Multiassay Profiling of a Focused Small Molecule Library Reveals Predictive Bidirectional Modulation of the lncRNA MALAT1 Triplex Stability In Vitro.

The rapidly accelerating characterization of RNA tertiary structures has revealed their pervasiveness and active roles in human diseases. Small molecule-mediated modulation of RNA tertiary structures constitutes an attractive avenue for the development of tools for therapeutically targeting and/or uncovering the pathways associated with these RNA motifs. This potential has been highlighted by targeting of the triple helix present at the 3'-end of the noncoding RNA MALAT1, a transcript implicated in several human diseases. This triplex has been reported to decrease the susceptibility of the transcript to degradation and promote its cellular accumulation. While small molecules have been shown to bind to and impact the stability of the MALAT1 triple helix, the small molecule properties that lead to these structural modulations are not well understood. We designed a library utilizing the diminazene scaffold, which is underexplored but precedented for nucleic acid binding, to target the MALAT1 triple helix. We employed multiple assays to holistically assess what parameters, if any, could predict the small molecule affinity and effect on triplex stability. We designed and/or optimized competition, calorimetry, and thermal shift assays as well as an enzymatic degradation assay, the latter of which led to the discovery of bidirectional modulators of triple helix stability within the scaffold-centric library. Determination of quantitative structure-activity relationships afforded predictive models for both affinity- and stability-based assays. This work establishes a suite of powerful orthogonal biophysical tools for the evaluation of small molecule:RNA triplex interactions that generate predictive models and will allow small molecule interrogation of the growing body of disease-associated RNA triple helices.

RT-qPCR as a screening platform for mutational and small molecule impacts on structural stability of RNA tertiary structures.

The exponential increase in the discovery and characterization of RNA tertiary structures has highlighted their active role in a variety of human diseases, yet often their interactome and specific function remain unknown. Small molecules offer opportunities to both decode these cellular roles and develop therapeutics, however there are few examples of small molecules that target biologically relevant RNA tertiary structures. While RNA triple helices are a particularly attractive target, discovery of triple helix modulators has been hindered by the lack of correlation between small molecule affinity and effect on structural modulation, thereby limiting the utility of affinity-based screening as a primary filtering method. To address this challenge, we developed a high-throughput RT-qPCR screening platform that reports on the effect of mutations and additives, such as small molecules, on the stability of triple helices. Using the 3'-end of the oncogenic long non-coding RNA MALAT1 as a proof-of-concept, we demonstrated the applicability of both a two-step and a one-pot method to assess the impact of mutations and small molecules on the stability of the triple helix. We demonstrated the adaptability of the assay to diverse RNA tertiary structures by applying it to the SARS-CoV-2 pseudoknot, a key viral RNA structure recently identified as an attractive therapeutic target for the development of antivirals. Employment of a functional high-throughput assay as a primary screen will significantly expedite the discovery of probes that modulate the structural landscape of RNA structures and, consequently, help gain insight into the roles of these pervasive structures.

Quantitative Structure-Activity Relationship (QSAR) Study Predicts Small-Molecule Binding to RNA Structure.

The diversity of RNA structural elements and their documented role in human diseases make RNA an attractive therapeutic target. However, progress in drug discovery and development has been hindered by challenges in the determination of high-resolution RNA structures and a limited understanding of the parameters that drive RNA recognition by small molecules, including a lack of validated quantitative structure-activity relationships (QSARs). Herein, we develop QSAR models that quantitatively predict both thermodynamic- and kinetic-based binding parameters of small molecules and the HIV-1 transactivation response (TAR) RNA model system. Small molecules bearing diverse scaffolds were screened against TAR using surface plasmon resonance. Multiple linear regression (MLR) combined with feature selection afforded robust models that allowed direct interpretation of the properties critical for both binding strength and kinetic rate constants. These models were validated with new molecules, and their accurate performance was confirmed via comparison to ensemble tree methods, supporting the general applicability of this platform.

Amilorides inhibit SARS-CoV-2 replication in vitro by targeting RNA structures.

The SARS-CoV-2 pandemic, and the likelihood of future coronavirus pandemics, emphasized the urgent need for development of novel antivirals. Small-molecule chemical probes offer both to reveal aspects of virus replication and to serve as leads for antiviral therapeutic development. Here, we report on the identification of amiloride-based small molecules that potently inhibit OC43 and SARS-CoV-2 replication through targeting of conserved structured elements within the viral 5'-end. Nuclear magnetic resonance­based structural studies revealed specific amiloride interactions with stem loops containing bulge like structures and were predicted to be strongly bound by the lead amilorides in retrospective docking studies. Amilorides represent the first antiviral small molecules that target RNA structures within the 5' untranslated regions and proximal region of the CoV genomes. These molecules will serve as chemical probes to further understand CoV RNA biology and can pave the way for the development of specific CoV RNA­targeted antivirals.

Small molecule targeting of biologically relevant RNA tertiary and quaternary structures.

Initial successes in developing small molecule ligands for non-coding RNAs have underscored their potential as therapeutic targets. More recently, these successes have been aided by advances in biophysical and structural techniques for identification and characterization of more complex RNA structures; these higher-level folds present protein-like binding pockets that offer opportunities to design small molecules that could achieve a degree of selectivity often hard to obtain at the primary and secondary structure level. More specifically, identification and small molecule targeting of RNA tertiary and quaternary structures have allowed researchers to probe several human diseases and have resulted in promising clinical candidates. In this review we highlight a selection of diverse and exciting successes and the experimental approaches that led to their discovery. These studies include examples of recent developments in RNA-centric assays and ligands that provide insight into the features responsible for the affinity and biological outcome of RNA-targeted chemical probes. This report highlights the potential and emerging opportunities to selectively target RNA tertiary and quaternary structures as a route to better understand and, ultimately, treat many diseases.

Regulation of MALAT1 triple helix stability and in vitro degradation by diphenylfurans.

Small molecule-based modulation of a triple helix in the long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) has been proposed as an attractive avenue for cancer treatment and a model system for understanding small molecule:RNA recognition. To elucidate fundamental recognition principles and structure-function relationships, we designed and synthesized nine novel analogs of a diphenylfuran-based small molecule DPFp8, a previously identified lead binder of MALAT1. We investigated the role of recognition modalities in binding and in silico studies along with the relationship between affinity, stability and in vitro enzymatic degradation of the triple helix. Specifically, molecular docking studies identified patterns driving affinity and selectivity, including limited ligand flexibility, as observed by ligand preorganization and 3D shape complementarity for the binding pocket. The use of differential scanning fluorimetry allowed rapid evaluation of ligand-induced thermal stabilization of the triple helix, which correlated with decreased in vitro degradation of this structure by the RNase R exonuclease. The magnitude of stabilization was related to binding mode and selectivity between the triple helix and its precursor stem loop structure. Together, this work demonstrates the value of scaffold-based libraries in revealing recognition principles and of raising broadly applicable strategies, including functional assays, for small molecule-RNA targeting.

Photoresponsive azo-combretastatin A-4 analogues.

Colchicine analogues in which an azo group is incorporated into a molecule containing the key pharmacophore of colchicine, have found particular utility as switchable tubulin binding chemotherapeutics. Combretastatin is a related compound containing a stilbene fragment that shows different bioactivity for the cis and trans isomers. We have performed cell assays on 17 new compounds structurally related to a previously reported azo-analogue of combretastatin. One of these compounds showed enhanced potency against HeLa (IC50 = 0.11 µM) and H157 cells (IC50 = 0.20 µM) for cell studies under 400 nm irradiation and the highest photoactivity (IC50 with irradiation/IC50 in dark = 550). We have performed docking and physicochemical studies of this new compound (7). Kinetic studies in water reveal a longer half-life for the cis isomer of 7 which may be one factor responsible for the better IC50 values in cell assays and the improved photoresponsive behavior.