Biology subjects

Peccati, F.

Publications and source records attributed to Peccati, F..

4 recordsLinked to original sources

bioRxiv2024

Epistasis in the receptor binding domain of contemporary H3N2 viruses that reverted to bind sialylated diLacNAc repeats

Since the introduction of H3N2 influenza A viruses in the human population, these viruses have continuously evolved to escape human immunity, with mutations occurring in and around the receptor binding site. This process, called antigenic drift, recently resulted in viruses that recognize elongated glycans that are not abundantly displayed in the human respiratory tract. Such receptor specificities hampered our ability to pick and propagate vaccine strains. Using ELISA, glycan array, tissue staining, flow cytometry, and hemagglutinin assays, this study revealed that the most recent H3N2 viruses have expanded receptor specificity by regaining effective recognition to shorter glycans. In recent H3 strains, Y159 and T160 are responsible for restricted binding to elongated glycans; in contemporary strains, however, Y159N and T160I dominate with a consequent loss of strength in receptor binding. Yet, effective receptor interaction is rescued by a remote mutation in the 190-helix, Y195F. The results demonstrate epistasis of critical residues in three of the four structural elements composing the HA receptor-binding site (the 130-loop, 150-loop, and 190-helix), which synergistically contribute to shape receptor binding specificity. Interestingly, a positive correlation exists between binding to an asymmetrical N-glycan containing an 2,6 sialylated tri-LacNAc arm and binding to human and ferret respiratory tract tissues. Together, these results elucidate the epistatic nature of receptor binding specificity during influenza A virus H3N2 evolution.

microbiology↗

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

Benzylic Radical Stabilization Permits Ether Formation During Darobactin Biosynthesis

The Gram-negative selective antibiotic darobactin A has attracted interest owing to its intriguing fused bicyclic structure and unique mode of action. Biosynthetic studies have revealed that darobactin is a ribosomally synthesized and post-translationally modified peptide (RiPP). During maturation, the darobactin precursor peptide (DarA) is modified by a radical S-adenosyl methionine (rSAM)-dependent enzyme (DarE) to contain ether and C-C crosslinks. In this work, we describe the enzymatic tolerance of DarE using a panel of DarA variants, revealing that DarE can install the ether and C-C crosslinks independently and in different locations on DarA. These efforts produced 57 darobactin variants, 50 of which were enzymatically modified. Several new variants with fused bicyclic structures were characterized, including darobactin W3Y, which replaces tryptophan with tyrosine at the twice-modified central position, and darobactin K5F, which displays a fused diether ring pattern. Three additional darobactin variants contained fused diether macrocycles, leading us to investigate the origin of ether versus C-C crosslink formation. Computational analyses found that more stable and long-lived C{beta} radicals found on aromatic amino acids correlated with ether formation. Further, molecular docking and calculated transition state structures provide support for the different indole connectivity observed for ether (Trp-C7) and C-C (Trp-C6) crosslink formation. We also provide experimental evidence for a {beta}-oxotryptophan modification, a proposed intermediate during ether crosslink formation. Finally, mutational analysis of the DarA leader region and protein structural predictions identified which residues were dispensable for processing and others that govern substrate engagement by DarE. Our work informs on darobactin scaffold engineering and sheds additional light on the underlying principles of rSAM catalysis.

biochemistry↗

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↗