Synthesis, biological evaluation and docking studies of silicon incorporated diarylpyrroles as MmpL3 inhibitors: An effective strategy towards development of potent anti-tubercular agents.

Growing global demand for new molecules to treat tuberculosis has created an urgent need to develop novel strategies to combat the menace. BM212 related compounds were found to be potent anti-TB agents and they inhibit mycolic acid transporter, MmpL3, a known potent drug target from Mycobacterium tuberculosis. In order to enhance their inhibitory potency, several silicon analogues of diarylpyrroles related to BM212 were designed, synthesized, and evaluated for anti-tubercular activities. In Alamar blue assay, most of the silicon-incorporated compounds were found to be more potent than the parent compound (BM212), against Mycobacterium tuberculosis (MIC = 1.7 µM, H37Rv). Docking results from the crystal structure of MmpL3 and silicon analogues as pharmacophore model also strongly correlate with the biological assays and suggest that the incorporation of silicon in the inhibitor scaffold could enhance their potency by stabilizing the hydrophobic residues at the binding pocket. The best docking hit, compound 12 showed an MIC of 0.1 µM against H37Rv with an acceptable in vitro ADME profile and excellent selectivity index. Overall, the present study indicates that, the designed silicon analogues, especially compound 12 could be a good inhibitor for an intrinsically flexible drug-binding pocket of MmpL3 and has potential for further development as anti-tubercular agents.

Postmodification of voxelotor (GBT 440) via [Rh]-catalyzed cross dehydrogenative coupling with olefins.

The directed Rh(III)-catalyzed cross dehydrogenative coupling of the pyrazole unit of the GBT-440 scaffold has been explored with simple as well as conjugated olefins to synthesize post-functionalized GBT-440 analogues. The screening of these synthesized compounds for improving the oxygen binding efficiency of the hemoglobin isolated from the sickled red blood cells revealed that some of these compounds are as good as GBT-440.

Expression of HER2 and EGFR Proteins in Advanced Stage High-grade Serous Ovarian Tumors Show Mutual Exclusivity.

Human epidermal growth factors play an important role in ovarian carcinogenesis and are evaluated for prognostic and possible therapeutic roles in high-grade serous ovarian malignancies. The present study was undertaken to evaluate the expression of human epidermal growth factor receptor 2 (HER2) and epidermal growth factor receptor (EGFR) in advanced stage serous carcinoma and their influence on prognosis. The expression of HER2 and EGFR was studied in 59 cases of stage III and IV ovarian serous carcinomas by immunohistochemistry and fluorescent in situ hybridization. Of the 48 interpretable tumors for HER2, 6 tumors (12.5%) were scored as positive, 14 (29%) as equivocal and 28 tumors (58.5%) were negative by immunohistochemistry, while only 2/48 (4%) showed frank amplification by fluorescent in situ hybridization with ≥4 copies per cell. HER2 gene expression measured by quantitative polymerase chain reaction had good positive correlation with both protein expression and gene amplification. Although EGFR expression was seen in 32% of tumors, none of the tumors positive for HER2 protein or gene amplification had co-expression of EGFR indicating mutual exclusivity of their expression. Gene expression of both proteins also confirmed their inverse correlation (Pearsons CC=-0.15, P=0.3). Further there was no influence of protein or gene expression of these markers on the overall survival. In conclusion, HER2 and EGFR are expressed in a small percentage of tumors and the mutual exclusivity of these markers precludes the possibility of dual targeting with anti-HER2 and anti-EGFR therapy in advanced stage high-grade serous ovarian carcinoma.

Overturning the Peribysin Family Natural Products Isolated from Periconia byssoides OUPS-N133: Synthesis and Stereochemical Revision of Peribysins A, B, C, F, and G.

Herein we report the stereochemical revision of peribysins A, B, C, F, and G, guided by enantiospecific total synthesis starting from (+)-nootkatone. Furthermore, we reconfirmed the absolute stereochemistry of peribysin Q. The highlights of the synthesis are enone transposition and kinetic iodination resulting in separation of diastereomers. Our findings coupled with synthetic and biological data previously reported by Danishefsky's group suggest that the original stereochemistries of peribysins A, B, C, F, and G were misassigned.

Total Synthesis and Biological Evaluation of Cell Adhesion Inhibitors Peribysin A and B: Structural Revision of Peribysin B.

Total synthesis of potent cell-adhesion inhibitors peribysins A and B has been accomplished for the first time in racemic form. A Diels-Alder/aldol sequence to build the skeleton and decoration of the desired functionalities of the targeted natural products using highly stereoselective operations are the highlights. The structures of synthesized peribysins were fully characterized using spectral data and single-crystal X-ray analysis. Through this total synthesis, the initially proposed structure of peribysin B has been revised. Furthermore, the cell-adhesion inhibition potential of the scaffold (two peribysins + three analogues) was confirmed using anti-adhesion assay.

Tripeptides derived from reactive centre loop of potato type II protease inhibitors preferentially inhibit midgut proteases of Helicoverpa armigera.

Potato type II protease inhibitors (Pin-II PIs) impede the growth of lepidopteran insects by inhibiting serine protease-like enzymes in the larval gut. The three amino acid reactive centre loop (RCL) of these proteinaceous inhibitors is crucial for protease binding and is conserved across the Pin-II family. However, the molecular mechanism and inhibitory potential of the RCL tripeptides in isolation of the native protein has remained elusive. In this study, six peptides corresponding to the RCLs of the predominant Pin-II PIs were identified, synthesized and evaluated for in vitro and in vivo inhibitory activity against serine proteases of the polyphagous insect, Helicoverpa armigera. RCL peptides with sequences PRN, PRY and TRE were found to be potent inhibitors that adversely affected the growth and development of H. armigera. The binding mechanism and differential affinity of the RCL peptides with serine proteases was delineated by crystal structures of complexes of the RCL peptides with trypsin. Residues P1 and P2 of the inhibitors play a crucial role in the interaction and specificity of these inhibitors. Important features of RCL peptides like higher inhibition of insect proteases, enhanced efficacy at alkaline gut pH, longer retention and high stability in insect gut make them suitable molecules for the development of sustainable pest management strategies for crop protection.

Modification of the sugar specificity of a plant lectin: structural studies on a point mutant of Erythrina corallodendron lectin.

A mutant of Erythrina corallodendron lectin was generated with the aim of enhancing its affinity for N-acetylgalactosamine. A tyrosine residue close to the binding site of the lectin was mutated to a glycine in order to facilitate stronger interactions between the acetamido group of the sugar and the lectin which were prevented by the side chain of the tyrosine in the wild-type lectin. The crystal structures of this Y106G mutant lectin in complex with galactose and N-acetylgalactosamine have been determined. A structural rationale has been provided for the differences in the relative binding affinities of the wild-type and mutant lectins towards the two sugars based on the structures. A hydrogen bond between the O6 atom of the sugars and the variable loop of the carbohydrate-binding site of the lectin is lost in the mutant complexes owing to a conformational change in the loop. This loss is compensated by an additional hydrogen bond that is formed between the acetamido group of the sugar and the mutant lectin in the complex with N-acetylgalactosamine, resulting in a higher affinity of the mutant lectin for N-acetylgalactosamine compared with that for galactose, in contrast to the almost equal affinity of the wild-type lectin for the two sugars. The structure of a complex of the mutant with a citrate ion bound at the carbohydrate-binding site that was obtained while attempting to crystallize the complexes with sugars is also presented.

Structure and sugar-specificity of basic winged-bean lectin: structures of new disaccharide complexes and a comparative study with other known disaccharide complexes of the lectin.

Crystal structures of the complexes of basic winged-bean agglutinin with the disaccharides Galalpha1-4Gal (galabiose), Galalpha1-6Glc (mellibiose) and Galalpha1-4Galbeta-Et have been determined and the complex with Galalpha1-2Gal has been modelled. The interactions of the nonreducing Gal with the lectin at the primary site are the same as those in the known complexes with disaccharides having the alpha1-->3 linkage. The second residue in Galalpha1-4Gal and Galalpha1-6Glc forms a water bridge to the lectin, while the ethyl group in Galalpha1-4Galbeta-Et makes nonpolar interactions. In complexes involving disaccharides with alpha1-3 linkages, which form part of the A and B blood-group substances, the second sugar residue forms a direct hydrogen bond to the variable loop in the binding site of the lectin. This in part explains the specificity of the lectin for the blood-group substances and also the higher affinity of alpha1-->3-linked disaccharides for the lectin compared with disaccharides involving other linkages. Including those reported here, 14 crystal structures involving the lectin, accounting for 54 crystallographically independent subunits, are available. A comparative study of these structures shows that the region involving the curved beta-sheet which nestles the metal ions is relatively rigid. The carbohydrate-binding region is perched on this region. The flat beta-sheet, which is involved in oligomerization and exhibits considerable variability in legume lectins, is relatively flexible. Indeed, the structures of basic winged-bean lectin have been of critical importance in establishing legume lectins as a family of proteins in which small alterations in essentially the same tertiary structure lead to large variations in quaternary association. They have also provided a structural explanation of the blood-group specificity of the lectin.

Generation of blood group specificity: new insights from structural studies on the complexes of A- and B-reactive saccharides with basic winged bean agglutinin.

Basic winged bean agglutinin binds A-blood group substance with higher affinity and B-blood group substance with lesser affinity. It does not bind the O substance. The crystal structures of the lectin, complexed with A-reactive and B-reactive di and tri saccharides, have been determined. In addition, the complexes of the lectin with fucosylated A-trisaccharides and B-trisaccharides and with a variant of the A-trisaccharide have been modeled. These structures and models provide valuable insights into the structural basis of blood group specificities. All the four carbohydrate binding loops of the lectin contribute to the primary combining site while the loop of variable length contributes to the secondary binding site. In a significant advance to the current understanding, the interactions at the secondary binding site also contribute substantially, albeit in a subtle manner, to determine the blood group specificity. Compared with the interactions of the B-trisaccharide with the lectin, the third sugar residue of the A-reactive trisacharide forms an additional hydrogen bond with a lysine residue in the variable loop. In the former, the formation of such a hydrogen bond is prevented by a shift in the orientation of third sugar resulting from an internal hydrogen bond in it. The formation of this bond is also facilitated by an interaction dependent change in the rotamer conformation of the lysyl residue of the variable loop. Thus, the difference in the interactions at the secondary site is generated by coordinated movements in the ligand as well as the protein. A comparison of the crystal structure and the model of the complex involving the variant of the A-trisaccharide results in the delineation of the relative contributions of the interactions at the primary and the secondary sites in determining blood group specificity.

Structural basis for the carbohydrate-specificity of basic winged-bean lectin and its differential affinity for Gal and GalNAc.

The crystal structure of the complexes of basic winged-bean lectin with galactose, 2-methoxygalactose, N-acetylgalactosamine and methyl-alpha-N-acetylgalactosamine have been determined. Lectin-sugar interactions involve four hydrogen bonds and a stacking interaction in all of the complexes. In addition, an N-H...O hydrogen bond involving the hydroxyl group at C2 exists in the galactose and 2-methoxygalactose complexes. An additional hydrophobic interaction involving the methyl group in the latter leads to the higher affinity of the methyl derivative. In the lectin-N-acetylgalactosamine complex the N-H...O hydrogen bond is lost, but a compensatory hydrogen bond is formed involving the O atom of the acetamido group. In addition, the CH(3) moiety of the acetamido group is involved in hydrophobic interactions. Consequently, the 2-methyl and acetamido derivatives of galactose have nearly the same affinity for the lectin. The methyl group alpha-linked to the galactose takes part in additional hydrophobic interactions. Therefore, methyl-alpha-N-acetylgalactosamine has a higher affinity than N-acetylgalactosamine for the lectin. The structures of basic winged-bean lectin-sugar complexes provide a framework for examining the relative affinity of galactose and galactosamine for the lectins that bind to them. The complexes also lead to a structural explanation for the blood-group specificity of basic winged-bean lectin.

Crystallization and preliminary X-ray crystallographic analysis of a galactose-specific lectin from Dolichos lablab.

The galactose-specific lectin from the seeds of Dolichos lablab has been crystallized using the hanging-drop vapour-diffusion technique. The crystals belong to space group P1, with unit-cell parameters a = 73.99, b = 84.13, c = 93.15 A, alpha = 89.92, beta = 76.01, gamma = 76.99 degrees. X-ray diffraction data to a resolution of 3.0 A have been collected under cryoconditions (100 K) using a MAR imaging-plate detector system mounted on a rotating-anode X-ray generator. Molecular-replacement calculations carried out using the available structures of legume lectins as search models revealed that the galactose-specific lectin from D. lablab forms a tetramer similar to soybean agglutinin; two such tetramers are present in the asymmetric unit.

Structural basis for the specificity of basic winged bean lectin for the Tn-antigen: a crystallographic, thermodynamic and modelling study.

The crystal structure of winged bean basic agglutinin in complex with GalNAc-alpha-O-Ser (Tn-antigen) has been elucidated at 2.35 angstroms resolution in order to characterize the mode of binding of Tn-antigen with the lectin. The Gal moiety occupies the primary binding site and makes interactions similar to those found in other Gal/GalNAc specific legume lectins. The nitrogen and oxygen atoms of the acetamido group of the sugar make two hydrogen bonds with the protein atoms whereas its methyl group is stabilized by hydrophobic interactions. A water bridge formed between the terminal oxygen atoms of the serine residue of the Tn-antigen and the side chain oxygen atom of Asn128 of the lectin increase the affinity of the lectin for Tn-antigen compared to that for GalNAc. A comparison with the available structures reveals that while the interactions of the glyconic part of the antigen are conserved, the mode of stabilization of the serine residue differs and depends on the nature of the protein residues in its vicinity. The structure provides a qualitative explanation for the thermodynamic parameters of the complexation of the lectin with Tn-antigen. Modeling studies indicate the possibility of an additional hydrogen bond with the lectin when the antigen is part of a glycoprotein.