Synthesis, oxidation potential and anti-mycobacterial activity of isoniazid and analogues: insights into the molecular isoniazid activation mechanism

Tuberculosis (TB) is one of the leading causes of death due to infectious diseases. Among the specific drugs currently employed to treat tuberculosis, isoniazid (INH), a pro-drug, attracts a great interest. However, Mycobacterium tuberculosis (MTB) clinical isolates resistant to INH are significantly increasing thus compromising the efficiency of TB-treatment. Concerning the mechanism of resistance to INH, it is nowadays well established that it mainly results from mutations in the katG genes encoding for INH enzymatic activator. Recently, it was proposed that mutation in katG induces differences in heme or side chain redox potential so that the enzyme loses its ability to oxidize INH to radical species. In this work, we synthesized and selected a series of INH analogues for a study of their oxidation potential and evaluation of their anti-TB efficiency toward MTB wild-type and drug resistant strains. On the contrary to what was postulated, no correlation exists between the easier oxidation of a molecule and its anti-MTB activity toward resistant strains. Based on experimental data and theoretical calculations, we proposed an activation mechanism for INH and analogues based on a one-electron oxidation step of the hydrazyl function at the proximal nitrogen followed by a radical transposition to the distal nitrogen, which then induces a b-homolytic cleavage of the C(=O)-N bond to afford diazene and the isonicotinoyl radical species

Mechanochemical Synthesis and Biological Evaluation of Novel Isoniazid Derivatives with Potent Antitubercular Activity.

A series of isoniazid derivatives bearing a phenolic or heteroaromatic coupled frame were obtained by mechanochemical means. Their pH stability and their structural (conformer/isomer) analysis were checked. The activity of prepared derivatives against Mycobacterium tuberculosis cell growth was evaluated. Some compounds such as phenolic hydrazine 1a and almost all heteroaromatic ones, especially 2, 5 and 7, are more active than isoniazid, and their activity against some M. tuberculosis MDR clinical isolates was determined. Compounds 1a and 7 present a selectivity index >1400 evaluated on MRC5 human fibroblast cells. The mechanism of action of selected hydrazones was demonstrated to block mycolic acid synthesis due to InhA inhibition inside the mycobacterial cell

A Phenotypic Based Target Screening Approach Delivers New Antitubercular CTP Synthetase Inhibitors

Despite its great potential, the target-based approach has been mostly unsuccessful in tuberculosis drug discovery, while whole cell phenotypic screening has delivered several active compounds. However, for many of these hits, the cellular target has not yet been identified, thus preventing further target-based optimization of the compounds. In this context, the newly validated drug target CTP synthetase PyrG was exploited to assess a target-based approach of already known, but untargeted, antimycobacterial compounds. To this purpose the publically available GlaxoSmithKline antimycobacterial compound set was assayed, uncovering a series of 4-(pyridin-2-yl)­thiazole derivatives which efficiently inhibit the <i>Mycobacterium tuberculosis</i> PyrG enzyme activity, one of them showing low activity against the human CTP synthetase. The three best compounds were ATP binding site competitive inhibitors, with <i>K</i>_i values ranging from 3 to 20 μM, but did not show any activity against a small panel of different prokaryotic and eukaryotic kinases, thus demonstrating specificity for the CTP synthetases. Metabolic labeling experiments demonstrated that the compounds directly interfere not only with CTP biosynthesis, but also with other CTP dependent biochemical pathways, such as lipid biosynthesis. Moreover, using a <i>M. tuberculosis pyrG</i> conditional knock-down strain, it was shown that the activity of two compounds is dependent on the intracellular concentration of the CTP synthetase. All these results strongly suggest a role of PyrG as a target of these compounds, thus strengthening the value of this kind of approach for the identification of new scaffolds for drug development

אסוקי הלכתא - פסחים

Abstract Mycobacterium tuberculosis, the etiological agent of the infectious disease tuberculosis, kills approximately 1.5 million people annually, while the spread of multidrug-resistant strains is of great global concern. Thus, continuous efforts to identify new antitubercular drugs as well as novel targets are crucial. Recently, two prodrugs activated by the monooxygenase EthA, 7947882 and 7904688, which target the CTP synthetase PyrG, were identified and characterized. In this work, microbiological, biochemical, and in silico methodologies were used to demonstrate that both prodrugs possess a second target, the pantothenate kinase PanK. This enzyme is involved in coenzyme A biosynthesis, an essential pathway for M. tuberculosis growth. Moreover, compound 11426026, the active metabolite of 7947882, was demonstrated to directly inhibit PanK, as well. In an independent screen of a compound library against PyrG, two additional inhibitors were also found to be active against PanK. In conclusion, these direct PyrG and PanK inhibitors can be considered as leads for multitarget antitubercular drugs and these two enzymes could be employed as a “double-tool” in order to find additional hit compounds

The EU approved antimalarial pyronaridine shows antitubercular activity and synergy with rifampicin, targeting RNA polymerase

The search for compounds with biological activity for many diseases is turning increasingly to drug repurposing. In this study, we have focused on the European Union-approved antimalarial pyronaridine which was found to have in vitro activity against Mycobacterium tuberculosis (MIC 5 mu g/mL). In macromolecular synthesis assays, pyronaridine resulted in a severe decrease in incorporation of C-14-uracil and C-14-leucine similar to the effect of rifampicin, a known inhibitor of M. tuberculosis RNA polymerase. Surprisingly, the co-administration of pyronaridine (2.5 mu g/ml) and rifampicin resulted in in vitro synergy with an MIC 0.0019-0.0009 mu g/mL. This was mirrored in a THP-1 macrophage infection model, with a 16-fold MIC reduction for rifampicin when the two compounds were co-administered versus rifampicin alone. Docking pyronaridine in M. tuberculosis RNA polymerase suggested the potential for it to bind outside of the RNA polymerase rifampicin binding pocket. Pyronaridine was also found to have activity against a M. tuberculosis clinical isolate resistant to rifampicin, and when combined with rifampicin (10% MIC) was able to inhibit M. tuberculosis RNA polymerase in vitro. All these findings, and in particular the synergistic behavior with the antitubercular rifampicin, inhibition of RNA polymerase in combination in vitro and its current use as a treatment for malaria, may suggest that pyronaridine could also be used as an adjunct for treatment against M. tuberculosis infection. Future studies will test potential for in vivo synergy, clinical utility and attempt to develop pyronaridine analogs with improved potency against M. tuberculosis RNA polymerase when combined with rifampicin