OR35

1:San Raffaele Scientific Institute; 2:University of Milan

The bedaquiline (BDQ)-based regimen for multidrug-resistant tuberculosis (MDR-TB), “BPaLM”, is facing limitation due to the increasing presence of Rv0678 mutation that reduce the regulation of mmpL5/S5 efflux pump. DprE1 inhibitors, a new promising class of anti-TB drugs, share the same resistance pathway as BDQ. This highlights the need to analyse their interactions with BDQ when co-administered. EUCAST protocol was used to establish BDQ and BTZ-043 minimal inhibitory concentrations (MICs) in both H37Rv (n=3) and resistant mutants (n=12). An EUCAST-based checkerboard assay was developed to investigate BDQ/BTZ-043 interactions. DMSO-soluble drugs were prepared in 7H9/OADC, and serial dilutions were performed in 96-well plates in 7H9/OADC ensuring a final 0.5% DMSO concentration. Delamanid, expected to be synergic with BDQ, was used as control. A computational analysis was used to explore BDQ/BTZ-043 interactions and mmpL5/S5 efflux pump's role to better understand the in-vitro results. Computational analyses reveal that DprE1 inhibitors possess high affinity with an mmpL5/S5 pump’s accessory pocket, this affinity increase when in hetero-(with BDQ) or homo-dimers and can be linked to mmpL5/S5 saturation. This suggest that free DprE1 inhibitors (e.g., unbound due to target mutation) may enhance BDQ activity. In-vitro experiments indicate that BTZ-043 cooperates with BDQ inducing a MIC shift upon combination, predominantly in strains with high-level resistance to BTZ-043. These results underline the importance of testing combination of anti-TB drugs to predict their interaction before market introduction. The efflux pumps’ role will be validated with the use of the FDA-approved efflux pump inhibitor Verapamil in combinatory experiments.