RNA-binding proteins (RBPs) are paramount effectors of gene expression, and their malfunction underlies the origin of many diseases. However, therapeutically targeting RBPs with small molecules has proven challenging due to highly polar orthosteric ribonucleic interactions and a lack of lipophilic cavities indicative of druggability. We present an integrated screening campaign integrating fragment-based differentiable design with molecular dynamics to discover a cryptic site enabling the discovery of a novel non-functional RBP binder. We demonstrate the physics-driven optimization of this hit molecule to induce the proximity of an E3 ligase, facilitating the proteosome-specific degradation of an RBP and restoring a tumor-suppressive miRNA.

Development and validation of high-specificity intracellular targeting reagents remain a bottleneck. Here we describe a yeast surface display-based nanobody screening approach that led to high-specificity nanobodies against RNA-binding proteins (RBPs)hnRNPA2B1 and TIA1. We demonstrate that nanobodies enable fluorescence imaging of RBPs in live cells. We also show that nanobody-E3 ligase adapter domain fusions can target RBPs for proteasomal degradation. We anticipate these reagents will greatly aid the study of endogenous RBP dynamics and provide a novel means of interrogating RBP function.

I will speak about our efforts in clarifying and identifying RNA-binding proteins as targets in oncology and neurodegeneration. My lab uses focused pooled CRISPR inhibition and knock-out approaches to identify RBPs as vulnerabilities in different diseases, initially in oncology. We then use cutting-edge genomic methods to study the molecular pathways by which RBPs affect gene expression. Small molecule discovery approaches are then applied to modulate these in cancer systems, like avatars.

Anima’s mRNA Lightning platform has generated many novel chemical entities that modulate mRNA translation, further contributing to the expanse of the RNA-targeting small molecule field. Anima's phenotypic screening approach systematically evaluates the impact of small molecules on mRNA translation into proteins. Combined with AI-driven MOA elucidation, Anima has identified and validated compounds that are binding to proteins which regulate mRNA translation, offering an opportunity for both tissue-selective and target-specific modulation. Anima’s lead programs in fibrosis and oncology have demonstrated efficacy in animal and patient-derived models, and are advancing towards preclinical development.

Using targeted lipid nanoparticles (tLNP), we were able to transiently reprogram T cells in vivo by delivering modified mRNA encoding a CAR against fibroblast activation protein (FAP). In a mouse model of heart disease, injection of these mRNA FAPCAR tLNPs effectively produced transient FAP CAR T cells, leading to the ablation of FAP+ pathogenic fibroblasts. This treatment resulted in the reduction of cardiac fibrosis and the restoration of cardiac function. The ability to produce transient, functional CAR T cells in vivo with mRNA addresses some of the biggest hurdles in cell therapy including manufacturing, scalability, and safety concerns.

RNA offers a broad array of folded, three-dimensional structures that mediate their functional roles. Our drug discovery platform at Arrakis Therapeutics is directed at the intervention of those functions to therapeutic benefit using drug-like small molecules that bind folded RNA structures. This presentation will touch on some of the unique challenges in building a broad and robust RNA-targeted small molecule platform and provide early data on specific RNA targets.

An increasing number of diseases are now being attributed to non-coding RNA and the ability to target them would vastly expand the chemical space for drug development. Here we devise a screening strategy based upon affinity-selection mass spectrometry and used it to identify and characterize small molecules that bind to a variety of non-coding RNAs. We found compounds acting upon the non-coding RNA prototype Xist that change RNA conformation and biological activities. Thus, RNA can be systematically targeted by drug-like compounds that disrupt RNA structure and epigenetic function.

We have analyzed patterns in both RNA-biased small molecule chemical space and RNA topological space privileged for differentiation. In a recent example, quantitative structure-activity relationships (QSAR) models have allowed prediction of small molecule binding and/or modulation of specific RNA structures. We have applied these and other principles to functionally modulate conformations of the 3’-triple helix of the long noncoding RNA MALAT1, as well as several viral RNA structures.

Utilizing small molecules to modulate splicing has emerged as a successful therapeutic approach to regulating protein expression. Here, three diseases where small molecule splicing modulators can be utilized are described: spinal muscular atrophy, familial dysautonomia, and Huntington’s disease. For each of these indications, I will discuss the correlation between pharmaceutical properties and pharmacokinetics, pharmacokinetics and pharmacodynamics, and the correlation between pharmacodynamics and efficacy.

Small RNAs include tRNA, snRNA, micro-RNA, tRNA fragments and others that constitute >90% of RNA copy numbers in a human cell and perform many essential functions. We developed a multiplex small RNA-seq library preparation method (MSR-seq) to investigate laboratory, clinical, and environmental samples. We applied MSR-seq to study stress response in human cells, infections in nasal cavity, and microbiomes that reveal the importance of simultaneous investigation of small RNAs and their modifications in response to varying biological conditions.

The past twenty years have seen an explosion of interest in the structure and function of RNA and DNA. While some 80% of the human genome is transcribed into RNA, just ~3% of those transcripts code for protein sequences. Here, we discuss our group’s efforts to target RNA and DNA with drug-like small molecules using a small molecule microarray (SMM) screening platform and the molecular basis for these interactions.