Novel Inhibitors for Isocitrate Lyase as a Potent Antitubercular Agent for Mycobacterium Tuberculosis

Introduction: Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB). TB claimed 1.4 million lives in 2019. It is estimated that one-quarter of the world’s population is infected with latent TB. Current first- and second-line drugs used to treat TB require adherence to an extended period of therapy (at least six months) and do not target latent TB. It has been shown that the enzyme isocitrate lyase (ICL) is essential for the survival and persistence of Mtb in latent TB. ICL plays an important role in two metabolic pathways required during TB latency, the glyoxylate and methylcitrate cycle. ICL is absent in humans, and therefore, it is considered a promising drug target. Herein, is reported a novel family of ICL inhibitors that is effective in vitro against both the enzyme and Mtb. Methods: The His-tagged ICL1 enzyme was expressed using E. coli as a host and purified by Ni-NTA chromatography. The recombinant enzyme was used to set up crystallisation trials and in vitro enzymatic assay was performed on ICL1 inhibitors. The synthesis of D/L-threo-2-methylisocitrate and the methylation of five other drug candidates were described. Both co-crystallisation and soaking techniques were performed to obtain an ICL1:CL-54-04 complex crystal, whose structure was solved. The CL-54 drug family was tested against Mtb. A checkerboard assay was utilised to test the combinatory effect of CL-54-04 with rifampicin or bedaquiline in both glycerol and propionate media. To investigate the role of acetate metabolism in drug tolerance, ΔpckA, ICL knock-down and ΔprpD Mtb mutants were assessed. Results: Seven new conditions to crystallise ICL1 were identified, and eleven drugs were tested against the isolated enzyme. A promising new family of drugs have been identified as the most potent ICL1 inhibitors reported to date. One of the analogues, CL-54-04, had a bacteriostatic effect at 10 μM and a bactericidal effect at 100 μM against Mtb in vitro. A drug combination screening of CL-54-04 with rifampicin or bedaquiline led to an additive effect in the checkerboard assay and a significant impact in the colony-forming unit assay. The role of fatty acid metabolism in drug tolerance was investigated by testing three central carbon metabolism mutant strains against isoniazid. Discussion: From the three CL-54 analogues tested, only CL-54-04 caused a bactericidal effect against Mtb. A solved ICL1:CL-54-04 complex crystal structure showed that the catalytic loop had a distinctive move of 13.8 Å, suggesting that the binding of ICL with CL-54-04 leads to a close conformation of the active site. A superimposition of the solved ICL1:CL-54-04 complex and the ICL1 structure alignment with 3-nitropropionate demonstrates that CL-54-04 inhibitor binds and causes the same conformational changes as 3-nitropropionate. Using metabolomics analysis, the combination of rifampicin and CL-54-04 causes an accumulation of the methylcitrate cycle metabolites in propionate media, suggesting that the ICL has been inhibited. Fatty acid as a sole carbon source showed to increase the drug tolerance of all three Mtb mutants against isoniazid. Conclusion: A new drug family has been identified as lead compounds against ICL. The estimated IC50 of CL-54-01 is approximately half of the 3-nitropropionamide, and it causes the exact conformational change to the enzyme as 3-nitropropionate (the analogue of 3-nitropropionamide). These findings suggest that this drug family are the most promising ICL1 inhibitors reported to date

Fatty acid metabolism of Mycobacterium tuberculosis: a double-edged sword

Mycobacterium tuberculosis can cocatabolize a range of carbon sources. Fatty acids are among the carbons available inside the host’s macrophages. Here, we investigated the metabolic changes of the fatty acid-induced dormancy-like state of M. tuberculosis and its involvement in the acquisition of drug tolerance. We conducted metabolomics profiling using a phosphoenolpyruvate carboxykinase (PEPCK)-deficient M. tuberculosis strain in an acetate-induced dormancy-like state, highlighting an overaccumulation of methylcitrate cycle (MCC) intermediates that correlates with enhanced drug tolerance against isoniazid and bedaquiline. Further metabolomics analyses of two M. tuberculosis mutants, an ICL knockdown (KD) strain and PrpD knockout (KO) strain, each lacking an MCC enzyme—isocitrate lyase (ICL) and 2-methylcitrate dehydratase (PrpD), respectively—were conducted after treatment with antibiotics. The ICL KD strain, which lacks the last enzyme of the MCC, showed an overaccumulation of MCC intermediates and a high level of drug tolerance. The PrpD KO strain, however, failed to accumulate MCC intermediates as it lacks the second step of the MCC and showed only a minor level of drug tolerance compared to the ICL KD mutant and its parental strain (CDC1551). Notably, addition of authentic 2-methylisocitrate, an MCC intermediate, improved the M. tuberculosis drug tolerance against antibiotics even in glycerol medium. Furthermore, wild-type M. tuberculosis displayed levels of drug tolerance when cultured in acetate medium significantly greater than those in glycerol medium. Taken together, the fatty acid-induced dormancy-like state remodels the central carbon metabolism of M. tuberculosis that is functionally relevant to acquisition of M. tuberculosis drug tolerance

All-sky search for gravitational-wave bursts in the second joint LIGO-Virgo run

We present results from a search for gravitational-wave bursts in the data collected by the LIGO and Virgo detectors between July 7, 2009 and October 20, 2010: data are analyzed when at least two of the three LIGO-Virgo detectors are in coincident operation, with a total observation time of 207 days. The analysis searches for transients of duration < 1 s over the frequency band 64-5000 Hz, without other assumptions on the signal waveform, polarization, direction or occurrence time. All identified events are consistent with the expected accidental background. We set frequentist upper limits on the rate of gravitational-wave bursts by combining this search with the previous LIGO-Virgo search on the data collected between November 2005 and October 2007. The upper limit on the rate of strong gravitational-wave bursts at the Earth is 1.3 events per year at 90% confidence. We also present upper limits on source rate density per year and Mpc^3 for sample populations of standard-candle sources. As in the previous joint run, typical sensitivities of the search in terms of the root-sum-squared strain amplitude for these waveforms lie in the range 5 10^-22 Hz^-1/2 to 1 10^-20 Hz^-1/2. The combination of the two joint runs entails the most sensitive all-sky search for generic gravitational-wave bursts and synthesizes the results achieved by the initial generation of interferometric detectors.Comment: 15 pages, 7 figures: data for plots and archived public version at https://dcc.ligo.org/cgi-bin/DocDB/ShowDocument?docid=70814&version=19, see also the public announcement at http://www.ligo.org/science/Publication-S6BurstAllSky

Search for gravitational waves from binary black hole inspiral, merger, and ringdown

We present the first modeled search for gravitational waves using the complete binary black-hole gravitational waveform from inspiral through the merger and ringdown for binaries with negligible component spin. We searched approximately 2 years of LIGO data, taken between November 2005 and September 2007, for systems with component masses of 1–99M⊙ and total masses of 25–100M⊙. We did not detect any plausible gravitational-wave signals but we do place upper limits on the merger rate of binary black holes as a function of the component masses in this range. We constrain the rate of mergers for 19M⊙≤m1, m2≤28M⊙ binary black-hole systems with negligible spin to be no more than 2.0  Mpc−3 Myr−1 at 90% confidence