The Structural Basis of Mycobacterium tuberculosis RpoB Drug-Resistant Clinical Mutations on Rifampicin Drug Binding

Abstract

Tuberculosis (TB), caused by the Mycobacterium tuberculosis infection, continues to be a leading cause of morbidity and mortality in developing countries. Resistance to the first-line anti-TB drugs, isoniazid (INH) and rifampicin (RIF), is a major drawback to effective TB treatment. Genetic mutations in the β-subunit of the DNA-directed RNA polymerase (rpoB) are reported to be a major reason of RIF resistance. However, the structural basis and mechanisms of these resistant mutations are insufficiently understood. In the present study, thirty drug-resistant mutants of rpoB were initially modeled and screened against RIF via a comparative molecular docking analysis with the wild-type (WT) model. These analyses prioritized six mutants (Asp441Val, Ser456Trp, Ser456Gln, Arg454Gln, His451Gly, and His451Pro) that showed adverse binding affinities, molecular interactions, and RIF binding hinderance properties, with respect to the WT. These mutant models were subsequently analyzed by molecular dynamics (MD) simulations. One-hundred nanosecond all-atom MD simulations, binding free energy calculations, and a dynamic residue network analysis (DRN) were employed to exhaustively assess the impact of mutations on RIF binding dynamics. Considering the global structural motions and protein–ligand binding affinities, the Asp441Val, Ser456Gln, and His454Pro mutations generally yielded detrimental effects on RIF binding. Locally, we found that the electrostatic contributions to binding, particularly by Arg454 and Glu487, might be adjusted to counteract resistance. The DRN analysis revealed that all mutations mostly distorted the communication values of the critical hubs and may, therefore, confer conformational changes in rpoB to perturb RIF binding. In principle, the approach combined fundamental molecular modeling tools for robust “global” and “local” level analyses of structural dynamics, making it well suited for investigating other similar drug resistance cases.

State of the Art on Developments of (Bio)Sensors and Analytical Methods for Rifamycin Antibiotics Determination

“…RIF has an effectiveness of about 95% in cases of TB caused by susceptible strains [ 47 ], with a daily oral administration of the standard 600 mg single dose [ 24 , 48 ] or of a dose corresponding to 10–20 mg RIF/kg body weight [ 49 , 50 ]. Alone or in combination with other drugs, it represents the main treatment for infections (produced by various microorganisms), such as TB ( Mycobacteria tuberculosis ), osteomyelitis ( Staphylococcus aureus ), meningococcal disease ( Neisseria meningitidis ), leprosy ( Mycobacterium leprae ), gonorrhea ( Neisseria gonorrhoeae ) [ 51 ], Legionnaire’s disease ( Legionella pneumophila) [ 19 , 28 ] and even HIV [ 52 , 53 ]. Under regulated dosage, RIF is also recommended against methicillin-resistant Staphylococcus aureus [ 19 ], to treat cholestatic pruritus and infections associated with prosthetic joints [ 54 ], such as chemoprophylaxis of the postoperative endophthalmitis [ 55 ], or against Brucellosis and meningitis caused by Streptococcus pneumoniae , Haemophilus influenzae Type b and Neisseria meningitides [ 19 ].…”

Investigation of Multi-Subunit Mycobacterium tuberculosis DNA-Directed RNA Polymerase and Its Rifampicin Resistant Mutants

“…The clinical effects of H451D, H451L, H451N and H451R were also reported through several studies encompassing multiple populations worldwide [ 28 ]. Several in silico studies have attempted to investigate the binding of RIF at the Mtb -RNAP β subunit, thus providing mechanistic insights into the delimiting effects of these mutations [ 29 , 30 , 31 , 32 ]. Some specifically revealed that D441V, H451D, H451R and H451Y mutations may affect RIF activity by altering the critical binding pocket interactions [ 29 , 30 , 31 , 32 ].…”

“…Several in silico studies have attempted to investigate the binding of RIF at the Mtb -RNAP β subunit, thus providing mechanistic insights into the delimiting effects of these mutations [ 29 , 30 , 31 , 32 ]. Some specifically revealed that D441V, H451D, H451R and H451Y mutations may affect RIF activity by altering the critical binding pocket interactions [ 29 , 30 , 31 , 32 ]. Moreover, although numerous RRDR and non-RRDR or distal Mtb -RNAP mutations have been reported, the majority of clinical cases of RIF inefficacy have been linked to high-confidence mutations mostly occurring at residue positions D441, H451 and S456 located within RRDR cluster I [ 28 ].…”