Biology subjects

Alfonso, P.

Publications and source records attributed to Alfonso, P..

6 recordsLinked to original sources

bioRxiv2026

Protein-ligand binding kinetics are primarily controlled by the protein, not the ligand.

Protein-ligand interactions underpin biological regulation and drug action, with both binding affinity and binding kinetics shaping functional outcomes. By analysing kinetic data for 4,311 protein-small-molecule pairs, we find that when association occurs below the diffusion-controlled limit, the rates of ligand association (kon) and dissociation (koff) are primarily determined by how the initial encounter complex reorganizes into the final bound state, and that this reorganization is governed chiefly by the intrinsic dynamic properties of the protein rather than by structural features of the ligand. Counterintuitively, therefore, koff exhibits minimal dependence on ligand structure, so that dissociation proceeds through protein-gated conformational transitions rather than through direct rupture of protein-ligand contacts. This mechanistic behaviour stands in marked contrast to that for protein-protein complexes, based on an analysis of 1,561 interactions. Together, these findings challenge prevailing assumptions regarding the molecular determinants of small-molecule binding kinetics, and have broad implications for rationally modulating protein-ligand interactions and drug-target residence times.

biochemistry↗

bioRxiv2026

Kinetic Fingerprints as Mechanistic and Clinical Roadmaps Across KIT Activation States

In cancer therapy, traditional approaches often overlook the dynamic nature of drug-target interactions. We introduce kinetic fingerprints as a mechanistically informative tool to guide kinase inhibitor design and predict clinical performance. Profiling 172 compounds across multiple KIT conformations, including the oncogenic D816V mutation, show that prolonged residence time determines therapeutic success, while mutations accelerating dissociation rates (koff) drive resistance, positioning koff as a robust predictor of clinical failure. Beyond efficacy and resistance, kinetic signatures map molecular behavior: fast-associating scaffolds engage readily populated KIT states, slow binders overcome conformational barriers like juxtamembrane repositioning, and extended residence times highlight ligands stabilizing regulatory elements (G-loop and regulatory spine). Kinetic profiling further unveils mechanisms invisible to conventional methods, such as drug-induced kinase degradation, and exposes selectivity dimensions beyond affinity: avapritinib exhibits durable KIT D816V engagement yet transient off-target binding. Our findings redefine the evaluation of KIT inhibitors, establishing a framework for rational, kinetics-guided drug discovery in KIT-driven cancers. Table of Contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=166 SRC="FIGDIR/small/726488v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@17f522borg.highwire.dtl.DTLVardef@1ebabc6org.highwire.dtl.DTLVardef@15a5e61org.highwire.dtl.DTLVardef@1ea5e0e_HPS_FORMAT_FIGEXP M_FIG Kinetic profiling of 172 compounds across KIT conformationsincluding D816Vreveals kinetic fingerprints that predict efficacy, selectivity, resistance, and inhibitors ability to stabilize key regulatory elements. Far from a secondary metric, binding kinetics provide mechanistic insights beyond affinity, offering a powerful framework for rational drug design in KIT-driven cancers. C_FIG

biochemistry↗

bioRxiv2025

Covalent targeting leads to the development of LIMK1 isoform-selective inhibitors

Selectivity for closely related isoforms of protein kinases is a major challenge in the design of drugs and chemical probes. Covalent targeting of unique cysteines is a potential strategy to achieve selectivity for highly conserved binding sites. Here, we used a pan-LIMK inhibitor to selectively probe LIMK1 over LIMK2 by targeting the LIMK1-specific cysteine C349 located in the glycine-rich loop region. Binding kinetics of both non-covalent and covalent LIMK inhibitors were investigated, and the fast on-rate and small size of type-I inhibitors were used in the design of a covalent LIMK1 inhibitor. The developed cell-active, isoform-selective LIMK1 inhibitor showed excellent proteome-wide selectivity in pull-down assays, enabling studies of LIMK1 isoform-selective functions in cellular model systems and providing a versatile chemical tool for studies of the LIMK signalling pathway.

biochemistry↗

bioRxiv2025

Repurposing of the RIPK1 selective benzooxazepin-4-one scaffold for the development of a type-III LIMK1/2 inhibitor

Benzoxazepinones have been extensively studied as exclusively selective RIP kinase 1 inhibitors. This scaffold binds as a type-III inhibitor targeting the C-out/DFG-out conformation. This inactive conformation results in a large expansion of the kinase back pocket, a conformation that has also been reported for LIM kinases. Scaffold hopping is common in the design of orthosteric kinase inhibitors, but has not been explored in the design of allosteric inhibitors, mainly due to the typically exclusive selectivity of type III inhibitors. Here, we hypothesized that the shared structural properties of LIMKs and RIPKs could lead to novel type III LIMK inhibitors using the benzoxazepinone scaffold. We report the discovery of a novel LIMK1/2 inhibitor that relies on this scaffold-based approach. The discovered compound 10 showed low nanomolar potency on LIMK1/2 and exceptional selectivity, as confirmed by a comprehensive selectivity panel with residual RIPK activity as the only off-target. The study provides one of the few examples for scaffold hopping for type-III inhibitors which are usually associated with exclusive target selectivity. Table of contents (TOC) graphics O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/636296v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@19bad48org.highwire.dtl.DTLVardef@18d8929org.highwire.dtl.DTLVardef@1280043org.highwire.dtl.DTLVardef@1296ddf_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

Unveiling the Hidden Viromes Across the Animal Tree of Life: Insights from a Taxonomic Classification Pipeline Applied to Invertebrates of 31 Metazoan Phyla

Invertebrates constitute the majority of animal species on Earth, including most disease-causing agents or vectors, with more diverse viromes when compared to vertebrates. Recent advancements in high-throughput sequencing have significantly expanded our understanding of invertebrate viruses, yet this knowledge remains biased toward a few well-studied animal lineages. In this study, we analyze invertebrate DNA and RNA viromes for 31 phyla using 417 publicly available RNA-Seq datasets from diverse environments in the marine-terrestrial and marine-freshwater gradients. This study aims to (i) estimate virome compositions at the family level for the first time across the Animal Tree of Life, including the first exploration of the virome in several phyla, (ii) quantify the diversity of invertebrate viromes and characterize the structure of invertebrate-virus interaction networks, and (iii) investigate host phylum and habitat influence on virome differences. Results showed that a set of few viral families of eukaryotes, comprising Retroviridae, Flaviviridae and several families of giant DNA viruses, were ubiquitous and highly abundant. Nevertheless, some differences emerged between phyla, revealing for instance a less diverse virome in Ctenophora compared to the other animal phyla. Compositional analysis of the viromes showed that the host phylum explained over five times more variance in composition than its habitat. Moreover, significant similarities were observed between the viromes of some phylogenetically related phyla, which could highlight the influence of co-evolution in shaping invertebrate viromes. ImportanceThis study significantly enhances our understanding of the global animal virome by characterizing the viromes of previously unexamined invertebrate lineages from a large number of animal phyla. It showcases the great diversity of viromes within each phylum and investigates the role of habitat shaping animal viral communities. Furthermore, our research identifies dominant virus families in invertebrates and distinguishes phyla with analogous viromes. This study sets the road towards a deeper understanding of the virome across the Animal Tree of Life.

ecology↗

bioRxiv2023

Evidence for gene transfer between mycoviruses and their host: Curvulaviridae as a case study

Gene transfer between distinct evolutionary lineages has been recognized as a frequent event occurring between viruses and their hosts. This phenomenon has been studied to some extent in animal and plant viruses, not so much in the case of mycoviruses, for which the evolutionary origins of their proteins remain poorly understood. In this study, we have tested the hypothesis of a mosaic origin for mycoviruses genomes, with the RNA-dependent RNA-polymerase (RdRp) being of viral origin and the coat protein (CP) resulting from one or more transfer events from the host genome. Firstly, phylogenetic trees were constructed for the RdRps and the CPs from a selection of viruses to address for possible incongruent evolutionary histories. Moreover, a PSI-BLAST search using the CP sequences from the different mycovirus groups retrieved hypothetical proteins (HP) with many orthologues in fungal genomes showing significant sequence homology with the CP from the members within the Curvulaviridae family. The structures of these HPs, predicted in silico using AlphaFold, tend to show high similarity with viral CPs suggesting the occurrence of gene transfer between viruses and fungi, although no clear function has been yet attributed to these genes in the host. Phylogenetic analyses suggest that this gene transfer could have occurred in multiple independent events. Additional selection analysis supports the notion that the most parsimonious explanation is the transfer of the HP from the host to an ancestral viral genome followed by fast evolution to accommodate the newly acquired protein to function as a CP.

microbiology↗