Essential but Not Vulnerable: Indazole Sulfonamides Targeting Inosine Monophosphate Dehydrogenase as Potential Leads against Mycobacterium tuberculosis.

A potent, noncytotoxic indazole sulfonamide was identified by high-throughput screening of >100,000 synthetic compounds for activity against Mycobacterium tuberculosis (Mtb). This noncytotoxic compound did not directly inhibit cell wall biogenesis but triggered a slow lysis of Mtb cells as measured by release of intracellular green fluorescent protein (GFP). Isolation of resistant mutants followed by whole-genome sequencing showed an unusual gene amplification of a 40 gene region spanning from Rv3371 to Rv3411c and in one case a potential promoter mutation upstream of guaB2 (Rv3411c) encoding inosine monophosphate dehydrogenase (IMPDH). Subsequent biochemical validation confirmed direct inhibition of IMPDH by an uncompetitive mode of inhibition, and growth inhibition could be rescued by supplementation with guanine, a bypass mechanism for the IMPDH pathway. Beads containing immobilized indazole sulfonamides specifically interacted with IMPDH in cell lysates. X-ray crystallography of the IMPDH-IMP-inhibitor complex revealed that the primary interactions of these compounds with IMPDH were direct pi-pi interactions with the IMP substrate. Advanced lead compounds in this series with acceptable pharmacokinetic properties failed to show efficacy in acute or chronic murine models of tuberculosis (TB). Time-kill experiments in vitro suggest that sustained exposure to drug concentrations above the minimum inhibitory concentration (MIC) for 24 h were required for a cidal effect, levels that have been difficult to achieve in vivo. Direct measurement of guanine levels in resected lung tissue from tuberculosis-infected animals and patients revealed 0.5-2 mM concentrations in caseum and normal lung tissue. The high lesional levels of guanine and the slow lytic, growth-rate-dependent effect of IMPDH inhibition pose challenges to developing drugs against this target for use in treating TB.

Structure, activity, and inhibition of the Carboxyltransferase ß-subunit of acetyl coenzyme A carboxylase (AccD6) from Mycobacterium tuberculosis.

In Mycobacterium tuberculosis, the carboxylation of acetyl coenzyme A (acetyl-CoA) to produce malonyl-CoA, a building block in long-chain fatty acid biosynthesis, is catalyzed by two enzymes working sequentially: a biotin carboxylase (AccA) and a carboxyltransferase (AccD). While the exact roles of the three different biotin carboxylases (AccA1 to -3) and the six carboxyltransferases (AccD1 to -6) in M. tuberculosis are still not clear, AccD6 in complex with AccA3 can synthesize malonyl-CoA from acetyl-CoA. A series of 10 herbicides that target plant acetyl-CoA carboxylases (ACC) were tested for inhibition of AccD6 and for whole-cell activity against M. tuberculosis. From the tested herbicides, haloxyfop, an arylophenoxypropionate, showed in vitro inhibition of M. tuberculosis AccD6, with a 50% inhibitory concentration (IC50) of 21.4 ± 1 µM. Here, we report the crystal structures of M. tuberculosis AccD6 in the apo form (3.0 Å) and in complex with haloxyfop-R (2.3 Å). The structure of M. tuberculosis AccD6 in complex with haloxyfop-R shows two molecules of the inhibitor bound on each AccD6 subunit. These results indicate the potential for developing novel therapeutics for tuberculosis based on herbicides with low human toxicity.

Cloning and characterization of bifunctional enzyme farnesyl diphosphate/geranylgeranyl diphosphate synthase from Plasmodium falciparum.

BACKGROUND: Isoprenoids are the most diverse and abundant group of natural products. In Plasmodium falciparum, isoprenoid synthesis proceeds through the methyl erythritol diphosphate pathway and the products are further metabolized by farnesyl diphosphate synthase (FPPS), turning this enzyme into a key branch point of the isoprenoid synthesis. Changes in FPPS activity could alter the flux of isoprenoid compounds downstream of FPPS and, hence, play a central role in the regulation of a number of essential functions in Plasmodium parasites. METHODS: The isolation and cloning of gene PF3D7_18400 was done by amplification from cDNA from mixed stage parasites of P. falciparum. After sequencing, the fragment was subcloned in pGEX2T for recombinant protein expression. To verify if the PF3D7_1128400 gene encodes a functional rPfFPPS protein, its catalytic activity was assessed using the substrate [4-14C] isopentenyl diphosphate and three different allylic substrates: dimethylallyl diphosphate, geranyl diphosphate or farnesyl diphosphate. The reaction products were identified by thin layer chromatography and reverse phase high-performance liquid chromatography. To confirm the product spectrum formed of rPfFPPS, isoprenic compounds were also identified by mass spectrometry. Apparent kinetic constants KM and Vmax for each substrate were determined by Michaelis-Menten; also, inhibition assays were performed using risedronate. RESULTS: The expressed protein of P. falciparum FPPS (rPfFPPS) catalyzes the synthesis of farnesyl diphosphate, as well as geranylgeranyl diphosphate, being therefore a bifunctional FPPS/geranylgeranyl diphosphate synthase (GGPPS) enzyme. The apparent KM values for the substrates dimethylallyl diphosphate, geranyl diphosphate and farnesyl diphosphate were, respectively, 68 ± 5 µM, 7.8 ± 1.3 µM and 2.06 ± 0.4 µM. The protein is expressed constitutively in all intra-erythrocytic stages of P. falciparum, demonstrated by using transgenic parasites with a haemagglutinin-tagged version of FPPS. Also, the present data demonstrate that the recombinant protein is inhibited by risedronate. CONCLUSIONS: The rPfFPPS is a bifunctional FPPS/GGPPS enzyme and the structure of products FOH and GGOH were confirmed mass spectrometry. Plasmodial FPPS represents a potential target for the rational design of chemotherapeutic agents to treat malaria.

Wild-type phosphoribosylpyrophosphate synthase (PRS) from Mycobacterium tuberculosis: a bacterial class II PRS?

The 5-phospho-α-D-ribose 1-diphosphate (PRPP) metabolite plays essential roles in several biosynthetic pathways, including histidine, tryptophan, nucleotides, and, in mycobacteria, cell wall precursors. PRPP is synthesized from α-D-ribose 5-phosphate (R5P) and ATP by the Mycobacterium tuberculosis prsA gene product, phosphoribosylpyrophosphate synthase (MtPRS). Here, we report amplification, cloning, expression and purification of wild-type MtPRS. Glutaraldehyde cross-linking results suggest that MtPRS predominates as a hexamer, presenting varied oligomeric states due to distinct ligand binding. MtPRS activity measurements were carried out by a novel coupled continuous spectrophotometric assay. MtPRS enzyme activity could be detected in the absence of P(i). ADP, GDP and UMP inhibit MtPRS activity. Steady-state kinetics results indicate that MtPRS has broad substrate specificity, being able to accept ATP, GTP, CTP, and UTP as diphosphoryl group donors. Fluorescence spectroscopy data suggest that the enzyme mechanism for purine diphosphoryl donors follows a random order of substrate addition, and for pyrimidine diphosphoryl donors follows an ordered mechanism of substrate addition in which R5P binds first to free enzyme. An ordered mechanism for product dissociation is followed by MtPRS, in which PRPP is the first product to be released followed by the nucleoside monophosphate products to yield free enzyme for the next round of catalysis. The broad specificity for diphosphoryl group donors and detection of enzyme activity in the absence of P(i) would suggest that MtPRS belongs to Class II PRS proteins. On the other hand, the hexameric quaternary structure and allosteric ADP inhibition would place MtPRS in Class I PRSs. Further data are needed to classify MtPRS as belonging to a particular family of PRS proteins. The data here presented should help augment our understanding of MtPRS mode of action. Current efforts are toward experimental structure determination of MtPRS to provide a solid foundation for the rational design of specific inhibitors of this enzyme.

Pyrimidin-2(1H)-ones based inhibitors of Mycobacterium tuberculosis orotate phosphoribosyltransferase.

Tuberculosis (TB) is an ancient human chronic infectious disease caused mainly by Mycobacterium tuberculosis. The emergence of strains resistant to first and second line anti-TB drugs, associated with the increasing number of TB cases among HIV positive subjects, and the large number of individuals infected with latent bacilli have urged the development of new strategies to treat TB. Enzymes of nucleotide metabolism pathways provide promising molecular targets for the development of drugs, aiming at both active and latent TB. The orotate phosphoribosyltransferase (OPRT) enzyme catalyzes the synthesis of orotidine 5'-monophosphate from 5'-phospho-α-d-ribose 1'-diphosphate and orotic acid, in the de novo pyrimidine synthesis pathway. Based on the kinetic mechanism and molecular properties, here we describe the design, selection and synthesis of substrate analogs with inhibitory activity of M. tuberculosis OPRT (MtOPRT) enzyme. Steady-state kinetic measurements were employed to determine the mode of inhibition of commercially available and chemically derived compounds. The 6-Hydroxy-2-oxo-1,2-dihydropyridine-4-carboxylic acid (6) chemical compound and its derivative, 3-Benzylidene-2,6-dioxo-1,2,3,6-tetrahydropyridine-4-carboxylic acid (13), showed enzyme inhibition constants in the submicromolar range. Isothermal titration calorimetry data indicated that binding of both compounds to MtOPRT have negative enthalpy and favorable Gibbs free energy probably due to their high complementarity to the enzyme's binding pocket. Improvement of compound 13 hydrophobic character by addition of an aromatic ring substituent resulted in entropic optimization, reflected on a thermodynamic discrimination profile characteristic of high affinity ligands. These inhibitors represent lead compounds for further development of MtOPRT inhibitors with increased potency, which may be tested as anti-TB agents.

Molecular, kinetic and thermodynamic characterization of Mycobacterium tuberculosis orotate phosphoribosyltransferase.

Tuberculosis (TB) is a chronic infectious disease caused mainly by Mycobacterium tuberculosis. The worldwide emergence of drug-resistant strains, the increasing number of infected patients among immune compromised populations, and the large number of latent infected individuals that are reservoir to the disease have underscored the urgent need of new strategies to treat TB. The nucleotide metabolism pathways provide promising molecular targets for the development of novel drugs against active TB and may, hopefully, also be effective against latent forms of the pathogen. The orotate phosphoribosyltransferase (OPRT) enzyme of the de novo pyrimidine synthesis pathway catalyzes the reversible phosphoribosyl transfer from 5'-phospho-α-D-ribose 1'-diphosphate (PRPP) to orotic acid (OA), forming pyrophosphate and orotidine 5'-monophosphate (OMP). Here we describe cloning and characterization of pyrE-encoded protein of M. tuberculosis H37Rv strain as a homodimeric functional OPRT enzyme. The M. tuberculosis OPRT true kinetic constants for forward reaction and product inhibition results suggest a Mono-Iso Ordered Bi-Bi kinetic mechanism, which has not been previously described for this enzyme family. Absence of detection of half reaction and isothermal titration calorimetry (ITC) data support the proposed mechanism. ITC data also provided thermodynamic signatures of non-covalent interactions between substrate/product and M. tuberculosis OPRT. These data provide a solid foundation on which to base target-based rational design of anti-TB agents and should inform us how to better design inhibitors of M. tuberculosis OPRT.

Molecular, kinetic, thermodynamic, and structural analyses of Mycobacterium tuberculosis hisD-encoded metal-dependent dimeric histidinol dehydrogenase (EC 1.1.1.23).

The emergence of drug-resistant strains of Mycobacterium tuberculosis, the major causative agent of tuberculosis (TB), and the deadly HIV-TB co-infection have led to an urgent need for the development of new anti-TB drugs. The histidine biosynthetic pathway is present in bacteria, archaebacteria, lower eukaryotes and plants, but is absent in mammals. Disruption of the hisD gene has been shown to be essential for M. tuberculosis survival. Here we present cloning, expression and purification of recombinant hisD-encoded histidinol dehydrogenase (MtHisD). N-terminal amino acid sequencing and electrospray ionization mass spectrometry analyses confirmed the identity of homogeneous MtHisD. Analytical gel filtration, metal requirement analysis, steady-state kinetics and isothermal titration calorimetry data showed that homodimeric MtHisD is a metalloprotein that follows a Bi Uni Uni Bi Ping-Pong mechanism. pH-rate profiles and a three-dimensional model of MtHisD allowed proposal of amino acid residues involved in either catalysis or substrate(s) binding.

Recombinant Escherichia coli GMP reductase: kinetic, catalytic and chemical mechanisms, and thermodynamics of enzyme-ligand binary complex formation.

Guanosine monophosphate (GMP) reductase catalyzes the reductive deamination of GMP to inosine monophosphate (IMP). GMP reductase plays an important role in the conversion of nucleoside and nucleotide derivatives of guanine to adenine nucleotides. In addition, as a member of the purine salvage pathway, it also participates in the reutilization of free intracellular bases. Here we present cloning, expression and purification of Escherichia coli guaC-encoded GMP reductase to determine its kinetic mechanism, as well as chemical and thermodynamic features of this reaction. Initial velocity studies and isothermal titration calorimetry demonstrated that GMP reductase follows an ordered bi-bi kinetic mechanism, in which GMP binds first to the enzyme followed by NADPH binding, and NADP(+) dissociates first followed by IMP release. The isothermal titration calorimetry also showed that GMP and IMP binding are thermodynamically favorable processes. The pH-rate profiles showed groups with apparent pK values of 6.6 and 9.6 involved in catalysis, and pK values of 7.1 and 8.6 important to GMP binding, and a pK value of 6.2 important for NADPH binding. Primary deuterium kinetic isotope effects demonstrated that hydride transfer contributes to the rate-limiting step, whereas solvent kinetic isotope effects arise from a single protonic site that plays a modest role in catalysis. Multiple isotope effects suggest that protonation and hydride transfer steps take place in the same transition state, lending support to a concerted mechanism. Pre-steady-state kinetic data suggest that product release does not contribute to the rate-limiting step of the reaction catalyzed by E. coli GMP reductase.

UMP kinase from Mycobacterium tuberculosis: Mode of action and allosteric interactions, and their likely role in pyrimidine metabolism regulation.

The pyrH-encoded uridine 5'-monophosphate kinase (UMPK) is involved in both de novo and salvage synthesis of DNA and RNA precursors. Here we describe Mycobacterium tuberculosis UMPK (MtUMPK) cloning and expression in Escherichia coli. N-terminal amino acid sequencing and electrospray ionization mass spectrometry analyses confirmed the identity of homogeneous MtUMPK. MtUMPK catalyzed the phosphorylation of UMP to UDP, using ATP-Mg²(+) as phosphate donor. Size exclusion chromatography showed that the protein is a homotetramer. Kinetic studies revealed that MtUMPK exhibits cooperative kinetics towards ATP and undergoes allosteric regulation. GTP and UTP are, respectively, positive and negative effectors, maintaining the balance of purine versus pyrimidine synthesis. Initial velocity studies and substrate(s) binding measured by isothermal titration calorimetry suggested that catalysis proceeds by a sequential ordered mechanism, in which ATP binds first followed by UMP binding, and release of products is random. As MtUMPK does not resemble its eukaryotic counterparts, specific inhibitors could be designed to be tested as antitubercular agents.

The conserved Lysine69 residue plays a catalytic role in Mycobacterium tuberculosis shikimate dehydrogenase.

BACKGROUND: The shikimate pathway is an attractive target for the development of antitubercular agents because it is essential in Mycobacterium tuberculosis, the causative agent of tuberculosis, but absent in humans. M. tuberculosis aroE-encoded shikimate dehydrogenase catalyzes the forth reaction in the shikimate pathway. Structural and functional studies indicate that Lysine69 may be involved in catalysis and/or substrate binding in M. tuberculosis shikimate dehydrogenase. Investigation of the kinetic properties of mutant enzymes can bring important insights about the role of amino acid residues for M. tuberculosis shikimate dehydrogenase. FINDINGS: We have performed site-directed mutagenesis, steady-state kinetics, equilibrium binding measurements and molecular modeling for both the wild-type M. tuberculosis shikimate dehydrogenase and the K69A mutant enzymes. The apparent steady-state kinetic parameters for the M. tuberculosis shikimate dehydrogenase were determined; the catalytic constant value for the wild-type enzyme (50 s-1) is 68-fold larger than that for the mutant K69A (0.73 s-1). There was a modest increase in the Michaelis-Menten constant for DHS (K69A = 76 microM; wild-type = 29 microM) and NADPH (K69A = 30 microM; wild-type = 11 microM). The equilibrium dissociation constants for wild-type and K69A mutant enzymes are 32 (+/- 4) microM and 134 (+/- 21), respectively. CONCLUSION: Our results show that the residue Lysine69 plays a catalytic role and is not involved in substrate binding for the M. tuberculosis shikimate dehydrogenase. These efforts on M. tuberculosis shikimate dehydrogenase catalytic mechanism determination should help the rational design of specific inhibitors, aiming at the development of antitubercular drugs.

A structural model for chorismate synthase from Mycobacterium tuberculosis in complex with coenzyme and substrate.

The enzymes of the shikimate pathway constitute an excellent target for the design of new antibacterial agents; chorismate synthase (CS) catalyzes the last step of this pathway. The prediction of Mycobacterium tuberculosis (MTB) CS three-dimensional structure and the geometric docking of the coenzyme FMN and the substrate EPSP were performed using the crystal structure of CS from Streptococcus pneumoniae as template. Energy minimization of the whole complex showed, as expected, that most of the template interactions are preserved in the MTB structure, except for HIS11, ARG139 and GLN255. However, novel interactions involving ARG111, GLY113 and SER317 were also observed.