Identifying small molecule binding ligands for the pup-ligase (PafA) of Mycobacterium tuberculosis.

Abstract

The rapid emergence of resistant TB strains (Mycobacterium tuberculosis (Mtb)) renders traditional treatment options ineffective and necessitates the generation of novel anti-TB drugs that possess innovative modes of action. The pup-ligase (PafA) of Mtb that solely mediates protein proteasomal removal via the pupylation cascade has recently been identified as a suitable target for TB drug development. A novel approach would be to recruit proteolysis targeting chimeras (PROTACs) technology as an alternative anti-TB treatment option by developing PROTAC-like molecules capable of recruiting the pupylation cascade. Therefore, the identification of novel PafA small-molecule binding ligands is an essential first step to establish possible new TB therapies. To this effect, PafA recombinant expression was successfully optimised in E. coli cells at 20°C for 20 h, where a 50-kDa protein was observed by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Moreover, the identity of the protein was confirmed via immunoblotting with anti-His antibodies and PafA subsequently purified via immobilized metal affinity chromatography (IMAC) to high purity. A thermal shift assay (TSA) of PafA against 48 small-molecule compounds from a chemical library pre-screened for non-specific inhibition activity was conducted. Seven Hit compounds were detected significantly binding PafA (P < 0.05), all inducing a > 5 °C increase of PafA melting temperature (Tm) upon binding. Future research on these novel PafA binding ligands will be to ascertain whether they possess inhibitor qualities. Additionally, they will be used in the synthesis of heterobifunctional molecules to create the first PROTAC-like molecules for targeted proteasomal degradation of essential Mtb proteins – a novel type of anti-TB drug.