Inhibitor Development for Bacterial N5-CAIR Mutase and MERS Coronavirus Papain-like Protease

The de novo purine biosynthesis pathway is an attractive target for antibacterial drug design, and PurE from this pathway has been identified to be crucial for Bacillus anthracis survival in serum. In this study, we adopted a fragment-based hit discovery approach, using three screening methods − saturation transfer difference nucleus magnetic resonance (STD-NMR), water-ligand observed via gradient spectroscopy (WaterLOGSY) NMR, and surface plasmon resonance (SPR), against B. anthracis PurE (BaPurE) to identify active site binding fragments by initially testing 352 compounds in a Zenobia fragment library. Competition STD NMR with the BaPurE product effectively eliminated non-active site binding hits from the primary hits, selecting active site binders only. Binding affinities (dissociation constant, KD) of these compounds varied between 234 and 301 μM. Based on test results from the Zenobia compounds, we subsequently developed and applied a streamlined fragment screening strategy to screen a much larger library consisting of 3,000 computationally pre-selected fragments. Fifteen final fragment hits were confirmed to exhibit binding affinities varying from 14 μM to 900 μM, which were categorized into five different basic scaffolds. All fifteen fragment hits have ligand efficiencies higher than 0.30. We demonstrated that at least two fragments from two different scaffolds exhibit inhibitory activity against the BaPurE enzyme. Middle East Respiratory Syndrome coronavirus (MERS-CoV) papain-like protease (PLpro) is known to be essential for viral replication, making it an attractive target in antiviral drug discovery. In our study, we explored and optimized the MERS-PLpro crystallization condition. Two structures were solved with resolutions at 2.59 Å and 1.79 Å, respectively. The overall structure of MERS-PLpro resembles that of SARS-PLpro. The MERS-PLpro blocking loop 2 (BL2) structure differs significantly from that of SARS-PLpro, where it plays a crucial role in inhibitor binding. Four SARS-PLpro lead inhibitors with IC50 values ranging from 0.2 to 2.0 µM were tested against MERS-PLpro, none of which were effective against MERS-PLpro. Structure and sequence alignments revealed that two residues, Y269 and Q270, responsible for inhibitor binding to SARS-PLpro were replaced by T274 and A275 in MERS-PLpro, eliminating critical binding interactions for similar types of inhibitors. High-throughput screening (HTS) of 25,000 compounds against both PLpro enzymes identified a small fragment-like noncovalent dual inhibitor. This newly identified compound acts as a competitive inhibitor with an IC50 of 6 µM against MERS-PLpro, indicating that it binds to the active site, whereas it acts as an allosteric inhibitor against SARS-PLpro with an IC50 of 11 µM. Docking studies were performed to predict the possible interactions between inhibitor and both SARS and MERS-PLpro. These results demonstrate that inhibitor recognition specificity of MERS-PLpro differs from that of SARS-PLpro. In addition, mutagenesis studies demonstrated that MERS-PLpro N109 is critical in maintaining the protease activity. N109 was suggested to stabilize the substrate intermediate by forming a hydrogen bond with it. MERS-PLpro N109D mutant exhibits minor enzyme activity. This result suggests that N109 is a critical residue for intermediate stabilization, probably through an H-bond formation with the side chain amine group of N109.