptase is frequently dysregulated in human cancers and matriptase has been reported to activate latent growth factors such as hepatocyte growth factor/scatter factor, and proteases such as urokinase plasminogen activator suggesting that matriptase inhibitors could have therapeutic potential in treatment of cancer. Here we report a structure-based approach which led to the discovery of selective and potent matriptase inhibitors with benzene as central core having 1,3,5 tri-substitution pattern. X-ray crystallography of one of the potent analogs in complex with matriptase revealed strong hydrogen bonding and salt-bridge interactions in the S1 pocket, as well as strong CH-π contacts between the P2/P4 cyclohexyl and Trp215 side-chain. An additional interaction of the pendant amine at cyclohexyl with Gln175 side-chain results in substantial improvement in matriptase inhibition and selectivity against other related serine proteases. Compounds 15 and 26 showed tumor growth inhibition in a subcutaneous DU-145 prostate cancer mouse model. These compounds could be useful as tools to further explore the biology of matriptase as a drug target.

Discovery of Pyridyl Bis(oxy)dibenzimidamide Derivatives as Selective Matriptase Inhibitors.

Matriptase belongs to trypsin-like serine proteases involved in matrix remodeling/degradation, growth regulation, survival, motility, and cell morphogenesis. Herein, we report a structure-based approach, which led to the discovery of sulfonamide and amide derivatives of pyridyl bis(oxy)benzamidine as potent and selective matriptase inhibitors. Co-crystal structures of selected compounds in complex with matriptase supported compound designing. Additionally, WaterMap analyses indicated the possibility of occupying a distinct pocket within the catalytic domain, exploration of which resulted in >100-fold improvement in potency. Co-crystal structure of 10 with matriptase revealed critical interactions leading to potent target inhibition and selectivity against other serine proteases.

Crystal structure of binary and ternary complexes of serine hydroxymethyltransferase from Bacillus stearothermophilus: insights into the catalytic mechanism.

Serine hydroxymethyltransferase (SHMT), a member of the alpha-class of pyridoxal phosphate-dependent enzymes, catalyzes the reversible conversion of serine to glycine and tetrahydrofolate to 5,10-methylene tetrahydrofolate. We present here the crystal structures of the native enzyme and its complexes with serine, glycine, glycine, and 5-formyl tetrahydrofolate (FTHF) from Bacillus stearothermophilus. The first structure of the serine-bound form of SHMT allows identification of residues involved in serine binding and catalysis. The SHMT-serine complex does not show any significant conformational change compared with the native enzyme, contrary to that expected for a conversion from an "open" to "closed" form of the enzyme. However, the ternary complex with FTHF and glycine shows the reported conformational changes. In contrast to the Escherichia coli enzyme, this complex shows asymmetric binding of the FTHF to the two monomers within the dimer in a way similar to the murine SHMT. Comparison of the ternary complex with the native enzyme reveals the structural basis for the conformational change and asymmetric binding of FTHF. The four structures presented here correspond to the various reaction intermediates of the catalytic pathway and provide evidence for a direct displacement mechanism for the hydroxymethyl transfer rather than a retroaldol cleavage.