Biology subjects

Manigrasso, J.

Publications and source records attributed to Manigrasso, J..

3 recordsLinked to original sources

bioRxiv2026

Conformational dynamics at the pre-miR-377 Dicer site governs selective small-molecule recognition

Small molecules that target precursor microRNAs (pre-miRNAs) offer a strategy to modulate microRNA biogenesis, but rational design is limited by the intrinsic conformational dynamics of RNA. Here, we combine site-specific nuclear magnetic resonance (NMR) spectroscopy with enhanced-sampling molecular dynamics (MD) simulations to define the conformational ensemble of pre-miR-377 and its recognition by the small-molecule binder C1. Our data reveals that the Dicer cleavage site contains a dynamic adenosine bulge in which U25 shuffles between A3 and A4 with a [~]9:1 population ratio, generating two interconverting RNA triple structures. NMR-informed MD and binding simulations show that C1 engages this dynamic region through a pathway-dependent mechanism that favors minor-groove entry, stabilizes intermediate, partially reorganized Dicer-site conformations, and redistributes the conformational ensemble away from processing-competent states. A conformationally locked mutant abolishes binding, indicating that recognition depends on bulge-enabled RNA dynamics rather than a single static structure. These results define the structural and dynamic basis of small-molecule recognition at the pre-miR-377 Dicer site and highlight the importance of RNA conformational ensembles in RNA-targeted drug discovery.

biophysics↗

bioRxiv2023

Targeting the conserved active site of splicing machines with specific and selective small molecule modulators

The self-splicing group II introns are bacterial and organellar ancestors of the nuclear spliceosome and retro-transposable elements of pharmacological and biotechnological importance. Integrating enzymatic, crystallographic, and simulation studies, we demonstrate how these introns recognize small molecules through their conserved active site. These RNA-binding small molecules selectively inhibit the two steps of splicing by adopting distinctive poses at different stages of catalysis, and by preventing crucial active site conformational changes that are essential for splicing progression. Our data exemplify the enormous power of RNA binders to mechanistically probe vital cellular pathways. Most importantly, by proving that the evolutionarily-conserved RNA core of splicing machines can recognize small molecules specifically, our work puts solid bases for the rational design of splicing modulators not only against bacterial and organellar introns, but also against the human spliceosome, which is a validated drug target for the treatment of congenital diseases and cancers.

biochemistry↗

bioRxiv2021

Structure-based Design of CDC42 Effector Interaction Inhibitors For the Treatment of Cancer

CDC42 family GTPases (RHOJ, RHOQ, CDC42) are upregulated but rarely mutated in cancer and control both the ability of tumor cells to invade surrounding tissues and the ability of endothelial cells to vascularize tumors. Here we use computer-aided drug design to discover a new chemical entity (ARN22089) that targets CDC42 GTPases and blocks CDC42 effector interactions without affecting the binding between closely related GTPases (RAC1, RAS, RAL) and their downstream effectors. Our lead compound has broad activity against a panel of cancer cell lines, inhibits S6 phosphorylation and MAPK activation, activates pro-inflammatory and apoptotic signaling, and blocks tumor growth and angiogenesis in three-dimensional vascularized microtumor models (VMT) in vitro. In addition, ARN22089 has a favorable pharmacokinetic profile and can inhibit the growth of BRAF mutant mouse melanomas and patient-derived xenografts in vivo. Taken together, this work identifies a promising new class of therapeutic agents that influence tumor growth by modulating CDC42 signaling in both the tumor cell and its microenvironment.

molecular biology↗