| Abstract | 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. |
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