Fragment Screening Hit Draws Attention to a Novel Transient Pocket Adjacent to the Recognition Site of the tRNA-Modifying Enzyme TGT

Abstract

Fragment-based lead discovery was applied to tRNA-guanine transglycosylase, an enzyme modifying post-transcriptionally tRNAs in Shigella, the causative agent of shigellosis. TGT inhibition prevents translation of Shigella’s virulence factor VirF, hence reducing pathogenicity. One discovered fragment opens a transient subpocket in the preQ1-recognition site by pushing back an aspartate residue. This step is associated with reorganization of further amino acids structurally transforming a loop adjacent to the recognition site by duplicating the volume of the preQ1-recognition pocket. We synthesized 6-carboxamido-, 6-hydrazido-, and 4-guanidino-benzimidazoles to target the opened pocket, including a dihydro-imidazoquinazoline with a propyn-1-yl exit vector pointing into the transient pocket and displacing a conserved water network. MD simulations and hydration-site analysis suggest water displacement to contribute favorably to ligand binding. A cysteine residue, exclusively present in bacterial TGTs, serves as gatekeeper of the transient subpocket. It becomes accessible upon pocket opening for selective covalent attachment of electrophilic ligands in eubacterial TGTs.

Fragment-to-Lead Medicinal Chemistry Publications in 2020

“… 22 While a string of other 2020 publications describe antimicrobial FBLD hit-finding and exploration studies, they do not meet all the criteria for F2L table inclusion. Examples include studies focused on bacterial targets such as metallo-β-lactamase(MBL)NDM-1, 54 dihydrofolate reductase from M. tuberculosis (MtDHFR), 55 M. tuberculosis MabA (FabG1) 56 and tRNA-modifying enzyme TGT, where a hit fragment opens a transient subpocket that can be exploited by a new series of ligands; 57 tRNA modification enzyme TrmD, 58 fungal targets such as glucosamine 6-phosphate N -acetyltransferase (Gna1), which is a key enzyme in the biosynthesis of an essential fungal cell-wall component; 59 parasite targets such as the cysteine protease enzyme cruzain; 60 allosteric binders for farnesyl pyrophosphate synthase of Trypanosoma brucei ; 61 bromodomain-containing factor 3 of Trypanosoma cruzi ; 62 membrane-bound pyrophosphatases (mPPases); virus targets (e.g., viral DNA-binding proteins Epstein–Barr nuclear antigen 1 (EBNA1) and latency-associated nuclear antigen (LANA); 63 and, as indicated above, several efforts to probe and target SARS-CoV-2 proteins. Another interesting study developed PqsR-targeting quorum-sensing inhibitors (a study that uses biophysical screening and enthalpic efficiency evaluations, and introduces relatively flexible linkers).…”

“…Another continuing theme is the identification of novel binding sites, such as that for tRNA-modifying enzyme TGT, where one fragment opens up a transient subpocket that can be exploited by newly designed ligands. Other studies that did not meet the lead criteria explore allosteric pockets in enzymes, e.g., for farnesyl pyrophosphate synthase of the parasite T. brucei …”

Application of Fragment-Based Drug Discovery to Versatile Targets

“…While aforementioned methods are useful in FBDD, other methods that are able to probe protein-ligand interactions have been utilized in FBDD. An anchoring approach was applied to develop protease inhibitors (Hassaan et al, 2020;Konstantinidou et al, 2020). Capillary electrophoresis was successfully applied to identify fragment hits binding to heat shock protein 90 ATPase (Austin et al, 2012).…”

“…It has been noted that the shift of Tm is proportional to the concentration or affinity of fragments in most cases, but it is not straightforward to correlate the shifts in Tms of compounds with their binding affinities. It is always a good strategy to confirm the identified hits through other biophysical methods (Cramer et al, 2017;Hassaan et al, 2020; Figure 2). It has been noted that other factors such as protein dynamics might influence Tm changes induced by ligand binding.…”