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New drugs to treat tuberculosis (TB) are urgently needed to combat the increase in resistance observed among the current first-line and second-line treatments. Here, we propose ketol-acid reductoisomerase (KARI) as a target for anti-TB drug discovery. Twenty-two analogues of IpOHA, an inhibitor of plant KARI, were evaluated as antimycobacterial agents. The strongest inhibitor of

The biosynthetic pathway for the branched-chain amino acids is present in plants, fungi and bacteria, but not in animals, making it an attractive target for herbicidal and antimicrobial drug discovery. Ketol-acid reductoisomerase (KARI; EC 1.1.1.86) is the second enzyme in this pathway, converting in a Mg(2+) - and NADPH-dependent reaction either 2-acetolactate or 2-aceto-2-hydroxybutyrate to their corresponding 2,3-dihydroxy-3-alkylbutyrate products. Here, we have determined the crystal structure of Mycobacterium tuberculosis (Mt) KARI, a class I KARI, with two magnesium ions bound in the active site. X-ray data were obtained to 1.0 Å resolution and the final model has an Rfree of 0.163. The structure shows that the active site is solvent-accessible with the two metal ions separated by 4.7 Å. A comparison of this structure with that of Mg(2+) -free Pseudomonas aeruginosa KARI suggests that upon magnesium binding no movement of the N domain relative to the C domain occurs. However, upon formation of the Michaelis complex, as illustrated in the structure of Slackia exigua KARI in complex with NADH.Mg(2+) . N-hydroxy-N-isopropyloxamate (IpOHA, a transition state analog), domain movements and reduction of the metal-metal distance to 3.5 Å are observed. This inherent flexibility therefore appears to be critical for initiation of the KARI-catalyzed reaction. This study provides new insights into the complex structural rearrangements required for activity of KARIs, particularly those belonging to class I, and provides the framework for the rational design of Mt KARI inhibitors that can be tested as novel antituberculosis agents.Coordinates and structure factors for the Mt KARI.Mg(2+) complex are available in the Protein Data Bank under accession number 4YPO.

Ketol-acid reductoisomerase (KARI) is an NAD(P)H and Mg2+-dependent enzyme of the branched-chain amino acid (BCAA) biosynthesis pathway. Here we describe the first crystal structures of Staphylococcus aureus (Sa) KARI, these in complex with two transition state analogs, cyclopropane-1,1-dicarboxylate (CPD) and N-isopropyloxalyl hydroxamate (IpOHA). These compounds bind competitively and in multi-dentate manner to KARI with Ki values of 2.73 μM and 7.9 nM, respectively; however, IpOHA binds slowly to the enzyme. Interestingly, intact IpOHA is present in only ~25% of binding sites, while its deoxygenated form is present in the remaining sites. This deoxy form of IpOHA binds rapidly to Sa KARI, but with much weaker affinity (Ki = 21 μM). Thus, our data pinpoint the origin of the slow binding mechanism of IpOHA. Furthermore, we propose that CPD mimics the early stage of the catalytic reaction (preceding the reduction step), while IpOHA mimics the late stage (after the reduction took place). These structural insights will guide strategies to design potent and rapidly binding derivatives of these compound for the development of novel biocides.

The design and synthesis of 4-alkynyl pyrazole derivatives has led to the identification of new class of PDE4 inhibitors. All these compounds were accessed for the first time via a facile Pd/C-CuI-PPh(3) mediated C-C bond forming reaction between an appropriate pyrazole iodide and various terminal alkynes. In vitro PDE4B inhibitory properties and molecular modeling studies of some of the compounds synthesized indicated that 4-alkynyl pyrazole could be a promising template for the discovery of novel PDE4 inhibitors.

Novel thieno[2,3-d]pyrimidines containing a cyclohexane ring fused with a six- or five-membered heterocyclic moiety along with a benzylic nitrile were designed as potential inhibitors of PDE4. Expeditious synthesis of these compounds was carried out via a multi-step sequence consisting of a few key steps such as Gewald reaction, Dieckmann type cyclisation and Krapcho decarboxylation. This newly developed strategy involved construction of the thienopyrimidine ring followed by the cyclohexanone moiety and subsequently the fused heterocyclic ring. A number of thieno[2,3-d]pyrimidine based derivatives were synthesized using this method some of which showed promising PDE4B inhibitory properties. One of them was tested for PDE4D inhibition in vitro and dose dependent inhibition of TNF-α. A few selected molecules were docked into the PE4B protein the results of which showed good overall correlations to their observed PDE4B inhibitory properties in vitro. The crystal structure analysis of representative compounds along with hydrogen bonding patterns and molecular arrangement present within the molecule is described.

A series of novel N-aryl substituted thieno[2,3-d]pyrimidin-4(3H)-ones were designed and synthesized as potential inhibitors of chorismate mutase. Synthesis of this class of compounds was carried out by using Cu-mediated C-N bond forming reaction between thieno[2,3-d]pyrimidin-4(3H)-ones and aryl boronic acids. The reaction can be performed in an open flask as the conversion was found to be not sensitive to the presence of air or atmospheric moisture. A range of compounds were prepared by using this method and single crystal X-ray diffraction study was performed using a representative compound. In vitro pharmacological data of some of the compounds synthesized along with dose response studies using active molecules are presented. In silico interactions of these molecules with chorismate mutase are also presented.

Novel polysubstituted pyrroles have been designed and accessed via a one-pot multicomponent reaction followed by Pd-mediated C-C bond forming reactions. All the compounds synthesized were tested for their PDE4B inhibitory properties in vitro and two of them obtained via Heck reaction showed significant inhibition. The docking results suggested that these alkenyl derivatives containing ester moiety interact well with the PDE4B protein in silico where the ester carbonyl oxygen played a key role. The pyrrole framework presented here could be a new template for the identification of small molecule based novel inhibitors of PDE4. The single crystal X-ray data of a representative compound is presented.

A number of novel 1,8-disubstituted 5,5-dimethyl-4,5-dihydro-1H-benzo[g]indazoles based on a conformationally restricted pyrazole framework have been designed as potential inhibitors of PDE4. All these compounds were readily prepared by using simple chemistry strategy. The in vitro PDE4B inhibitory properties and molecular modeling studies of some of the compounds synthesized along with the X-ray single crystal data of a representative compound is presented.