Spring 2021 challenge: phase 2 contenstant

Discovery of Selective Binding Inhibitors Targeting cFRS for Malaria Resistance

Jeevan Patra, University of Petroleum and Energy Studies (Dr. Smriti Arora), Dehradun, India
We pre-dominantly focused on the mutations in cytosolic tRNA synthetase of Plasmodium vivax conferring drug resistance. We created the mutation in P. vivax cytosolic phenylalanine tRNA synthetase (cFRS) in silico (L544V) reported already to cause drug resistance. We superimposed the structures of mutated P. vivax (cFRS) and of homo sapiens (HsFRS) to avert cytotoxicity. This mutated PDB of P. vivax was superimposed onto HsFRS PDB to study novel scaffolds as the inhibitors. After initial virtual screening, a refinement was done for pose conformers which were focused mainly at the active sites (L-Phe, ATP and auxiliary), L544V, S545 (active binding for plasmodium inhibitory) and other active hydrophobic residues. Lead optimization and binding affinity studies were performed for the improvement in binding conformers. The best conformers were further scrutinized as per the Optibrium and ADMET profiling. These top drug-like molecules are being subjected to wet lab screening.

After 3 months, Jeevan has achieved the following goals:

  1. Malarial pathogen is becoming severely drug resistant and the disease is significant burden to many countries especially tropical countries like India. In this work, we have developed mutated model of cFRS plasmodium using PDB 7BY6 (residue L554V). The mutated model was confirmed for conformational flexibility (RMSF) and found to be stable without any restraints. The confirmed model was further superimposed on HsFRS (3L4G) for studying its cytotoxicity. From medicinal chemistry perspective, drug filters (MONA) were applied to co-crystal structure and it was deduced that a compound with lesser molecular obesity could occupy all three active sites with better efficacy, overcome resistance with little or no cytotoxicity. Based on similarities with template (FB9), a virtual library (infiniSee) was designed with about 5000 leads (In-stock/On-demand synthesis). The natural Trojan-Horse based inhibitors were also considered based recently reported perspective (Travin et al. 2021).
  2. The designed library was probed against the defined binding site of the superimposed proteins (mutated Pv cFRS and Hs FRS). Using the recently published data, the active site (S545) and surrounding residues were predetermined which are responsible for its putative activity. Few best leads had low binding affinity range but could not fit into binding pocket and had poor Optibrium properties as well. The top 50 leads were optimized using the empirical point of randomization and structural modifications (core hopping and fragment builder of SeeSAR) to curtail these pitfalls. From the set of newly optimized series, all 20 newly optimized molecules shown to be within the pico-molar to sub-picomolar range. These drug-like molecules overcame resistance, had optimized ADMET (ADMET 2.0), and better binding affinities. They were also found to show better ligand efficiency values and none of them had clash strains and tolerance effect. These molecules were further verified using MM-GBSA.
  3. The protocol for carrying out the functional studies, Plasmodium inhibitory activity, and cytotoxicity effect over Hs is well established (Sharma et al. 2021), which been outsourced. The top 20 molecules were retro-synthesised. Due to lack of availability of reagents, and building blocks few of the molecules were initiated for on-demand synthesis from the WuxiLabNetwork and few will be procured from Enamine Ltd. Based on the wet lab screening analysis, iterative medicinal chemistry optimization will be performed for the improvement. The SeeSAR module will support us for lead optimization in core hopping and R-group enumerations for binding affinity improvements. The optimized putative inhibitor with sub-nanomolar (EC50 <500nM) will be studied for the pharmacological properties using MMVsolapredictor for further translational research. All these research deliverables are anticipated to be completed soon.
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