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

Abstract

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.

OverexpressedMalat1Drives Metastasis through Inflammatory Reprogramming of Lung Adenocarcinoma Microenvironment

“…Given the recurrent overexpression of Malat1 across many cancer types, it is tempting to speculate that Malat1 overexpression in pre-metastatic disease may serve as a valuable prognostic marker for Ccl2 therapy. Conversely, given the risks of Ccl2 therapy (Bonapace et al, 2014), targeting Malat1 via antisense oligonucleotides (ASOs) (Arun et al ., 2016; Gutschner et al ., 2013) or ENE-targeting compounds (Abulwerdi et al, 2019; Zafferani et al, 2022) may be an attractive alternative due lack of anticipated side-effects. Finally, it will also be important to determine whether Malat1 overexpression is a marker of immunosuppression and whether Malat1 inhibition may improve the performance of immune checkpoint inhibitors.…”

Locked Nucleic Acid Oligonucleotides Facilitate RNA•LNA-RNA Triple-Helix Formation and Reduce MALAT1 Levels

“…The small molecules niclosamide and tyrphostin 9 reduce MALAT1 levels via glycogen synthase kinase-3 beta (GSK3B) and heterogeneous nuclear ribonucleoproteins (hnRNPs) K and C [21]. However, most small molecules have targeted the MALAT1 triple helix [22][23][24][25][26][27][28][29]. These small molecules belong to the classes of diphenylfuran derivatives [22,24], imidazole derivatives [23], diminazene derivatives [26], flavonoids [27] and aromatic heterocyclic compounds [25,29].…”

“…However, most small molecules have targeted the MALAT1 triple helix [22][23][24][25][26][27][28][29]. These small molecules belong to the classes of diphenylfuran derivatives [22,24], imidazole derivatives [23], diminazene derivatives [26], flavonoids [27] and aromatic heterocyclic compounds [25,29]. Although most small molecules have been shown to modulate the stability of the MALAT1 triple helix in a test tube, the aromatic imidazole-derived compound 5 decreases endogenous MALAT1 by 54% in MMTV-PyMT tumors and 38% in mammary tumor organoid branching, while MENβ levels do not change significantly [23].…”

Recognizing the power of machine learning and other computational methods to accelerate progress in small molecule targeting of RNA

“…One strategy to continue the exploration, validation, and expansion of RNA-privileged chemical space is to generate synthetic SM libraries, and we chose to use scaffolds with a known propensity to bind nucleic acids and demonstrated clinical utility. [35][36][37] Compared to current commercially available libraries, which generally occupy a similar subsection of R-BIND chemical space, we can generate and analyze synthetic libraries that complete coverage and push the boundaries of R-BIND space. Continuous synthetic tuning of these scaffolds has led to the discovery of lead molecules for chemical probe development of medicinally relevant RNA targets, including viral and long noncoding RNA structures.…”