Molecular Insights into De Novo Small-Molecule Recognition by an Intron RNA Structure

Chemistry Medicinal Chemistry Biophysics Biochemistry Life Sciences
RNA targeting small-molecule recognition cryoEM splicing inhibitor RNA-ligand interactions

Academic Background

RNA, as a carrier of genetic information and functional molecule, has long been considered an “undruggable” target. In recent years, with advances in RNA structural biology, scientists have begun exploring the development of small-molecule drugs targeting RNA. However, this field faces three core challenges: (1) lack of systematic understanding of RNA-ligand recognition principles; (2) difficulties in high-resolution structural determination of large RNA-small molecule complexes; and (3) limited screening methods for functional RNA ligands.

This study focuses on group I introns, a unique RNA structure widely present in pathogenic fungi. By integrating high-throughput screening, medicinal chemistry, and cryo-EM technology, the researchers achieved de novo ligand design and high-resolution structural determination for a large catalytic RNA for the first time. This work provides an important molecular mechanism template for RNA-targeted drug development.

Source of the Paper

This research was led by Professor Anna Marie Pyle’s team at Yale University, with Tianshuo Liu, Ling Xu, and Kevin Chung as co-first authors, in collaboration with HHMI and New England Discovery Partners. The paper was published on May 8, 2025, in PNAS (vol. 122, no. 19), titled “Molecular insights into de novo small-molecule recognition by an intron RNA structure.”

Detailed Research Workflow

1. High-Throughput Screening Platform

Research Subject: Group IA1 intron (c.a.mtlsu) in the mitochondria of Candida albicans.

Innovative Methods: - Developed a molecular beacon fluorescence detection system: Real-time monitoring of splicing activity by specifically recognizing exon ligation sequences produced by self-splicing. - Screened the Enamine RNA-targeted compound library (~100,000 compounds).

Key Data: - Initial hit rate: 0.2% - Lead compound Z3686288076 showed an IC50 of 0.84 μM - Kinetic analysis confirmed it as a competitive inhibitor of guanosine monophosphate (GMP) (Ki = 0.67 μM)

2. Structure-Activity Relationship (SAR) Study

Medicinal Chemistry Optimization: - Systematic modification of the lead compound’s three-ring structure (rings A/B/C) - Evaluated activity using radioactive splicing assays

Key Findings: - Stereochemistry and cyclic structure of ring B were non-essential (compounds 9-10) - Terminal primary amine was a critical pharmacophore (compounds 7/8/14 lost activity) - N1 atom in ring A was indispensable (compound 16 showed 200-fold reduced activity) - 2-Aminopyrimidine served as the core recognition scaffold (compound 23 was inactive)

Optimization Results: - Simplified compound 11 (ethylene diamine replacing ring B) improved activity to 0.21 μM - Best-performing compound 17 achieved an IC50 of 86 nM

3. Cryo-EM Structure Determination

Sample Preparation: - Constructed A9U mutant to stabilize the P1 helix - Prepared RNA-ligand complexes under Mg²⁺ and Ca²⁺ conditions

Data Collection: - Titan Krios 300kV microscope - Falcon 4i direct electron detector - Total datasets: 16,185 (Ca²⁺) and 8,983 (Mg²⁺) micrographs

Structure Determination: - cryoSPARC 4.4.1 processing pipeline - Local resolution reached 2.25 Å (ligand-binding region) - Ligand Q-score: 0.79 (expected 0.65 @ 2.5 Å)

Major Findings

1. Molecular Mechanism of Ligand Recognition

Base Mimicry: - 2-Aminopyrimidine mimics guanine, forming a base triple with G154:C258 - Terminal amine forms an electrostatic network with A152/A253 phosphates (distance Å)

Metal Ion Plasticity: - Catalytic metal Mc was absent due to ligand geometry changes - Structural metal Me coordinated a water molecule to form hydrogen bonds with the ligand’s bridging nitrogen - Explained the loss of activity in compound 13 (S replacing N)

2. RNA Conformational Dynamics

ωG Gating Mechanism: - The terminal ωG nucleotide (G316) formed a non-canonical base triple - Steric effects stabilized ligand binding

P1 Helix Displacement: - Scissile phosphate moved by 4.6 Å - 5’ exon folded into a triple-helix structure - Revealed the structural basis for splicing inhibition

3. Unique Structural Modules

P7ext Scaffolding Function: - P11 pseudoknot stabilized the P7ext-P6 junction - A-minor motifs mediated long-range P7ext-P9.1 interactions - Four-strand non-canonical base-stacking platform (P3/P7/P7ext/J8/7)

Conclusions and Significance

Scientific Impact

  1. RNA-Targeting Design Principles: Established aminopyrimidine-ethylenediamine as a universal pharmacophore for RNA targeting.
  2. Dynamic Recognition Mechanism: Revealed RNA specificity through metal ion coordination and conformational plasticity.
  3. Structural Biology Breakthrough: Achieved the first 2.4 Å resolution structure of a large RNA-small molecule complex.

Practical Value

  1. Antifungal Drug Development: Designed specific inhibitors targeting conserved group I introns in pathogenic fungi.
  2. RNA-Targeting Platform: Established a complete workflow from screening to structural determination.
  3. Computational Biology Support: Provided high-quality template data for RNA structure prediction and virtual screening.

Research Highlights

  1. Methodological Innovation: First application of cryo-EM for high-resolution RNA-small molecule complex determination.
  2. Dynamic Perspective Breakthrough: Captured ligand-induced changes in metal ion coordination and RNA conformation.
  3. Translational Medical Value: Demonstrated the feasibility of rational design for functional RNA-targeted drugs.