Identification of mycobacterial efflux pump inhibiting compounds from Berberis holstii Engl. and determination of their mechanism of action

Mycobacterium tuberculosis (Mtb), the causative agent for tuberculosis (TB), is the leading infectious killer of humankind. In 2023, an estimated 10.8 million new cases of TB and 1.25 million deaths were reported globally. Sub-Saharan Africa faces a heavy TB burden, worsened by high HIV-AIDS prevalence and rising drug resistance, making novel anti-TB therapies a pressing priority. This study investigated the efflux inhibition (EI) activity of compounds characterised from Berberisholstii Engl. extracts. Following in vitro evaluation of the antimycobacterial activity of aqueous and organic extracts against the non-pathogenic mycobacterial model, Mycobacterium smegmatis (Msm), and Mtb. Thereafter, molecular docking of several compounds identified in chromatographic fractions onto Rv1258c, MmpS5-MmpL5 and Rv2333c Mtb efflux pumps (EPs) was undertaken to infer their binding modes and affinities. Subsequently, fractions containing potentially active compounds were tested in combination with spectinomycin (SPEC) and fractional inhibitory concentration index (FICI) values determined. Validation of Mtb efflux pump inhibition was performed using CRISPRi knockdown strains of Rv1258c and Rv2333c. MeOH extracts of the root and the stem bark, and aqueous extracts of the roots, exhibited minimum inhibitory concentration (MIC99) values of 906.25μg/ml, 4500 μg/ml and 1875 μg/ml, respectively, against wild-type Msm. On the other hand, MeOH extract of the leaves and aqueous extract of the stem bark had MIC99 values of 60.43 μg/ml and 1.74 μg/ml respectively, against wild-type Mtb. Molecular docking of chillanamine, isoboldine, berberrubine, reticuline, N-methylcoclaurine, thalifoline, apoglaziovine, and orientine onto Mtb Rv1258c, MmpS5-MmpL5 and Rv2333c revealed string binding affinities of <-5 kcal/mol. Of the seven fractions containing the bioactive compounds evaluated in checkerboard assays, six were synergistic with SPEC (FICI <0.5), with two fractions lowering the MIC99 of SPEC by 8-fold against wild-type Mtb. This study identified phytochemicals with probable Mtb efflux pump inhibition, warranting further investigation for potential use in TB combination therapy.