Identification of novel diphenyl urea inhibitors of Mt-GuaB2 active against Mycobacterium tuberculosis

Abstract

In contrast with most bacteria, which harbour a single inosine monophosphate dehydrogenase (IMPDH) gene, the genomic sequence of Mycobacterium tuberculosis H37Rv predicts three genes encoding IMPDH: guaB1, guaB2 and guaB3. These three genes were cloned and expressed in Escherichia coli to evaluate functional IMPDH activity. Purified recombinant Mt-GuaB2, which uses inosine monophosphate as a substrate, was identified as the only active GuaB orthologue in M. tuberculosis and showed optimal activity at pH 8.5 and 37 6C. Mt-GuaB2 was inhibited significantly in vitro by a panel of diphenyl urea-based derivatives, which were also potent anti-mycobacterial agents against M. tuberculosis and Mycobacterium smegmatis, with MICs in the range of 0.2-0.5 mg ml "1 . When Mt-GuaB2 was overexpressed on a plasmid in trans in M. smegmatis, a diphenyl urea analogue showed a 16-fold increase in MIC. Interestingly, when Mt-GuaB orthologues (Mt-GuaB1 and 3) were also overexpressed on a plasmid in trans in M. smegmatis, they also conferred resistance, suggesting that although these Mt-GuaB orthologues were inactive in vitro, they presumably titrate the effect of the inhibitory properties of the active compounds in vivo.

Inosine 5′-Monophosphate Dehydrogenase Inhibitors as Antimicrobial Agents: Recent Progress and Future Perspectives

“…In the literature precedence of diphenylureas (DPUs) as IMPDH inhibitors, a set of 16 DPUs for inhibition of Mt-GuaB2 was screened [31]. Three compounds 26-28 from the set exhibited >90% inhibition at 1 mM concentration and were potent anti-TB agents against Mtb and M. smegmatis, with MICs ranging from 0.2-0.5 μg/ml.…”

Genetic Approaches to Facilitate Antibacterial Drug Development

“…This type of experiment has helped to clarify the MOA of INH (isoniazid), which was debated to impair one of two enzymes involved in fatty acid biosynthesis, InhA or KasA. Strains that overexpressed these proteins were analyzed for their susceptibilities to INH and the KasA inhibitor thiolac- (Feng and Barletta 2003), platensimycin to KasA and KasB ), ethambutol to the arabinosyltransferases EmbB and EmbC (Goude et al 2009), and certain diphenylurea compounds to inosine monophosphate dehydrogenase (Olaleye et al 2010;Usha et al 2011). …”

“…If the inhibitor binding site is conserved, overexpression of any member of such a protein family will increase the MIC even when only one family member is the physiological target. This has been discussed for inosine-5 0 -monophosphate (IMP) dehydrogenases (GuaB1/2/ 3) (Usha et al 2011), ketoacyl-ACP synthases (KasA/B) ), and methionine aminopeptidases (MapA/B) (Olaleye et al 2010). In some of these cases, the MIC may have increased because the overproduced protein sequestered the inhibitor from its physiological target.…”

Novel inhibitors of Mycobacterium tuberculosis GuaB2 identified by a target based high-throughput phenotypic screen

“…The rate limiting two-step catalysis of IMP to XMP conversion by IMPDH requires nicotinamide adenine dinucleotide (NAD + ) as a cofactor, which is first reduced to NADH by a dehydrogenation reaction, forming an intermediate covalent bond between IMPDH and XMP, followed by a hydrolysis reaction that breaks the covalent attachment and releases XMP12. GuaB2 is the only catalytically active and essential IMPDH shown by transposon site hybridization (TraSH) to be required for viability of Mtb 1314. Inhibition of GuaB2 results in depletion in guanine nucleotides for DNA and RNA synthesis1516.…”