New Mutations in the Mycobacterial ATP Synthase: New Insights into the Binding of the Diarylquinoline TMC207 to the ATP Synthase C-Ring Structure

Abstract

TMC207 is a new antituberculous drug belonging to the diarylquinoline class which very efficiently inhibits the ATP synthase of mycobacteria such as Mycobacterium tuberculosis, one of the most important pathogens in the world. In order to map the amino acid residues involved in the binding of the drug, we have selected in vitro TMC207-resistant mutants from M. tuberculosis and diverse atypical mycobacteria. Six distinct mutations, Asp28¡Gly, Asp28¡Ala, Leu59¡Val, Glu61¡Asp, Ala63¡Pro, and Ile66¡Met, have been identified in the subunit c forming a C ring in the ATP synthase. They were studied by evaluating the levels of resistance that they confer in the selected clones and by using an isogenic complementation system in Mycobacterium smegmatis. The rates of increase of TMC207 MIC values (8-to 133-fold) were interpreted by constructing by homology modeling a structure of the mycobacterial C ring which was used for docking simulations with TMC207. Our results suggest that the residues found to be mutated in the resistant clones, together with a tyrosine specifically conserved at position 64 in mycobacteria, define a cleft located between two adjacent c subunits in the C ring. This cleft, which encompasses the proton-binding site (Glu61), is well fitted to bind TMC207 at the level of the bromoquinoline moiety, with the drug being anchored by several ionic, hydrogen, and halogen bonds with residues Glu61, Tyr64, and Asp28, respectively. These data shed light on the molecular interactions allowing TMC207 to bind specifically and efficiently at the level of the proton-binding site of the mycobacterial C ring.

Primary Clofazimine and Bedaquiline Resistance among Isolates from Patients with Multidrug-Resistant Tuberculosis

“…In addition to identifying one mutation, c158t (S53L), that was previously reported (11), we identified three new mutations scattered across the Rv0678 gene (Table 2). Several target-based resistance mutations in the atpE gene have been described in bedaquiline-resistant strains previously selected in vitro (16)(17)(18)), but we did not find any atpE mutations in these isolates. Almeida et al (14) discovered mutations in pepQ that confer low-level resistance to both clofazimine (0.5 to 1 g/ml) and bedaquiline (0.12 to 0.25 g/ml) in M. tuberculosis, but we did not find pepQ mutations in the five clinical isolates with clofazimine MICs of Ն1.2 g/ml.…”

“…As mutations in Rv0678 are a major mechanism of clofaziminebedaquiline cross-resistance (11) and several target-based resistance mutations in the atpE gene have been described in bedaquiline-resistant strains selected in vitro (16)(17)(18), we sequenced the Rv0678 and atpE genes. All four clofazimine-bedaquiline crossresistant strains had a mutation in the Rv0678 gene.…”

In Vitro Susceptibility Testing of Bedaquiline against Mycobacterium avium Complex

“…The gene encoding the subunit of mycobacterial ATP is called atpE, and its amino acid sequence is known to be highly conserved in isolates of Mycobacterium tuberculosis (3). Studies by Segala and colleagues used multiple mutations in subunit C in the mycobacterial C ring to show its importance in the binding of bedaquiline (9). There appears to be no cross-resistance with standard antituberculous agents, although there is cross-resistance with clofazimine, a drug used primarily for leprosy but occasionally for MDR-TB, and with some nontuberculous mycobacteria (NTM) (10,11).…”

Potenciais alvos moleculares para o desenvolvimento de novos fármacos antituberculose

“…Apesar dos recentes avanços, 17,18 infelizmente já foram relatas cepas resistentes a estas novas moléculas, devido ao fato das bactérias estarem em constante evolução, [19][20][21] reforçando a urgente necessidade do desenvolvimento de novos fármacos para o tratamento da tuberculose. 2 Todavia, após a bedaquilina houve um aumento notável no número de artigos reportando compostos com potente atividade antituberculose.…”