Evaluation of a novel inhibitor of aspartate semialdehyde dehydrogenase as a potent antitubercular agent against Mycobacterium tuberculosis.

The in vitro activity of IMB-XMA0038, a novel inhibitor targeting Mycobacterial tuberculosis (Mtb) aspartate semialdehyde dehydrogenase, was evaluated. Minimum inhibitory concentrations (MICs) of IMB-XMA0038 were against 20 Mtb isolates, including H37Rv (ATCC 27294), ten clinical pan-sensitive isolates, and nine clinical multidrug-resistant (MDR) isolates. In addition, minimum bactericidal concentrations (MBCs) were also determined against the H37Rv and 6 MDR isolates (the background information is same as above in order). A model was generated to evaluate IMB-XMA0038 activity against dormant Mtb. The post-antibiotic effect (PAE), an important indicator of antimicrobial drug dosing schedules to obtain efficacy, was determined based on time required for regrowth of Mtb to 50% of the OD600max value after treatment with various concentrations of IMB-XMA0038 and INH. In addition, interactions between IMB-XMA0038 and other anti-tuberculosis drugs, measured using a checkerboard assay, revealed that IMB-XMA0038 MICs of 0.5-1 µg/mL could be achieved in combinations. Synergistic effects were observed for IMB-XMA0038 when used together with almost all other anti-tuberculosis drugs against most Mtb isolates. IMB-XMA0038 exhibited greater activity than rifampin against Mtb under hypoxic conditions, as reflected by CFU decreases of 1.1-log-unit versus 0.8-log-unit, respectively, for IMB-XMA0038 and rifampin concentrations of 4 × MIC. IMB-XMA0038-induced PAEs (9, 10, 11 days) were comparable to INH PAEs (10, 11, 12 days). These findings suggest that addition of IMB-XMA0038 to current therapeutic regimens could be useful to improve the efficacy of treatments for drug-resistant and drug-susceptible TB.

IMB-XMA0038, a new inhibitor targeting aspartate-semialdehyde dehydrogenase of Mycobacterium tuberculosis.

The emergence of drug-resistant tuberculosis (TB) constitutes a major challenge to TB control programmes. There is an urgent need to develop effective anti-TB drugs with novel mechanisms of action. Aspartate-semialdehyde dehydrogenase (ASADH) is the second enzyme in the aspartate metabolic pathway. The absence of the pathway in humans and the absolute requirement of aspartate in bacteria make ASADH a highly attractive drug target. In this study, we used ASADH coupled with Escherichia coli type III aspartate kinase (LysC) to establish a high-throughput screening method to find new anti-TB inhibitors. IMB-XMA0038 was identified as an inhibitor of MtASADH with an IC50 value of 0.59 µg/mL through screening. The interaction between IMB-XMA0038 and MtASADH was confirmed by surface plasmon resonance (SPR) assay and molecular docking analysis. Furthermore, IMB-XMA0038 was found to inhibit various drug-resistant MTB strains potently with minimal inhibitory concentrations (MICs) of 0.25-0.5 µg/mL. The conditional mutant strain MTB::asadh cultured with different concentrations of inducer (10-5 or 10-1 µg/mL pristinamycin) resulted in a maximal 16 times difference in MICs. At the same time, IMB-XMA0038 showed low cytotoxicity in vitro and vivo. In mouse model, it encouragingly declined the MTB colony forming units (CFU) in lung by 1.67 log10 dosed at 25 mg/kg for 15 days. In conclusion, our data demonstrate that IMB-XMA0038 is a promising lead compound against drug-resistant tuberculosis.

Identification of inhibitors targeting polyketide synthase 13 of Mycobacterium tuberculosis as antituberculosis drug leads.

Polyketide synthase 13 (Pks13) is an essential enzyme in the synthesis of mycolic acids in Mtb. Therefore, Pks13 is a promising drug target for tuberculosis treatment. We used a structure-guided approach to identify novel chemotype inhibitors of Pks13 and assessed them using a Pks13 enzymatic assay and surface plasmon resonance. The structure-activity relationships (SAR) results demonstrated that the substituents at the 2, 5, and 6 positions of the 4H-chromen-4-one scaffold are critical for maintaining the MIC. Compound 6e with 2-hydroxyphenyl at the 2 position of the 4H-chromen-4-one scaffold, exhibited potent activity against Mtb H37Rv (MIC = 0.45 µg/mL) and displayed good Pks13 affinity and inhibition (IC50 = 14.3 µM). This study described here could provide an avenue to explore a novel inhibitor class for Pks13 and aid the further development of antituberculosis drugs.

Long non-coding RNA UCA1 upregulates KIF20A expression to promote cell proliferation and invasion via sponging miR-204 in cervical cancer.

Cervical cancer is a female cancer with the second highest motility over the world. It is urgent to find new therapeutic methods based on long-coding RNAs and microRNAs. UCA1 was proved to be related with many human cancer types, but limited researches have been performed for the inner associations between UCA1 and cervical cancer. Eighty females who were undergoing surgeries were recruited for study in our research. We took the cervical cancer tissues and cells from them. Massive experiments and analysis were conducted to investigate the gene expressions and protein expressions about UCA1, KIF20A, and miR-204 in normal cells and cancer cells. The techniques contain real-time PCR, migration/invasion assay, western blot, in vivo experiments, and so on.We found that UCA1 expression was greatly up-regulated in cervical cancer tissues and cell lines. Our in vitro assays revealed that the suppressing of UCA1 could reduce cervical cancer cells proliferation, migration, and invasion. In addition, we found that lncRNA UCA1 could sponge miR-204 and promote the proliferation and invasion of cervical cancer cells via the up-regulating of KIF20A expression. As a result, the inhibiting of UCA1 could lower cervical cancer (CC) cells growth rate in vivo.Our results identified that UCA1 could serve as an oncogene in cervical cancer cell progression through the modulating of miR-204/KIF20A axis. It gives novel insights to the searching of novel therapeutic methods for cervical cancer.

Digging Deeper to Save the Old Anti-tuberculosis Target: D-Alanine-D-Alanine Ligase With a Novel Inhibitor, IMB-0283.

The emergence of drug-resistant Mycobacterium tuberculosis (Mtb) has hampered treatments for tuberculosis, which consequently now require novel agents to overcome such drug resistance. The genetically stable D-alanine-D-alanine ligase A (DdlA) has been deemed as an excellent therapeutic target for tuberculosis. In the present study, a competitive inhibitor (IMB-0283) of DdlA was obtained via high-throughput screening. The minimum inhibitory concentrations (MIC) of IMB-0283 for the standard and clinical drug-resistant Mtb strains ranged from 0.25 to 4.00 µg/mL, whereas the conventional inhibitor of DdlA, D-cycloserine (DCS), only inhibited the growth of the standard Mtb strain at 16 µg/mL. The lethal effect of IMB-0283 on Mtb was found to act intracellularly in a DdlA-dependent manner. Specifically, IMB-0283 prevented the synthesis of neonatal cell walls but did not damage mature cell walls. Compared with those of DCS, IMB-0283 exhibited lower cytotoxicity and a higher selective index (SI). At the same dosages of treatment, IMB-0283 reduced bacterial load (log CFU/mL) in an acute animal model from 5.58 to 4.40, while DCS did not yield any such treatment efficacy. Taken together, the lower cytotoxicity and more efficacious in vivo activity of IMB-0283 suggest that it is a promising lead compound for antituberculosis drug development.

IMB-SD62, a triazolothiadiazoles derivative with promising action against tuberculosis.

One lead 3,6-disubstituted 1,2,4-triazolo[3,4-b][1,3,4]thiadiazole was identified as an inhibitor of shikimate dehydrogenase with antitubercular activity. Following up this compound, we optimized the lead through systematic modification of the 3 and 6 positions. The antitubercular activities in vitro, shikimate dehydrogenase inhibitory activities and cytotoxicity of derivatives were determined. We found IMB-SD62 with lower cytotoxicity and better activity. Thus, we studied the in vivo efficacy of IMB-SD62 against Mycobacterium tuberculosis and pharmacokinetics of IMB-SD62. In vivo acute M. tuberculosis H37Rv infection assay, IMB-SD62 showed antitubercular activity with the mean lung CFU counts decreasing 1.7 lg. The plasma pharmacokinetics study in rats showed that the oral bioavailability of IMB-SD62 was 14% and the half time was 1.05 h. The results of tissue distribution indicated that IMB-SD62 was mainly absorbed by liver and lung. In vitro metabolism study suggested that the metabolic ways of IMB-SD62 were dealkylated, oxidized and demethylated. CYP enzyme inhibition of IMB-SD62 in human liver microsomes was also evaluated. IMB-SD62 showed barely inhibition on CYP3A4 and CYP2D6. The excretion study manifested that IMB-SD62 was mainly eliminated by fecal excretion in rats. We concluded that based on these pharmaceutical properties, IMB-SD62 has the potential to be developed into new TB drug.

Plasmid to generate Mycobacteria mutants.

The generation of conditional mutants has been an effective approach to studying bacteria and validating drug targets, and mutants of Mycobacteria are no exception. However unlike other bacteria, there is still a paucity of available tools for Mycobacteria. We constructed a new plasmid containing tetracycline-repressive expression system (TetRr1.7) and Xer Site-Specific recombinase system to generate label-free controllable expression strains. The plasmid was subsequently used to construct a strain of M. tuberculosis expressing the only copy of D-alanine:D-alanine ligase under the control of the tetracycline-repressive promoter. The results showed that the mutant strain lost the ability of colony formation, became more sensitive to D-cycloserine and the cell wall of the mutant strain was disrupted when anhydrotetracycline was added to the medium. Taken together these observations, confirmed that the expression of D-alanine:D-alanine ligase was tightly controlled by the promoter. In conclusion, the new plasmid is a convenient tool for constructing stable conditional mutant strains in Mycobacteria and can be used for future target identification.

Identification of a novel inhibitor of isocitrate lyase as a potent antitubercular agent against both active and non-replicating Mycobacterium tuberculosis.

OBJECTIVE: Screen and identify novel inhibitors of isocitrate lyase (ICL) as potent antitubercular agents against Mycobacterium tuberculosis and determine their inhibitory characteristics, antitubercular activities and mechanisms of action. METHODS: Recombinant ICL of M. tuberculosis was expressed and purified, which was used for high-throughput screening (HTS) and the following experiments. A total of 71,765 compounds were screened to identify ICL inhibitors which were then evaluated for their roles as potent antitubercular agents. To determine the inhibitory characteristics of the agents against latent M. tuberculosis in persistent infections, a macrophage model (mouse J774A.1 cell) infected with Mycobacterium marinum BAA-535 strain was built and assessed. The potent antitubercular agents were identified using the macrophage model. Then, the inhibitory intensity and mode of the agents that exhibit on ICL protein of M. tuberculosis were analyzed, and the interaction mechanisms were preliminarily clarified according to the parameters of enzyme kinetics, circular dichroism experiments, fluorescence quenching assay, and molecular docking. RESULTS: The previously established ICL inhibitor screening model was evaluated to be suitable for HTS assay. Of the 71,765 compounds, 13 of them were identified to inhibit ICL effectively and stably. IMBI-3 demonstrated the most significant inhibitory activity with IC50 of 30.9 µmol/L. Its minimum inhibitory concentration (MIC) for M. tuberculosis, including extensively drug-resistant tuberculosis (XDR-TB) and multidrug-resistant tuberculosis (MDR-TB), were determined in the range of 0.25-1 µg/mL. When IMBI-3 is used in combination with isoniazid, the colony-forming units (CFU) counting of latent M. tuberculosis in J774A.1 macrophage cells decreased significantly as IMBI-3 concentration increased. The inhibition mode of IMBI-3 on ICL was probably competitive inhibition with an inhibition constant (Ki) of approximate 1.85 µmol/L. The interaction between IMBI-3 and ICL of M. tuberculosis was also confirmed by circular dichroism experiments and fluorescence quenching assay. And seven possible active amino acids of ICL of M. tuberculosis were identified in the active site through molecular docking. CONCLUSION: IMBI-3, a novel potent antitubercular agent targeting ICL of M. tuberculosis, was identified and evaluated. It inhibited both log-phase M. tuberculosis in vitro and dormant M. tuberculosis in macrophages. It was the first representative compound of this family with the ICL enzyme inhibition and antimycobacterial activities.

Identification and Validation of Aspartic Acid Semialdehyde Dehydrogenase as a New Anti-Mycobacterium Tuberculosis Target.

Aspartic acid semialdehyde dehydrogenase (ASADH) lies at the first branch point in the essential aspartic acid biosynthetic pathway that is found in bacteria and plants but is absent from animals. Mutations in the asadh gene encoding ASADH produce an inactive enzyme, which is lethal. Therefore, in this study, we investigated the hypothesis that ASADH represents a new anti-Mycobacterium tuberculosis (MTB) target. An asadh promoter-replacement mutant MTB, designated MTB::asadh, in which asadh gene expression is regulated by pristinamycin, was constructed to investigate the physiological functions of ASADH in the host bacteria. Bacterial growth was evaluated by monitoring OD600 and ASADH expression was analyzed by Western blotting. The results showed that the growth and survival of MTB::asadh was completely inhibited in the absence of the inducer pristinamycin. Furthermore, the growth of the mutant was rigorously dependent on the presence of the inducer in the medium. The starved mutant exhibited a marked reduction (approximately 80%) in the cell wall materials compared to the wild-type, in addition to obvious morphological differences that were apparent in scanning electron microscopy studies; however, with the addition of pristinamycin, the cell wall contents and morphology similar to those of the wild-type strain were recovered. The starved mutant also exhibited almost no pathogenicity in an in vitro model of infection using mouse macrophage J774A.1 cells. The mutant showed a concentration-dependent recovery of pathogenicity with the addition of the inducer. These findings implicate ASADH as a promising target for the development of novel anti-MTB drugs.

Construction of small plasmid vectors for use in genetic improvement of the extremely acidophilic Acidithiobacillus caldus.

The genetic improvement of biomining bacteria including Acidithiobacillus caldus could facilitate the bioleaching process of sulfur-containing minerals. However, the available vectors for use in A. caldus are very scanty and limited to relatively large broad-host-range IncQ plasmids. In this study, a set of small, mobilizable plasmid vectors (pBBR1MCS-6, pMSD1 and pMSD2) were constructed based on plasmid pBBR1MCS-2, which does not belong to the IncQ, IncW, or IncP groups. The function of the tac promoter on 5.8-kb pMSD2 was determined by inserting a kanamycin-resistant reporter gene. The resulting recombinant pMSD2-Km was successfully transferred by conjugation into A. caldus MTH-04 with transfer frequency of 1.38±0.64×10(-5). The stability and plasmid copy number of pMSD2-Km in A. caldus MTH-04 were 75±2.7% and 5-6 copies per cell, respectively. By inserting an arsABC operon into pMSD2, an arsenic-resistant recombinant pMSD2-As was constructed and transferred into A. caldus MTH-04 by conjugation. The arsenic tolerance of A. caldus MTH-04 containing pMSD2-As was obviously increased up to 45mM of NaAsO2. These vectors could be applied in genetic improvement of A. caldus as well as other bioleaching bacteria.

Identification and validation of a novel lead compound targeting 4-diphosphocytidyl-2-C-methylerythritol synthetase (IspD) of mycobacteria.

Tuberculosis is a serious threat to world-wide public health usually caused in humans by Mycobacterium tuberculosis (M. tuberculosis). It exclusively utilizes the methylerythritol phosphate (MEP) pathway for biosynthesis of isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP), the precursors of all isoprenoid compounds. The 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase (IspD; EC 2.7.7.60) is the key enzyme of the MEP pathway. It is also of interest as a new chemotherapeutic target, as the enzyme is absent in mammals and ispD is an essential gene for growth. A high-throughput screening method was therefore developed to identify compounds that inhibit IspD. This process was applied to identify a lead compound, domiphen bromide (DMB), that may effectively inhibit IspD. The inhibitory action of DMB was confirmed by over-expressing or down-regulating IspD in Mycobacterium smegmatis (M. smegmatis), demonstrating that DMB inhibit M. smegmatis growth additionally through an IspD-independent pathway. This also led to higher levels of growth inhibition when combined with IspD knockdown. This novel IspD inhibitor was also reported to exhibit antimycobacterial activity in vitro, an effect that likely occurs as a result of perturbation of cell wall biosynthesis.