A new strategy for hit generation: Novel in cellulo active inhibitors of CYP121A1 from Mycobacterium tuberculosis via a combined X-ray crystallographic and phenotypic screening approach (XP screen).

There is a pressing need for new drugs against tuberculosis (TB) to combat the growing resistance to current antituberculars. Herein a novel strategy is described for hit generation against promising TB targets involving X-ray crystallographic screening in combination with phenotypic screening. This combined approach (XP Screen) affords both a validation of target engagement as well as determination of in cellulo activity. The utility of this method is illustrated by way of an XP Screen against CYP121A1, a cytochrome P450 enzyme from Mycobacterium tuberculosis (Mtb) championed as a validated drug discovery target. A focused screening set was synthesized and tested by such means, with several members of the set showing promising activity against Mtb strain H37Rv. One compound was observed as an X-ray hit against CYP121A1 and showed improved activity against Mtb strain H37Rv under multiple assay conditions (pan-assay activity). Data obtained during X-ray crystallographic screening were utilized in a structure-based campaign to design a limited number of analogues (less than twenty), many of which also showed pan-assay activity against Mtb strain H37Rv. These included the benzo[b][1,4]oxazine derivative (MIC90 6.25 µM), a novel hit compound suitable as a starting point for a more involved hit to lead candidate medicinal chemistry campaign.

Comparative fitness analysis of D-cycloserine resistant mutants reveals both fitness-neutral and high-fitness cost genotypes.

Drug resistant infections represent one of the most challenging medical problems of our time. D-cycloserine is an antibiotic used for six decades without significant appearance and dissemination of antibiotic resistant strains, making it an ideal model compound to understand what drives resistance evasion. We therefore investigated why Mycobacterium tuberculosis fails to become resistant to D-cycloserine. To address this question, we employed a combination of bacterial genetics, genomics, biochemistry and fitness analysis in vitro, in macrophages and in mice. Altogether, our results suggest that the ultra-low rate of emergence of D-cycloserine resistance mutations is the dominant biological factor delaying the appearance of clinical resistance to this antibiotic. Furthermore, we also identified potential compensatory mechanisms able to minimize the severe fitness costs of primary D-cycloserine resistance conferring mutations.

Uncoupling conformational states from activity in an allosteric enzyme.

ATP-phosphoribosyltransferase (ATP-PRT) is a hexameric enzyme in conformational equilibrium between an open and seemingly active state and a closed and presumably inhibited form. The structure-function relationship of allosteric regulation in this system is still not fully understood. Here, we develop a screening strategy for modulators of ATP-PRT and identify 3-(2-thienyl)-L-alanine (TIH) as an allosteric activator of this enzyme. Kinetic analysis reveals co-occupancy of the allosteric sites by TIH and L-histidine. Crystallographic and native ion-mobility mass spectrometry data show that the TIH-bound activated form of the enzyme closely resembles the inhibited L-histidine-bound closed conformation, revealing the uncoupling between ATP-PRT open and closed conformations and its functional state. These findings suggest that dynamic processes are responsible for ATP-PRT allosteric regulation and that similar mechanisms might also be found in other enzymes bearing a ferredoxin-like allosteric domain.Active and inactive state ATP-phosphoribosyltransferases (ATP-PRTs) are believed to have different conformations. Here the authors show that in both states, ATP-PRT has a similar structural arrangement, suggesting that dynamic alterations are involved in ATP-PRT regulation by allosteric modulators.

Protein CoAlation: a redox-linked post-translational modification.

Regulation of metabolic pathways by signal transduction and transcriptional cascades can alter cellular levels of metabolites. Metabolites themselves can also have regulatory activity as shown in a new study published in the Biochemical Journal Tsuchiya et al. describe a novel antibody and mass spectrometry-based method for identifying proteins that are reversibly modified with Coenzyme A (CoA). Analysis of the 'CoAlated proteome' under conditions of oxidative and metabolic stress revealed a bias towards the modification of metabolic enzymes by CoA. Furthermore, CoAlation was shown to alter the activity of target proteins. These results suggest that CoAlation is a widespread post-translational modification that may have important roles in the metabolic response to stress.

Thiophenecarboxamide Derivatives Activated by EthA Kill Mycobacterium tuberculosis by Inhibiting the CTP Synthetase PyrG.

To combat the emergence of drug-resistant strains of Mycobacterium tuberculosis, new antitubercular agents and novel drug targets are needed. Phenotypic screening of a library of 594 hit compounds uncovered two leads that were active against M. tuberculosis in its replicating, non-replicating, and intracellular states: compounds 7947882 (5-methyl-N-(4-nitrophenyl)thiophene-2-carboxamide) and 7904688 (3-phenyl-N-[(4-piperidin-1-ylphenyl)carbamothioyl]propanamide). Mutants resistant to both compounds harbored mutations in ethA (rv3854c), the gene encoding the monooxygenase EthA, and/or in pyrG (rv1699) coding for the CTP synthetase, PyrG. Biochemical investigations demonstrated that EthA is responsible for the activation of the compounds, and by mass spectrometry we identified the active metabolite of 7947882, which directly inhibits PyrG activity. Metabolomic studies revealed that pharmacological inhibition of PyrG strongly perturbs DNA and RNA biosynthesis, and other metabolic processes requiring nucleotides. Finally, the crystal structure of PyrG was solved, paving the way for rational drug design with this newly validated drug target.

Kinetic mechanism and inhibition of Mycobacterium tuberculosis D-alanine:D-alanine ligase by the antibiotic D-cycloserine.

D-cycloserine (DCS) is an antibiotic that is currently used in second-line treatment of tuberculosis. DCS is a structural analogue of D-alanine, and targets two enzymes involved in the cytosolic stages of peptidoglycan synthesis: alanine racemase (Alr) and D-alanine:D-alanine ligase (Ddl). The mechanisms of inhibition of DCS have been well-assessed using Alr and Ddl enzymes from various bacterial species, but little is known regarding the interactions of DCS with the mycobacterial orthologues of these enzymes. We have over-expressed and purified recombinant Mycobacterium tuberculosis Ddl (MtDdl; Rv2981c), and report a kinetic examination of the enzyme with both its native substrate and DCS. MtDdl is activated by K(+), follows an ordered ter ter mechanism and displays distinct affinities for D-Ala at each D-Ala binding site (K(m,D-Ala1) = 0.075 mm, K(m,D-Ala2) = 3.6 mm). ATP is the first substrate to bind and is necessary for subsequent binding of D-alanine or DCS. The pH dependence of MtDdl kinetic parameters indicate that general base chemistry is involved in the catalytic step. DCS was found to competitively inhibit D-Ala binding at both MtDdl D-Ala sites with equal affinity (K(i,DCS1) = 14 µm, K(i,DCS2) = 25 µm); however, each enzyme active site can only accommodate a single DCS molecule at a given time. The pH dependence of K(i,DCS2) revealed a loss of DCS binding affinity at high pH (pK(a) = 7.5), suggesting that DCS binds optimally in the zwitterionic form. The results of this study may assist in the design and development of novel Ddl-specific inhibitors for use as anti-mycobacterial agents.