Nag S. Kumar

Inhibition of recombinant human maltase glucoamylase by salacinol and derivatives

Inhibitors targeting pancreatic α‐amylase and intestinal α‐glucosidases delay glucose production following digestion and are currently used in the treatment of Type II diabetes. Maltase‐glucoamylase (MGA), a family 31 glycoside hydrolase, is an α‐glucosidase anchored in the membrane of small intestinal epithelial cells responsible for the final step of mammalian starch digestion leading to the release of glucose. This paper reports the production and purification of active human recombinant MGA amino terminal catalytic domain (MGAnt) from two different eukaryotic cell culture systems. MGAnt overexpressed in Drosophila cells was of quality and quantity suitable for kinetic and inhibition studies as well as future structural studies. Inhibition of MGAnt was tested with a group of prospective α‐glucosidase inhibitors modeled after salacinol, a naturally occurring α‐glucosidase inhibitor, and acarbose, a currently prescribed antidiabetic agent. Four synthetic inhibitors that bind and inhibit MGAnt activity better than acarbose, and at comparable levels to salacinol, were found. The inhibitors are derivatives of salacinol that contain either a selenium atom in place of sulfur in the five‐membered ring, or a longer polyhydroxylated, sulfated chain than salacinol. Six‐membered ring derivatives of salacinol and compounds modeled after miglitol were much less effective as MGAnt inhibitors. These results provide information on the inhibitory profile of MGAnt that will guide the development of new compounds having antidiabetic activity.

Identification of Pyruvate Kinase in Methicillin-Resistant Staphylococcus aureus as a Novel Antimicrobial Drug Target

ABSTRACT Novel classes of antimicrobials are needed to address the challenge of multidrug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA). Using the architecture of the MRSA interactome, we identified pyruvate kinase (PK) as a potential novel drug target based upon it being a highly connected, essential hub in the MRSA interactome. Structural modeling, including X-ray crystallography, revealed discrete features of PK in MRSA, which appeared suitable for the selective targeting of the bacterial enzyme. In silico library screening combined with functional enzymatic assays identified an acyl hydrazone-based compound (IS-130) as a potent MRSA PK inhibitor (50% inhibitory concentration [IC50] of 0.1 μM) with >1,000-fold selectivity over human PK isoforms. Medicinal chemistry around the IS-130 scaffold identified analogs that more potently and selectively inhibited MRSA PK enzymatic activity and S. aureus growth in vitro (MIC of 1 to 5 μg/ml). These novel anti-PK compounds were found to possess antistaphylococcal activity, including both MRSA and multidrug-resistant S. aureus (MDRSA) strains. These compounds also exhibited exceptional antibacterial activities against other Gram-positive genera, including enterococci and streptococci. PK lead compounds were found to be noncompetitive inhibitors and were bactericidal. In addition, mutants with significant increases in MICs were not isolated after 25 bacterial passages in culture, indicating that resistance may be slow to emerge. These findings validate the principles of network science as a powerful approach to identify novel antibacterial drug targets. They also provide a proof of principle, based upon PK in MRSA, for a research platform aimed at discovering and optimizing selective inhibitors of novel bacterial targets where human orthologs exist, as leads for anti-infective drug development.

Design and synthesis of an all-in-one 3-(1,1-difluoroprop-2-ynyl)-3H-diazirin-3-yl functional group for photo-affinity labeling

A novel radioisotope-free photo-affinity probe containing the 3-(1,1-difluoroprop-2-ynyl)-3H-diazirin-3-yl functional group was designed and synthesized. This very compact functionality is envisaged to allow photochemically-induced coupling of a compound to its target followed by click reaction coupling with an azido-biotin reagent in order to facilitate purification of the labeled target. In a proof-of-concept study we have shown that 3-(1,1-difluoroprop-2-ynyl)-3H-diazirin-3-yl functional group could be photolyzed to efficiently furnish the methanol adduct 23 and that the generated highly unstable carbene does not react with the neighboring acetylene moiety. A subsequent click reaction with the azido-biotin derivative 25 proceeded smoothly to give triazole 26. This chemical probe should thus be of unique value for facilitating identification of the molecular structure of the target of a bioactive compound.

Binding of sulfonium-ion analogues of di-epi-swainsonine and 8-epi-lentiginosine to Drosophila Golgi α-mannosidase II: The role of water in inhibitor binding

Retaining glycosidases operate by a two‐step catalytic mechanism in which the transition states are characterized by buildup of a partial positive charge at the anomeric center. Sulfonium‐ion analogues of the naturally occurring glycosidase inhibitors, swainsonine and 8‐epi‐lentiginosine, in which the bridgehead nitrogen atom is replaced by a sulfonium‐ion, were synthesized in order to test the hypothesis that a sulfonium salt carrying a permanent positive charge would be an effective glycosidase inhibitor. Initial prediction based on computational docking indicated three plausible binding modes to Drosophila Golgi α‐mannosidase II (dGMII), the most likely being close to that of swainsonine. Observation of the binding of di‐epi‐thioswainsonine and 8‐epi‐thiolentiginosine to dGMII from crystallographic data, however, revealed an orientation different from swainsonine in the active site. Screening these two compounds against dGMII shows that they are inhibitors with IC50 values of 2.0 and 0.014 mM, respectively. This dramatic difference in affinity between the two compounds, which differ by only one hydroxyl group, is rationalized in terms of bound water molecules and the water molecule substructure in the active site, as identified by comparison of high resolution X‐ray crystal structures of several dGMII‐inhibitor complexes. Proteins 2008.

Novel EP4 Receptor Agonist‐Bisphosphonate Conjugate Drug (C1) Promotes Bone Formation and Improves Vertebral Mechanical Properties in the Ovariectomized Rat Model of Postmenopausal Bone Loss

Current treatments for postmenopausal osteoporosis aim to either promote bone formation or inhibit bone resorption. The C1 conjugate drug represents a new treatment approach by chemically linking the antiresorptive compound alendronate (ALN) with the anabolic agent prostanoid EP4 receptor agonist (EP4a) through a linker molecule (LK) to form a conjugate compound. This enables the bone‐targeting ability of ALN to deliver EP4a to bone sites and mitigate the systemic side effects of EP4a, while also facilitating dual antiresorptive and anabolic effects. In vivo hydrolysis is required to release the EP4a and ALN components for pharmacological activity. Our study investigated the in vivo efficacy of this drug in treating established bone loss using an ovariectomized (OVX) rat model of postmenopausal osteopenia. In a curative experiment, 3‐month‐old female Sprague‐Dawley rats were OVX, allowed to lose bone for 7 weeks, then treated for 6 weeks. Treatment groups consisted of C1 conjugate at low and high doses, vehicle‐treated OVX and sham, prostaglandin E2 (PGE2), and mixture of unconjugated ALN‐LK and EP4a to assess the effect of conjugation. Results showed that weekly administration of C1 conjugate dose‐dependently increased bone volume in trabecular bone, which partially or completely reversed OVX‐induced bone loss in the lumbar vertebra and improved vertebral mechanical strength. The conjugate also dose‐dependently stimulated endocortical woven bone formation and intracortical resorption in cortical bone, with high‐dose treatment increasing the mechanical strength but compromising the material properties. Conjugation between the EP4a and ALN‐LK components was crucial to the drugs anabolic efficacy. To our knowledge, the C1 conjugate represents the first time that a combined therapy using an anabolic agent and the antiresorptive compound ALN has shown significant anabolic effects which reversed established osteopenia.

Cheminformatics-driven discovery of selective, nanomolar inhibitors for staphylococcal pyruvate kinase.

We have recently mapped the protein interaction network of methicillin-resistant Staphylococcus aureus (MRSA), which revealed its scale-free organization with characteristic presence of highly connected hub proteins that are critical for bacterial survival. Here we report the discovery of inhibitors that are highly potent against one such hub target, staphylococcal pyruvate kinase (PK). Importantly, the developed compounds demonstrate complete selectivity for the bacterial enzyme compared to all human orthologues. The lead 91nM inhibitor IS-130 has been identified through ligand-based cheminformatic exploration of a chemical space around micromolar hits initially generated by experimental screening. The following crystallographic study resulted in identification of a tetrameric MRSA PK structure where IS-130 is bound to the interface between the proteins subunits. This newly described binding pocket is not present in otherwise highly similar human orthologues and can be effectively utilized for selective inhibition of bacterial PK. The following synthetic modifications of IS-130, guided by structure-based molecular modeling, resulted in the development of MRSA PK inhibitors with much improved antimicrobial properties. Considering a notable lack of recent reports on novel antibacterial targets and cognate antibacterial compounds, this study provides a valuable perspective on the development of a new generation of antimicrobials. Equally noteworthy, the results of the current work highlight the importance of rigorous cheminformatics-based exploration of the results of high-throughput experiments.