Pentameric ligand-gated ion channels (pLGICs) are ubiquitous neurotransmitter receptors in Bilateria, with a small number of known prokaryotic homologues. Here we describe a new inventory and phylogenetic analysis of pLGIC genes across all kingdoms of life. Our main finding is a set of pLGIC genes in unicellular eukaryotes, some of which are metazoan-like Cys-loop receptors, and others devoid of Cys-loop cysteines, like their prokaryotic relatives. A number of such “Cys-less” receptors also appears in invertebrate metazoans. Together, those findings draw a new distribution of pLGICs in eukaryotes. A broader distribution of prokaryotic channels also emerges, including a major new archaeal taxon, Thaumarchaeota. More generally, pLGICs now appear nearly ubiquitous in major taxonomic groups except multicellular plants and fungi. However, pLGICs are sparsely present in unicellular taxa, suggesting a high rate of gene loss and a non-essential character, contrasting with their essential role as synaptic receptors of the bilaterian nervous system. Multiple alignments of these highly divergent sequences reveal a small number of conserved residues clustered at the interface between the extracellular and transmembrane domains. Only the “Cys-loop” proline is absolutely conserved, suggesting the more fitting name “Pro loop” for that motif, and “Pro-loop receptors” for the superfamily. The infered molecular phylogeny shows a Cys-loop and a Cys-less clade in eukaryotes, both containing metazoans and unicellular members. This suggests new hypotheses on the evolutionary history of the superfamily, such as a possible origin of the Cys-loop cysteines in an ancient unicellular eukaryote. Deeper phylogenetic relationships remain uncertain, particularly around the split between bacteria, archaea, and eukaryotes.
Fragment-based lead discovery is becoming an increasingly popular strategy for drug discovery. Fragment screening identifies weakly binding compounds that require optimization to become high-affinity leads. As design of leads from fragments is challenging, reliable computational methods to guide optimization would be invaluable. We evaluated using molecular dynamics simulations and the free energy perturbation method (MD/FEP) in fragment optimization for the A2A adenosine receptor, a pharmaceutically relevant G protein-coupled receptor. Optimization of fragments exploring two binding site subpockets was probed by calculating relative binding affinities for 23 adenine derivatives, resulting in strong agreement with experimental data (R2 = 0.78). The predictive power of MD/FEP was significantly better than that of an empirical scoring function. We also demonstrated the potential of the MD/FEP to assess multiple binding modes and to tailor the thermodynamic profile of ligands during optimization. Finally, MD/FEP was applied prospectively to optimize three nonpurine fragments, and predictions for 12 compounds were evaluated experimentally. The direction of the change in binding affinity was correctly predicted in a majority of the cases, and agreement with experiment could be improved with rigorous parameter derivation. The results suggest that MD/FEP will become a powerful tool in structure-driven optimization of fragments to lead candidates.
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