Identifying small molecule binding ligands for the pup-ligase (PafA) of Mycobacterium tuberculosis.
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Date
2022
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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.
Description
Masters Degree. University of KwaZulu-Natal, Pietermaritzburg.