Repurposing chlorpromazine and its metabolites for antituberculosis drug discovery

Doctoral Thesis


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University of Cape Town

New chemotherapeutics are urgently needed to combat Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB). The development of compounds that could potentiate the activity of known antimycobacterial drugs is a relatively unexplored approach to new TB drug discovery. This study aimed to generate metabolites of chlorpromazine (CPZ), a phenothiazine with demonstrated in vitro activity against Mtb, and to investigate their potential utility in combination with anti-TB drugs. 7-HydroxyCPZ (M2), CPZ-N-oxide (M3), CPZ sulfoxide (M1), nor-CPZ (M5), nor-CPZ sulfoxide (M6b) and CPZ-N-S-dioxide (M4b) were generated from CPZ using various biotransformation systems and identified by Liquid Chromatography - Mass Spectrometry (LC/MS). The identity of M2 was confirmed with reference to a 7-hydroxyCPZ standard. M3, M1, M5, M6b and M4b were synthesized de novo and used to identify the metabolites generated in the biotransformation samples. Individually, CPZ and its metabolites (M2, M3, M5) were weakly active (MIC99 >50μM) against M. smegmatis (Msm) and Mtb while M1, M6b & M4b did not exhibit a MIC99 even at very high concentrations. Generally, an improvement in activity was observed where CPZ or its metabolites were used in combination with known anti-TB drugs. The combinations that exhibited a fractional inhibition concentration index (FICI) of < 0.5 were defined as synergistic. A combination of M2 and spectinomycin (SPEC) exhibited the highest synergism against Msm (FICI 0.19) and Mtb (FICI 0.13). In vitro assays established that CPZ and M2 are bactericidal against Mtb whereas M3 and M5 are bacteriostatic on their own. In combination assays, the use of RIF with M3 and M5, bedaquiline (BDQ) with M2, and SPEC with M3 were bactericidal. At 140μM, CPZ and M1, M2, M3 treated samples exhibited a 2-fold up-regulation of the cydA (Rv1623c) gene which encodes an essential subunit of the cytochrome bd-type menaquinol oxidase in Mtb. The same observation was made for RIF/M2 and RIF/M5 treated samples. These results suggest that the metabolites retain the mechanism of action (MoA) as the parental CPZ. The Mtb 16S rRNA gene, rrs (MTB000019) was identified as the biological target for SPEC. This brought into perspective the underlying mechanisms at play when SPEC is used in combination with CPZ, its metabolites or other drugs, against mycobacteria. This study establishes the utility of combination assays in confirming the active metabolite(s) of known drugs and provides proof of concept data to support follow-up investigations of CPZ and its metabolites as potential compounds for novel combination therapies for anti-TB drug development.

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