Enhanced identification of small molecules binding to hnRNPA1 via cryptic pockets mapping coupled with X-Ray fragment screening

TY - JOUR

T1 - Enhanced identification of small molecules binding to hnRNPA1 via cryptic pockets mapping coupled with X-Ray fragment screening

AU - Dunnett, Louise

AU - Das, Sayan

AU - Venditti, Vincenzo

AU - Prischi, Filippo

N1 - Copyright © 2025 The Authors. Published by Elsevier Inc. All rights reserved.

PY - 2025/4

Y1 - 2025/4

N2 - The human heterogeneous nuclear ribonucleoprotein (hnRNP) A1 is a prototypical RNA-binding protein essential for regulating a wide range of post-transcriptional events in cells. As a multifunctional protein with a key role in RNA metabolism, deregulation of its functions has been linked to neurodegenerative diseases, tumor aggressiveness, and chemoresistance, which has fuelled efforts to develop novel therapeutics that modulate its RNA-binding activities. Here, using a combination of molecular dynamics simulations and graph neural network pocket predictions, we showed that hnRNPA1 N-terminal RNA-binding domain (unwinding protein 1 [UP1]) contains several cryptic pockets capable of binding small molecules. To identify chemical entities for the development of potent drug candidates and experimentally validate identified druggable hotspots, we carried out a large fragment screening on UP1 protein crystals. Our screen identified 36 hits that extensively sample UP1 functional regions involved in RNA recognition and binding as well as map hotspots onto novel protein interaction surfaces. We observed a wide range of ligand-induced conformational variation by stabilization of dynamic protein regions. Our high-resolution structures, the first of an hnRNP in complex with a fragment or small molecule, provide rapid routes for the rational development of a range of different inhibitors and chemical tools for studying molecular mechanisms of hnRNPA1-mediated splicing regulation.

AB - The human heterogeneous nuclear ribonucleoprotein (hnRNP) A1 is a prototypical RNA-binding protein essential for regulating a wide range of post-transcriptional events in cells. As a multifunctional protein with a key role in RNA metabolism, deregulation of its functions has been linked to neurodegenerative diseases, tumor aggressiveness, and chemoresistance, which has fuelled efforts to develop novel therapeutics that modulate its RNA-binding activities. Here, using a combination of molecular dynamics simulations and graph neural network pocket predictions, we showed that hnRNPA1 N-terminal RNA-binding domain (unwinding protein 1 [UP1]) contains several cryptic pockets capable of binding small molecules. To identify chemical entities for the development of potent drug candidates and experimentally validate identified druggable hotspots, we carried out a large fragment screening on UP1 protein crystals. Our screen identified 36 hits that extensively sample UP1 functional regions involved in RNA recognition and binding as well as map hotspots onto novel protein interaction surfaces. We observed a wide range of ligand-induced conformational variation by stabilization of dynamic protein regions. Our high-resolution structures, the first of an hnRNP in complex with a fragment or small molecule, provide rapid routes for the rational development of a range of different inhibitors and chemical tools for studying molecular mechanisms of hnRNPA1-mediated splicing regulation.

UR - http://www.scopus.com/inward/record.url?scp=105000321129&partnerID=8YFLogxK

U2 - 10.1016/j.jbc.2025.108335

DO - 10.1016/j.jbc.2025.108335

M3 - Article

C2 - 39984046

SN - 0021-9258

VL - 301

SP - 108335

JO - The Journal of biological chemistry

JF - The Journal of biological chemistry

IS - 4

M1 - 108335

ER -