Identification of anti-Mycobacterium tuberculosis agents targeting the interaction of bacterial division proteins FtsZ and SepFe

Tuberculosis (TB) is one of the deadly diseases caused by Mycobacterium tuberculosis (Mtb), which presents a significant public health challenge. Treatment of TB relies on the combination of several anti-TB drugs to create shorter and safer regimens. Therefore, new anti-TB agents working by different mechanisms are urgently needed. FtsZ, a tubulin-like protein with GTPase activity, forms a dynamic Z-ring in cell division. Most of FtsZ inhibitors are designed to inhibit GTPase activity. In Mtb, the function of Z-ring is modulated by SepF, a FtsZ binding protein. The FtsZ/SepF interaction is essential for FtsZ bundling and localization at the site of division. Here, we established a yeast two-hybrid based screening system to identify inhibitors of FtsZ/SepF interaction in M. tuberculosis. Using this system, we found compound T0349 showing strong anti-Mtb activity but with low toxicity to other bacteria strains and mice. Moreover, we have demonstrated that T0349 binds specifically to SepF to block FtsZ/SepF interaction by GST pull-down, fluorescence polarization (FP), surface plasmon resonance (SPR) and CRISPRi knockdown assays. Furthermore, T0349 can inhibit bacterial cell division by inducing filamentation and abnormal septum. Our data demonstrated that FtsZ/SepF interaction is a promising anti-TB drug target for identifying agents with novel mechanisms.

Esteroide e Triterpenos de espécies de Qualea - Bioatividade sobre Mycobacterium tuberculosis / Sterol and Triterpenes from Qualea species: Bioactivity on Mycobacterium tuberculosis

O extrato clorofórmico das cascas de Qualea parviflora (Vochysiaceae) foi fracionado em cromatografia em coluna usando sílica gel para fornecer triterpenos (lupeol, lupenona, betulina, ácido epibetulínico, e friedelina) e um esteroide (β-sitosterol). Os compostos β- sitosterol, lupenona e lupeol foram identificados também em Q. grandiflora e Q. multiflora, enquanto friedelina foi detectada apenas em Q. multiflorautilizando a cromatografia gasosa acoplada à espectrometria de massas. A atividade anti Mycobacterium tuberculosis do extrato clorofórmico e dos compostos isolados foi determinada por MABA e o valor da CIM variou de 250,0 a 31,2 μg/mL. Esta investigação constitui o primeiro relato do estudo químico e antitubercular de compostos apolares das espécies de Qualea.

The chloroform extract of bark of the tropical tree Qualea parviflora (Vochysiaceae) was fractionated by column chromatography on silica gel, yielding triterpenes (lupeol, lupenone, betulin, epibetulinic acid and friedelin) and a steroid (β-sitosterol). β-sitosterol, lupenone and lupeol were also identified in Q. grandiflora and Q. multiflora, while friedelin was detected only in Q. Multiflora, by means of gas chromatography-mass spectrometry. The anti-Mycobacterium tuberculosis activity of the chloroform extract and isolated compounds was assayed by MABA and MIC values ranged from 250.0 to 31.2 μg/mL. This study is the first to investigate the chemistry and antitubercular activity of apolar compounds from Qualeaspecies.

Assessment of the Activity of Selected Indian Medicinal Plants against Mycobacterium tuberculosis: A Preliminary Screening Using the Microplate Alamar Blue Assay.

Aim: Identification of anti-Mycobacterium tuberculosis agents of plant origin, against sensitive and multidrug resistant (MDR) strains. Study Design: Assessing anti-M. tuberculosis activity of five Indian medicinal plants, which have been reported in traditional literature for various uses including respiratory ailments. Place and Duration of Study: Mumbai, India; May 2009 – December 2011. Methodology of Study: The reference strain (H37Rv), three susceptible and three MDR clinical isolates of M. tuberculosis were used. Acetone, ethanol and aqueous extracts (prepared sequentially) of Acorus calamus L. (rhizome), Andrographis paniculata Nees. (leaf), Ocimum sanctum L. (leaf), Piper nigrum L. (seed) and Pueraria tuberosa DC. (tuber) were tested at 1, 10 and 100 μg/ml using the Microplate Alamar Blue Assay. The active extracts were assessed for cytotoxicity on the human lung epithelial cell line (A549) using the neutral red assay and a phytochemical analysis was made using High Performance Thin Layer Chromatography (HPTLC). Results: Among the plants tested, the acetone extract of P. nigrum appears promising. It was effective against H37Rv, all susceptible isolates and one MDR isolate at 100 μg/ml. The ethanol extract caused some inhibition of growth, though less than the cut-off of 99%. A combination of acetone and ethanol extracts at 50 μg/ml each was effective against all isolates tested. The known active phytoconstituent of P. nigrum, piperine (also an efflux pump inhibitor), was effective against H37Rv in the presence of suboptimal concentration of Rifampicin, but not against the clinical isolates tested. Presence of piperine in the acetone and ethanol extracts was confirmed by HPTLC. Extracts of P. nigrum and piperine were not cytotoxic to the A549 cell line. Conclusion: Amongst the five plants tested, P. nigrum was active. The acetone extract may have active components in addition to piperine. It is possible that the class and expression of efflux pumps in H37Rv is different from that in the clinical isolates, and hence piperine did not inhibit these isolates. Thus, it is necessary to screen clinical isolates in addition to reference strains. The observation of the increased efficacy of the combination of acetone and ethanol extracts is interesting.