Torres-Mozas, A. | Search bioRxiv

bioRxiv2024

Synthesis and pharmacological characterization of UVI3502, a novel cannabinoid receptor 1 (CB1) antagonist/inverse agonist

The endocannabinoid (eCB) system regulates several brain functions and is implicated in neurological disorders. The pharmacological blockade of cannabinoid receptors has a therapeutic potential for various cognitive deficits, but also produces severe psychiatric side effects. Hence, new cannabinoid compounds that potentiate therapeutic effects, while minimizing toxicity, are required. In this study, we synthesized and characterized a novel antagonist/inverse agonist of CB1 receptors. UVI3502 showed affinity for two [3H]CP55,940 binding sites (IC50Hi 0.47 {+/-} 1.94 nM and IC50Lo 1470 {+/-} 1.80 nM). Subsequent binding assays performed in CB1 and CB2 overexpressing membranes determined that the low affinity binding site corresponded to CB1, but the high-affinity binding site of UVI3502 did not correspond to CB2 and the possibility of it corresponding to GPR55 was analyzed. The affinity of UVI3502 for CB1 receptors was further confirmed with neuroanatomical specificity by autoradiography in key brain areas, in which functional [35S]GTP{gamma}S assays demonstrated that UVI3502 behaved as an antagonist/inverse agonist of CB1 receptors, blocking the stimulation evoked by potent cannabinoid receptor agonist CP55,940 and decreasing basal [35S]GTP{gamma}S binding. The in silico characterization of the binding to CB1 receptor through molecular docking and molecular dynamics suggests that this activity is explained by the planar and rigid structure of UVI3502, which is optimal for interactions with the inactive state of the receptor. These results indicate that UVI3502 is a novel antagonist/inverse agonist of CB1 receptors, making it a compelling candidate for pharmacologically blocking cannabinoid receptors in the central nervous system. Significance StatementUVI3502 is a novel antagonist/inverse agonist of CB1 receptors, with almost no affinity for CB2 receptors and an additional high-affinity binding site for a third, cannabinoid-like receptor, potentially GPR55. In relevant brain areas for learning and memory processes with a high expression of CB1, UVI3502 blocks the stimulation evoked by the cannabinoid receptor agonist CP55,940, rendering it as an interesting compound for the pharmacological blockade of cannabinoid receptors in the central nervous system.

pharmacology and toxicology↗

bioRxiv2023

Thermodynamic Stabilization of Human Frataxin

Recombinant proteins and antibodies are routinely used as drugs to treat prevalent diseases such as diabetes or cancer, while enzyme replacement and gene therapies are the main therapeutic intervention lines in rare diseases. In protein-based therapeutics, optimized in vivo stability is key as intrinsic denaturation and intracellular proteostatic degradation will limit potency, particularly in treatments requiring a sustained action, while clearance mechanisms may limit the amount of circulating protein. In vivo stability is ultimately correlated with the intrinsic thermodynamic stability of the biomolecule, but this is difficult to optimize because it often goes at the expense of reducing protein activity. Here, we have used in silico engineering approaches to thermodynamically stabilize human frataxin, a small mitochondrial protein that acts as an allosteric activator for the biosynthesis of Fe-S clusters, whose genetically-driven impairment results in a rare disease known as Friedreich ataxia. Specifically, we developed an efficient thermostability engineering computational approach that combines information on amino acid conservation, the Rosetta energy function, and two recent artificial intelligence tools - AlphaFold and ProteinMPNN - to produce thermodynamically stabilized variants of human frataxin. Such protein variants rescued the large destabilization exerted by well-known pathological mutations, with an increase over 20 {degrees}C in the melting temperature and a thermodynamic stabilization of more than 3 kcal{middle dot}mol-1 at the physiological temperature. This stability surplus is translated into an enhanced resistance to proteolysis, while maintaining the protein fully functional. This case-study highlights the power of our combined computational approach to generate optimized variants, adequate for protein-based therapeutics.

biophysics↗