Identifying prospective inhibitors against LdtMt5 from Mycobacterium tuberculosis as a potential drug target.
Sabe, Victor Tinashe.
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Tuberculosis (TB) caused by the bacterium, Mycobacterium tuberculosis (M.tb) has resulted in an unprecedented number of deaths over centuries. L,D-transpeptidase enzymes are known to play a crucial role in the biosynthesis of the cell wall, which confers resistance to most antibiotics. These enzymes catalyze the 3→3 peptidoglycan cross-links of the M.tb cell wall. Specific β-lactam antibiotics (carbapenems) have been reported to inhibit cell wall polymerization of M.tb and they inactivate L,D-transpeptidases through acylation. L,Dtranspeptidase 5 (LdtMt5) is a unique paralog and a vital protein in maintaining integrity of the cell wall specifically in peptidoglycan metabolism therefore making it an important protein target. Carbapenems inhibit LdtMt2, but do not show reasonable inhibitory activities against LdtMt5. We therefore sought to perform virtual screening in order to acquire potential inhibitors against LdtMt5 and to investigate the affinity and to calculate the binding free energies between LdtMt5 and potential inhibitors. Furthermore, we sought to investigate the nature of the transition state involved in the catalytic reaction mechanism; to determine the activation free energies of the mechanism using ONIOM through the thermodynamics and energetics of the reaction path and lastly to express, purify and perform inhibition studies on LdtMt5. A total of 12766 compounds were computationally screened from the ZINC database to identify potential leads against LdtMt5. Docking was performed using two different software programs. Molecular dynamics (MD) simulations were subsequently performed on compounds obtained through virtual screening. Density functional theory (DFT) calculations were then carried out to understand the catalytic mechanism of LdtMt5 with respect to β-lactam derivatives using a hybrid ONIOM quantum mechanics/molecular mechanics (QM/MM) method. LdtMt5 complexes with six selected β-lactam compounds were evaluated. Finally, a lyophilised pET28a-LdtMt5 was used to transform E. coli strain BL21 (DE3) and SDS-PAGE was used to verify the purity, molecular weight and protein profile determination. Finally, an in vitro binding thermodynamics analysis using isothermal titration calorimetry (ITC) was later on performed on a single compound (the strongest binder) from the final set, in a bid to further validate the calculated binding energy values. A number of compounds from four different antimicrobial classes (n = 98) were obtained from the virtual screening and those with docking scores ranging from -7.2 to -9.9 kcal mol-1 were considered for MD analysis (n = 37). A final set of 10 compounds which exhibited the greatest affinity, from four antibiotic classes was selected and Molecular Mechanics/Generalized Born iii Surface Area (MM-GBSA) binding free energies (ΔGbind) from the set were characterised. The calculated binding free energies ranged from -30.68 to -48.52 kcal mol-1 . The β-lactam class of compounds demonstrated the highest ΔGbind and also the greatest number of potential inhibitors. The DFT activation energies (∆G # ) obtained for the acylation of LdtMt5 by the six selected β-lactams were calculated as 13.67, 20.90, 22.88, 24.29, 27.86 and 28.26 kcal mol-1 . The ∆G# results from the 6-membered ring transition state (TS) revealed that all selected six βlactams were thermodynamically more favourable than previously calculated activation energy values for imipenem and meropenem complexed with LdtMt5. The results are also comparable to those observed for LdtMt2, however for compound 1 the values are considerably lower than those obtained for meropenem and imipenem in complex with LdtMt2, thus suggesting in theory that compound 1 is a more potent inhibitor of LdtMt5. We also report the successful expression and and purification of LdtMt5, however the molecule selected for the in vitro inhibition study gave a poor result. On further review, we concluded that the main cause of this outcome was due to the relatively low insolubility of the compound. The outcome of this study provides insight into the design of potential novel leads for LdtMt5. Our screening obtained ten novel compounds from four different antimicrobial classes. We suggest that further in vitro binding thermodynamics analysis of the novel compounds from the four classes, including the carbapenems be performed to evaluate inhibition of these compounds on LdtMt5. If the experimental observations suggest binding affinity to the protein, catalytic mechanistic studies can be undertaken. These results will also be used to verify or modify our computational model.