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. 2025 Sep 15;147(38):34271–34282. doi: 10.1021/jacs.5c06802

Discovery of RNA-Reactive Small Molecules Guides the Design of Electrophilic Modules for RNA-Specific Covalent Binders

Noah A Springer †,‡, Patrick R A Zanon †, Amirhossein Taghavi †, Kisu Sung †, Matthew D Disney †,‡,*
PMCID: PMC12464989  PMID: 40951988

Abstract

RNA is a key drug target that can be modulated by small molecules; however, covalent binders of RNA remain largely unexplored. Using a high-throughput mass spectrometry screen of 2,000 electrophilic compounds, we identified ligands that react with RNA in a binding-dependent manner. RNA reactivity was influenced by both the reactive group and the noncovalent RNA-binding scaffold. In addition to known RNA-reactive electrophiles such as N-acylimidazoles and bis­(2-chloroethyl)­amines, covalent screening enabled the surprising discovery that common thiol-reactive electrophiles (chloroacetamide) and rarely characterized electrophiles (3-chloropivalamide) cross-linked to RNA. These results suggest that electrophiles commonly used for protein targeting can also covalently modify RNA, potentially contributing to both on- and off-target effects. This insight enabled the design of an RNA-specific covalent compound by modifying a bis-benzimidazole scaffold, originally identified to bind DNA, to react selectively with the expanded triplet repeat RNA, r­(CUG)exp, that causes myotonic dystrophy type 1 (DM1). Selectivity appears to arise from differences in the RNA and DNA binding modes, revealing that proper positioning of the electrophile toward the nucleophilic guanine residue is important for efficient covalent bond formation. Overall, this study highlights the potential of rationally designing covalent RNA-targeting small molecules.

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Introduction

RNA structure, both in noncoding (nc) and coding transcripts, plays a crucial role in its function and dysfunction. , Thus, one way to modulate RNA function is by targeting its structure with small molecules. Various strategies, such as sequence-based design, , structure-based design, − and high-throughput screening (HTS), − have been employed to identify and optimize small molecules for RNA targeting. While most bioactive compounds act as simple RNA binders, heterobifunctional compounds such as degraders (direct − or enzyme-mediated , ) and covalent ligands , have also emerged as RNA-targeted modalities. Previous efforts to develop covalent RNA-targeting compounds have primarily focused on well-known nucleic acid-reactive electrophiles, such as nitrogen mustards , or N-acylimidazoles, , attached to noncovalent binding elements that confer affinity for specific RNAs. This study develops a methodology to rapidly identify small molecules that form stable, covalent adducts with RNA structures (Figure A).

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Identification and application of compounds covalently targeting RNA. (A) This work seeks to develop a screening methodology to identify both RNA-reactive electrophiles and specific RNA-binding moieties. (B) Irreversible covalent bond formation of small molecules targeting RNA confers improved potency and selectivity.

A matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF)-based mass spectrometry (MS) screening approach was established, enabling the screening of a covalent compound library. This library included diverse electrophiles (e.g., α,β-unsaturated amides, chloroacetamides, imines) and potential RNA-binding elements. For this proof-of-concept study, the structure of a trinucleotide repeat expansion, r­(CUG)exp (where “exp” denotes an expanded repeat), associated with myotonic dystrophy type 1 (DM1), − was chosen as a test case. In DM1 patients, r­(CUG)exp is found in the 3′ untranslated region (UTR) of the dystrophia myotonica protein kinase (DMPK) mRNA. , This repeat expansion forms an array of 1 × 1 nucleotide UU internal loops (5’CUG/3’GUC; where the loop nucleotides are underlined) that bind with high affinity to RNA-binding proteins (RBPs), such as muscleblind-like protein 1 (MBNL1). − Sequestration of MBNL1 in toxic nuclear foci prevents it from performing its canonical function in alternative pre-mRNA splicing, leading to widespread dysregulation of alternative splicing in genes implicated in DM1 pathology, including the muscle-specific chloride ion channel 1 (CLCN1) and the insulin receptor (IR). −

Herein, the reactivity of 2,000 small molecules was comprehensively evaluated, identifying several compounds that bind to and react with validated structural models of r­(CUG)exp. − A mildly reactive electrophile capable of alkylating guanosine was discovered, and its nucleic acid reactivity as well as its reactivity with thiols were profiled, which demonstrated specific reaction with the RNA repeat. Attaching this covalent module to a r­(CUG)exp-binding small molecule imparted a significant improvement in potency and selectivity.

Results

Development of Methods to Identify Small Molecule–RNA Adducts

Two methods amenable for high-throughput screening were developed to detect adducts between RNA and small molecules: a gel-shift assay, where covalent modification of the RNA affects the electrophoretic mobility of the RNA, and a MALDI-TOF mass spectrometry assay. To benchmark these approaches, a series of commercially available compounds (1–9) was selected based on the known reactivity of nitrogen mustards toward nucleobases (Figure A). − These compounds contain a (2-chloroethyl)­amine moiety that generates a reactive aziridinium electrophile in situ that reacts with biological nucleophiles. − The small molecule panel includes the cancer chemotherapeutic chlorambucil (1), which has previously been shown to react with RNA in vitro and in cells when attached to a noncovalent RNA-binding compound, uramustine (2), bendamustine (3), melphalan (4), and estramustine (5). Additionally, phenoxybenzamine (6), Astrazon Red 6B (7), Astrazon Pink FG (8), and a benzofuran mustard derivative (9), which have no known nucleic acid-alkylating ability, were also studied. The different aromatic or heterocyclic substructures found in this panel may bind to RNA and facilitate a proximity-induced reaction, particularly the nitrogen-containing heteroaromatic moieties found in 2, 3, 7, and 8, which are similar to known RNA binders. Each compound was tested for binding to and reacting with a r­(CUG)exp mimic, in which two 1 × 1 nucleotide UU internal loops were placed into a stem loop structure (RNA1, Figure B). This minimized design enables its use in a variety of assays.

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Development of methodologies to identify small molecules that react with RNA. (A) Structures of the nine nitrogen mustard derivatives tested in both the electrophoretic mobility shift and MALDI-TOF mass spectrometry assays. Circles next to the compounds are sized according to their activity in each assay, a proxy for reactivity, where larger circles represent higher activity. “No Rxn” is used to denote compounds that showed no adduct formation in the corresponding assay. (B) Left: Secondary structure of RNA1 used for screening. The RNA contains two 1 × 1 nucleotide UU internal loops found in the trinucleotide repeat expansion r­(CUG)exp, as well as base pairs at the 5′ and 3′ ends and a GNRA tetraloop for stable folding. Right: Results from screening using the gel shift assay by capillary electrophoresis for one of the hit compounds (6). Hits were defined as compounds that reduced the electrophoretic mobility of RNA1 with signal intensities >4-fold higher than a DMSO (vehicle) control. (C) MALDI-TOF mass spectrum for the reaction of 6 with RNA1. Each peak is labeled with its corresponding mass (m/z) and the stoichiometry of adduct formation with 6.

The first method explored to assess covalent adduct formation with the r­(CUG) repeat RNA was an electrophoretic mobility shift assay, where covalent modification could change the electrophoretic mobility of the RNA target, akin to a previously reported method used to identify and study reactive compounds targeting a riboswitch. After incubating RNA1 (20 μM) with each nitrogen mustard (2 mM; 4 h at 37 °C), the change in electrophoretic mobility was assessed by capillary electrophoresis (CE) on a fragment analyzer. Of the nine compounds tested, five (1–4, 6) significantly changed the electrophoretic mobility of the RNA by more than 4-fold above background signal from a DMSO control, with 2 and 4 showing the greatest extent of modification (Figures B and ). The same five compounds were validated as RNA-reactive via a secondary assessment of reaction by denaturing polyacrylamide gel electrophoresis (dPAGE) and subsequent SYBR staining (). Interestingly, phenoxybenzamine (6), an FDA-approved α-adrenergic receptor inhibitor, induced shifts in the mobility of RNA1 (Figure B), despite no prior direct evidence of nucleic acid alkylation for this drug. Neither the dPAGE nor CE methods detected adduct formation for 5 and 7–9, despite the high concentration of the small molecules (2 mM) present in the assays; thus they were scored as unreactive with RNA1.

As an orthogonal method to assess covalent RNA adducts, the same reaction mixtures were subjected to MALDI-TOF mass spectrometry (Figure C). By directly observing the mass to charge ratio of the RNA species, any covalent modification will result in a new peak, reducing the potential for false negatives. Additionally, by observing the mass of the covalent adduct on the RNA, potential mechanisms of covalent adduct formation can be determined (such as the mass of the expected leaving group for substitution reactions), reducing the potential for false positives. All compounds that induced gel shifts also formed detectable adducts by MALDI-TOF. The most potent alkylators, 2 and 4, formed between one to seven adducts per RNA molecule, with no observable unmodified RNA remaining (). The other three hits from the CE and dPAGE assays – 6, 3, and 1 – resulted in stoichiometries of adduct formation of up to five, four, and three adducts per RNA, respectively (). Interestingly, three additional nitrogen mustards (7–9) that were inactive in the dPAGE and CE assays alkylated RNA1 as determined by MALDI-TOF MS (Figures A and ). Indeed, eight of the nine nitrogen mustards alkylated RNA1, with 5 being the only exception (Figure A). The electron-withdrawing carbamate group of 5 likely prevents the formation of the reactive aziridinium intermediate and therefore diminishes the likelihood it will react with RNA. Previous studies suggest that the mechanism of action of estramustine (5), unlike other anticancer nitrogen mustards, is based on antitubulin activity rather than DNA interstrand cross-linking. − Collectively, these data demonstrate that MALDI-TOF analysis has several advantages over electrophoretic mobility shift assays for discovering small molecules that react with RNA, particularly in its ability to assess the stoichiometry of modification and detect low-abundance modifications that do not result in an apparent gel-shift.

Development and Implementation of a High-Throughput Screen of Small Molecules That React with RNA Using MALDI-TOF

For proteins, specific noncovalent interactions between a covalent ligand and its binding pocket enable adequate positioning of the electrophile to covalently engage the protein nucleophile. − Further, covalency is commonly used to identify fragments that bind biomolecules with low affinity. − We envisioned that covalent compounds might also act similarly for RNA targets and that by screening a collection of such small molecules, new RNA-reactive modules as well as noncovalent RNA-binding elements might be discovered.

To adapt the MALDI-TOF method for high-throughput screening, an efficient, reproducible, and small-scale method for removing salt and other contaminants from RNA samples was required. Here, solid-phase reverse immobilization (SPRI)-based magnetic beads, commonly used for the preparation of sequencing libraries, were chosen. These beads use polyethylene glycol (PEG) and high salt concentrations to precipitate RNA onto carboxy-coated paramagnetic beads. Optimization of the methodincluding the addition of isopropanol to facilitate precipitation of small RNA constructs, increasing the number of washes to thoroughly remove salt and PEG contaminants, and miniaturizing the sample volumeenabled screening and RNA purification from 5 μL reactions in 384-well plates (). Full details for this method can be found in the

A 2,000-compound electrophile library (Extended Data Table S1) was carefully designed from both the perspective of the electrophile and potential noncovalent RNA-binding element. In particular, each compound harbors a module with potential to react with biomolecules and noncovalent binding elements to facilitate interaction with RNA (Figures A and ). Various electrophiles are represented in the covalent library, ranging from known nucleic-acid reactive N-acylimidazoles and bis­(2-chloroethyl)­amines to traditionally protein-targeted chloroacetamides , and α,β-unsaturated amides (Figures A and ). Various small molecules that bind to r­(CUG)exp have been reported in the literature, ,− and the library designed herein shares structural similarities with them. The library contains 98 unique nitrogen-containing heteroaromatic ring systems, commonly found in RNA-binding small molecules, with benzothiazole, pyridine, pyrazole, benzoxazole, and indole among the most common in the library (). Thus, these molecules might induce covalency via induced proximity, rather than nonspecific reactivity. The physicochemical properties of the library overlap with a subset of the chemical space occupied by small molecules in the DrugBank database, as assessed by Uniform Manifold Approximation and Projection (UMAP) analysis (Figure A). Additionally, most compounds in the library (97%) follow Lipinski’s Rule of 5 and have a topological polar surface area (TPSA) of less than 140 Å2 (99.8%) (Figures A and ). ,,

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Composition and screening of a 2,000-compound covalent library. (A) Left: The library is composed of both traditionally protein-targeted electrophiles and those known to react with RNA. Right: Uniform Manifold and Projection (UMAP) structural analysis of the library shows that the electrophile library (blue) overlaps with the properties of a subset of the DrugBank library. Histograms for several physicochemical properties of the electrophile library show that most compounds fall within desirable drug-like values of molecular weight <500 Da, cLogP < 5, and TPSA < 140 Å2. (B) Screening of the electrophile library for reacting with RNA1 identified 34 replicable, validated hits. Of these, 24 compounds contained a 3-chloropivalamide electrophile as the reactive component. While the RNA-binding motifs varied, several of the most potent covalent compounds contained heteroaromatic rings common among RNA-binding modules, with their structures shown to the right.

Adduct formation was assessed by incubating each compound (1 mM) with RNA1 (20 μM; 12 h at 37 °C). Long incubation times were used to maximize adduct formation. After cleanup with SPRI magnetic beads, covalent adducts of 42 small molecules with RNA1 were detected by MALDI-TOF, affording a hit rate of 2.1% (Extended Data Table S1 and Figure S5). Of these, 34 were validated by replicating the assay (81% validated; Extended Data Table S2, Figures B and ). To identify the small molecules that react with RNA1 to the greatest extent, the 34 validated hits were studied in a dose response at concentrations of 10, 100, and 1000 μM. From this collection, 18 compounds formed covalent adducts at doses lower than 1 mM ().

Of the 34 validated hits, three were chloroacetamides, which were previously unknown to react with RNA, , though chloroacetamides have been used to chemically cross-link interactions formed between RNA and RBPs. Two compounds containing known RNA-reactive electrophiles – one nitrogen mustard and one N-acylimidazole – reacted with RNA1.

Most strikingly, 24 of the small molecules that formed covalent adducts with RNA1 contained a 3-chloropivalamide which underwent substitution of the chloride substituent (Figure B). This was a particularly surprising result, as neopentyl halides are classically considered to be weakly reactive electrophiles toward SN2 reactions. , Though mechanistic studies were not performed, a possible mechanism involving an oxetane intermediate via the carbonyl oxygen of the amide may explain this reactivity (). Only 49 of the 2000 small molecules in the covalent library contained this reactive module; the difference between the hit rate of 3-chloropivalamides targeting RNA1 (24/49; 49%) as compared to other electrophiles in the library (10/1951; 0.5%) is statistically significant (p < 0.0001). A series of structurally similar 3-chloropivalamides containing a central 4-(piperazin-1-yl)­pyrimidine motif showed structure-dependent differences in reactivity (), suggesting that factors beyond the electrophile’s intrinsic reactivity, such as noncovalent affinity toward the RNA target or differences in solubility, are important for covalent bond formation with RNA1.

Compound 10 showed the greatest extent of adduct formation to RNA1 in the primary screen (1.03 for 10, 0.14 ± 0.15 on average for the other hits; ), secondary validation (1.74 for 10, 0.21 ± 0.23 on average for other validated hits; Extended Data Table S2), and dose–response studies (1.52 for 10, 0.15 ± 0.17 on average for other validated hits at 1 mM; ), as determined by the ratio in peak intensities of RNA containing the adduct to unmodified RNA. However, later analysis revealed the activity in these assays is derived from two degradation products of 10: 10g and 10h (see Figure S7 and Supplementary Note). Both 10g and 10h showed time-dependent reaction with RNA1 over the span of 23 h, with the (R) enantiomer (10h) resulting in a modestly higher extent of reaction at extended time points (0.97 ± 0.08 ratio of peak intensities vs 0.67 ± 0.02; ). This analysis revealed that changes to the noncovalent RNA binding element alone are sufficient to enhance or attenuate covalent binding to the RNA targeting, demonstrating the critical importance of both the electrophile and noncovalent affinity elements for successful covalent adduct formation.

To identify how the 3-chloropivalamide electrophile reacts with RNA, 10h-modified RNA1 was digested to nucleosides, revealing the guanine base as the sole target (). Although unequivocal assignment could not be made from the mass spectra, the N-7 position is known to be intrinsically prone to modification by many electrophiles including nitrogen mustard derivatives, dimethyl sulfate (DMS), , and diethyl pyrocarbonate (DEPC). Furthermore, guanosines in the r­(CUG)exp structure are engaged in Watson–Crick pairing, leaving N-7 accessible in the major groove. As many ligands that bind to RNA recognize the major groove, a proximity-induced reaction of 10h with N7-G could be rationalized. To support the N-7 of guanine as the reactive site, RNA1 modified with 10g was reduced with sodium borohydride, which induces an abasic site at guanines alkylated at the N-7 position. , MALDI-TOF spectra of the reduced RNA showed the disappearance of peaks corresponding to the covalent adducts of 10g and the appearance of a previously unobserved peak corresponding to an abasic site generated from the loss of a guanine (). This same peak was not observed when RNA1 lacking the covalent adducts was treated identically (; DMSO-treated samples), suggesting the reduction-induced abasic site requires covalent modification of the RNA.

Studying Structure–Reactivity Relationships of the 3-Chloropivalamide Electrophile with Diverse Nucleophiles

The reactivity profile of the 3-chloropivalamide electrophile was more thoroughly characterized by studying its reaction with various nucleophiles, including thiols and nucleobases. Analysis of the attachment points of the electrophile to the putative noncovalent RNA-binding modules in the library suggested that reactivity depended on the amide substitution. For example, none of the eight anilides reacted with RNA1, while all seven of the methyl-piperazine derivatives formed adducts (). Although these differences could be due to variations in the noncovalent RNA-binding components, it is likely that the amide properties influence the electrophile’s reactivity, as has been reported for other electrophiles such as chloroacetamides.

To assess effects on the reactivity of 3-chloropivalamides, eight electrophiles lacking a noncovalent RNA recognition element (11–18) were synthesized with varying amide substructures, including primary and secondary alkyl amines and aniline (Figure ). To study the rate of reaction of the electrophiles with guanosine, a colorimetric model system with 4-(4-Nitrobenzyl)­pyridine (NBP) was utilized (Figures and ). NBP is an aromatic nitrogen nucleophile commonly used to detect and assess alkylating agents due to its similar reactivity with N7-G, as indicated by their nearly identical Swain-Scott nucleophilicity values.

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Assessing the intrinsic reactivity of the 3-chloropivalamide electrophile. Top: Eight compounds containing cyclic secondary amines, primary amines, and aniline were coupled to the 3-chloropivalamide electrophile to assess the influence of the amide substitution on the reactivity of the electrophile. Their intrinsic reactivity was compared to the promiscuously reactive electrophile iodoacetamide (IA). Left: The reactivity of the compounds was measured by three assays: (i) a colorimetric assay with NBP was used to assess reactivity toward guanosine-like nucleophiles; (ii) the aqueous stability of the compounds was assessed using LC/MS; and (iii) the reactivity toward thiols was assessed using a colorimetric assay with Ellman’s reagent (DTNB). Right: The piperazine and piperidine amides (11–15) were 200-fold more selective toward guanosine-like nucleophiles (NBP) than thiols (DTNB) relative to iodoacetamide.

Reactions between the electrophiles 11–18 as well as iodoacetamide and NBP showed that the piperazine and piperidine amides (11–15) were the most reactive, while other derivatives (16–18) showed negligible alkylation after 28 h (Figures and ). To calculate second order rate constants for the reaction with NBP, the hydrolytic stability for each electrophile (as measured by LC-MS; Figures and ) was determined, where half-lives ranged from 2 h to >4 d (). After incorporating hydrolytic stability, rate constants for the reactions of 11–15 with NBP ranged from 2 × 10–4 M–1s–1 to 7 × 10–4 M–1s–1, which is 2–6-fold more reactive than iodoacetamide (1.2 ± 0.1 × 10–4 M–1s–1).

Nucleophilic thiols such as glutathione and reactive cysteines are other potential targets for the 3-chloropivalamide electrophile in cells. To assess intrinsic thiol reactivity, a colorimetric assay with 5,5-dithiobis­(2-nitrobenzoic acid) (DTNB) was used (Figures and ; ). To benchmark reactivity, studies were first conducted with the promiscuous thiol-reactive compound iodoacetamide, affording a second order rate constant of 2.55 ± 0.04 M–1 s–1, in agreement with a previously reported value (2.6 ± 0.1 M–1s–1). The amide substituents within 11–18 significantly influenced thiol reactivity by more than 60-fold, with second-order rate constants ranging from 6.1 ± 0.1 × 10–2 M–1 s–1 to <1 × 10–3 M–1 s–1 (Figures and ; ). In line with the NBP reactivity assay, piperazine and piperidine amides (11–15) were the most reactive, followed by the anilide (16) and secondary amides (17, 18), where 18 showed no detectable reactivity. These trends are largely consistent with those observed for chloroacetamides and correlate with the reactivity of the hits that emerged from the reactivity profiles of the 2,000-member covalent compound library toward RNA1 (). Furthermore, a previous screen of more than 750 chloroacetamides revealed that the vast majority of chloroacetamides are 4- to 40-fold less reactive toward DTNB than iodoacetamide (median reactivity 26-fold less reactive), though this varied with the type of amine. The chloroacetamide library contained piperazine and piperidine amides, as well as aromatic and alkyl primary amines, much like compounds 11–18. In contrast, the 3-chloropivalamide electrophiles 11–18 are 40- to >2,000-fold less reactive toward thiols than iodoacetamide (Figure S12 and Table S1); thus, they are less reactive toward thiols than most of the chloroacetamides tested previously, all falling below the median reactivity of the chloroacetamide library. When the relative reactivity of iodoacetamide and 11–15 toward both guanosine-like (NBP) and thiol (DTNB) nucleophiles are compared, a more than 200-fold shift in selectivity toward guanosine-like nucleophiles was observed for the 3-chloropivalamide electrophile ( and Figure ). Collectively, these studies suggest that the 3-chloropivalamide electrophiles possess a more selective reactivity profile toward guanine-like nucleophiles than α-haloacetamides, providing a potential for application as RNA-selective covalent compounds in a cellular environment.

Using Covalent Reactivity to Inform Design of Noncovalent r­(CUG)exp Binders

The covalent screen revealed not only RNA-reactive electrophiles, but also binding elements that confer modest noncovalent affinity toward the RNA. The binding of a noncovalent derivative of 10g, 10i, to RNA1 was studied by nuclear magnetic resonance (NMR) spectroscopy, which revealed weak binding of the compound to the RNA by Carr–Purcell–Meiboom–Gill (CPMG) NMR (300 μM of 10i and 5 μM of RNA1; ) and by monitoring the imino protons of RNA1 (100 μM of 10i and 50 μM of RNA1; ). This modest noncovalent affinity to the target RNA likely explains the approximately 5-fold improvement of adduct formation with 10g compared to 11, which lacks the noncovalent affinity motif (). The identified binding core of 10g was used to develop noncovalent ligands targeting r­(CUG)exp. Since the 3-chloropivalamide electrophile reacts with guanine in the major groove of RNA1, attaching moieties at this site with the potential to form hydrogen bonds in the major groove could improve noncovalent affinity for the RNA target. Indeed, of the four compounds synthesized (10j–m), two (10l and 10m) show improved binding relative to the original binding core, 10i (, see Supplementary Note for further details). In summary, covalent screening identified a weak binder of r­(CUG)exp, whose affinity for the RNA target was improved by utilizing information about the positioning of the electrophilic moiety within the major groove, potentially forming base triple-like interactions that occur naturally , and have been used to engineer triple helical structures. − Additional studies of this approach and this compound set are required to better understand how to utilize this scaffold to target r­(CUG)exp and provide potentially bioactive ligands.

Enhancing Reactivity Using Known r­(CUG)exp-Binding Ligands

Given the high concentrations (>1 mM) and low extent of reactivity (incomplete modification after 24 h) of 10g and 10h with RNA1, a previously validated r­(CUG)exp-binding compound, H 90 (a derivative of the DNA-binding small molecule Hoechst), was conjugated to the 3-chloropivalamide electrophile to improve the extent of covalent adduct formation (Figure A). A high affinity nucleic acid binder, Hoechst recognizes the minor groove of DNA , as well as noncanonically paired internal loop regions in RNA, particularly the 1 × 1 nucleotide UU internal loop found in r­(CUG)exp among a variety of other RNA targets. H was used as a module in a series of multivalent ligands that specifically bind r­(CUG)exp with high affinity and specificity. While an H homodimer alleviates DM1-associated phenotypes in a cellular model, H itself is biologically inert.

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Developing a more potent covalent r­(CUG)exp binder. (A) A previously validated r­(CUG)exp binder was appended to both the identified electrophile 11 and a noncovalent control lacking the chloride to generate the r­(CUG)exp-targeting compounds 20 and 19, respectively. (B) Left: Compound 20 dose-dependently formed adducts with RNA2 (1 μM). Right: A representative mass spectrum showing near-complete reaction of 20 (10 μM) with RNA2 (1 μM). (C) Compound 20 (20 μM) reacted to a much lesser extent with RNA3 (20 μM), which lacks the 1 × 1 UU loops, and DNA1 (20 μM) than with RNA2 (20 μM). (D) Low reactivity toward DNA may be explained by differential modes of binding. The H parent compound is a known minor groove binder in DNA (Left), but molecular docking of 19 to a model of r­(CUG)exp (Right) suggests that the electrophile can be presented to the major groove, where the proposed reactive N7-guanine resides.

Due to the favorable reactivity profile of 11 in the NBP alkylation assay (), a conjugate between H and 11, compound 20, as well as a noncovalent control 19, were synthesized (Figure A). A longer r­(CUG) repeat construct (r­(CUG)12; RNA2) was chosen to better represent the biologically relevant r­(CUG)exp structure (Figure ). [Note: this RNA was not used in the library screen because it requires more stringent washing necessary for clear detection by MALDI-TOF MS due to its larger molecular weight]. Compound 20, like 10g, specifically modified the guanine bases of RNA2 (Figures S8 and S15A). Compound 20 also showed dose-dependent labeling of RNA2; in particular, at 10 μM of 20, nearly complete alkylation of RNA2 (1 μM) was observed (Figures B and S15B), a higher extent of adduct formation than observed with 1 mM of 10g or 10h with RNA1 (). The greater extent of adduct formation at a 100-fold lower concentration of compound represents a more than 100-fold improvement of in vitro potency of the r­(CUG)-targeting compounds. The gel-shift assay by dPAGE was utilized to confirm the high extent of labeling at low concentrations. Nearly 50% of RNA2 (1 μM) showed reduced mobility when treated with 20 (5 μM). When the same samples were analyzed by MALDI-TOF, nearly 80% of the RNA was modified (). Though both methods confirm a high degree of labeling of RNA2 with 20, the differences in stoichiometry may arise from differences in staining of modified and unmodified RNA, comigration of modified and unmodified RNA, as was observed for some of the nitrogen mustards (Figures and ), or differences in ionization efficiency of unmodified and modified RNA by MALDI-TOF.

Importantly, the noncovalent RNA-binding component of 20 was necessary to facilitate these covalent adducts, as no reaction of 11, which lacks the RNA-binding H component, with RNA2 was observed until at least 2 mM of the small molecule was added (). Additionally, this reaction was selective for RNAs containing the 1 × 1 UU motif, as the fully base-paired RNA3 (0.17 ± 0.04 adducts per RNA) showed significantly attenuated reactivity toward 20 than RNA2 (0.93 ± 0.07 adducts per RNA, Figure C). Further, when 20 was incubated with a mixture of RNA2 and RNA3, only covalent adducts to RNA2 were observed (). The selectivity of the reaction of 20 with RNA2 was examined in the presence of 1 or 10 mM glutathione, a highly abundant thiol nucleophile present in cells. Under these conditions, only a modest (∼14%) decrease in reaction with RNA2 was observed (), suggesting that these compounds could be valuable for studying cellular models of DM1 by specifically targeting r­(CUG)exp and potentially improving DM1-associated defects.

Because 20 contains a binding core derived from the DNA-binding Hoechst dye, the ability of the compound to react with DNA was studied. Two DNA constructs (DNA1 and DNA2) were designed which contain both the preferred Hoechst binding sites (A/T stretches) and adjacent guanines for potential covalent adduct formation (Figures C and ). The binding of 19 (nonreactive to prevent potential interference of reactivity in the assay) to DNA1, DNA2, and RNA2 was evaluated by monitoring change in the intrinsic fluorescence of 19, resulting in binding EC50s of 250 nM (95% confidence interval (CI): 205–295 nM), 74 nM (95% CI: 53–98 nM), and >20 μM, respectively (). While the observed binding of 19 to RNA2 is weaker than previously observed for H, , these differences in affinity may be explained by differences in the compound structure (such as the additional steric bulk of the methylpiperazine of 19) or differences in the RNA target tested, as longer repeats such as those used previously may bind H more potently and cooperatively. However, despite the higher EC50 for 19 binding to RNA2 compared to both DNA oligonucleotides, the propensity of 20 to form covalent adducts with RNA2 (0.93 ± 0.06 adducts per RNA) is 13 times higher than with DNA1 (0.073 ± 0.004 adducts per DNA) (Figure C). This difference in reactivity is likely not due to differences in intrinsic reactivity of the electrophile toward DNA or RNA, as the electrophile lacking the RNA binder (11) reacts with DNA1 only approximately ∼ 2-fold less than with RNA2, despite RNA2 containing 2.5 times more potentially reactive guanine residues (15 vs 6) than DNA1 (Figure S15D,H).

To gain insight into these observations, molecular modeling was used. These studies revealed that 19 binds to the 1 × 1 nucleotide UU internal loops and can display the electrophilic site into the major groove of r­(CUG) repeats, in contrast to binding in the DNA minor groove , (Figure D). The lowest free energy pose of 19 bound to a r­(CUG) repeat containing RNA places the electrophilic site in proximity to the N-7 position of guanine at the base pair adjacent to the binding site (Figure D). Out of docked poses generated using AutoDock-GPU, the electrophile is positioned into the major groove for half of them, including 6 out of the 8 lowest free energy poses (). In contrast, when 19 is docked to a previously solved structure of DNA-bound Hoechst 33258 (PDB: 8BNA), all docked structures position the molecule and electrophilic site within the minor groove (). Further, the predicted binding free energies are ∼ 8 kcal/mol lower for docking of 19 to DNA (−18.92 kcal/mol) than the model r­(CUG) RNA (−10.45 kcal/mol), supporting the measured EC50s (). Thus, despite higher affinity, noncovalent binding with DNA, the lack of significant reactivity for 20 with DNA is likely due to the positioning of the reactive module in the minor groove, away from the reactive site, presumably the N-7 of guanine. In contrast, the binding of 20 to the RNA positions the reactive module near the guanine N-7 to facilitate reactivity. These results suggest that the specificity of a reactive, RNA-targeting small molecule is influenced by both its noncovalent affinity toward its target and its binding mode, wherein both occupancy and proper positioning of the reactive group are essential for robust, selective modification of RNA.

Discussion

Covalent approaches to target biomolecules have transformed chemical biology and medicinal chemistry. A wide variety of protein-targeted covalent binders have expanded the druggable space, informed biology, and garnered US Food and Drug Administration (FDA) approval. , Developing such approaches broadly for RNA targets could therefore have significant potential. Yet, there have been relatively few studies of small molecules that covalently target RNA. ,, One of the challenges with specific reactivity with RNA targets is that the bases have relatively similar reactivity profiles, unlike amino acid side chains which vary significantly. The development and optimization of a MALDI-TOF MS screening methodology allowed a broader view of the potential for RNA to be covalently targeted. Indeed, many compounds that have been used for specific protein reactivity also react with RNA, suggesting that RNA should be considered an on- or off-target in these and other screens, as has been previously put forward for noncovalent ligands.

Via an unbiased screen of a diverse covalent library, a 3-chloropivalamide electrophile was identified as a common motif among hit small molecules that react with RNA. Using these results coupled with chemical synthesis and reactivity assessment elucidated characteristics of a molecule that can provide covalent ligands for RNA targets. Importantly, attachment of this electrophile to a dual DNA- and RNA-binding compound afforded potent and selective reaction with RNA in vitro, particularly the 1 × 1 nucleotide UU internal loop that is present in r­(CUG)exp. This conversion was possible by exploiting the differences in binding mode between the targets, where the electrophile is positioned nearby an N-7 of guanosine in the RNA but not in the DNA target (Figures , S15, and S16). Previous work from our lab developed covalent and dimeric bis-benzimidazole-based ligands targeting r­(CUG)exp that utilized the nitrogen mustard chlorambucil as the electrophilic warhead, rather than the 3-chloropivalamides reported herein. , Despite higher intrinsic reactivity of the nitrogen mustards relative to the 3-chloropivalamides (see Chlorambucil-Alkyne in compared to 11 in Figure S15D), both prior reports utilizing the chlorambucil warhead demonstrate selective recognition of r­(CUG)exp RNA in cells, perhaps due to the use of higher affinity dimeric ligands that enabled use of low concentrations of the covalent ligand. While the monomeric bis-benzimidazole ligand 20 reported herein also demonstrates selective covalent modification in vitro at low μM concentrations (Figures and ), it has not been examined for transcriptome-wide selectivity. Given the lower intrinsic reactivity of the 3-chloropivalamide warhead, however, it holds potential for more selective modification, provided proper orientation and positioning of the electrophile in the major groove would remain when incorporated into dimeric bis-benzimidazole ligands. Thus, positional reactivity could be a general strategy for improving the selectivity of binding small molecules, as observed for targeted degradation approaches, either directly or via enzymatic recruitment.

Further, the concentration needed for more than one modification of RNA with the 3-chloropivalamide electrophile dropped from 1 to 2 mM for initial hits 10g and 10h to ∼ 5 μM for 20. A similar gain in potency has been observed for other electrophiles targeting RNA, including the N-acylimidazoles in SHAPE reagents. Despite their initial use as structure probing reagents at high mM concentrations, N-acylimidazole electrophiles have since been used for targeted covalent modification at μM concentrations in vitro and in cells. , Similarly, the nitrogen mustards, particularly chlorambucil (1), have been used as a warhead for targeted covalent modification at μM or nM concentrations in DM1 model cell lines, , despite requiring mM concentrations for unguided reactivity with RNA (Figures , S1, and S2). Further optimization of these 3-chloropivalamide electrophiles – in particular, attachment to higher affinity RNA binders and targeted orientation of the electrophile toward a nearby guanine nucleophile – may enable greater potency gains for the development of RNA-targeting covalent ligands.

Covalent screening in the format presented herein could have broader implications. By studying a larger and more diverse covalent compound library, it is possible to identify not only reactive molecules but also compounds that bind RNA, including those of low affinity or short residence times. Moreover, information about binding sites and the positioning of reactive molecules can guide the design of compounds that recognize the target noncovalently, leading to improved activity.

Conclusion

Herein, we described the development of a high-throughput mass spectrometry screen to identify ligands that react with RNA in a binding-dependent manner, including the surprising discovery that 3-chloropivalamide electrophiles cross-linked to RNA. By appending a dual DNA- and RNA-binding small molecule ligand to the 3-chloropivalamide electrophile, selective RNA modification was achieved by exploiting differences in the binding mode of the ligand. There are likely many additional approaches that can emerge based on these studies and earlier ones on covalent chemistry for RNA. , The development of covalent binders for RNA targets could not only provide bioactive compounds, but also be used to study the molecular recognition of RNA structures in cells in an unbiased way, enabling lead optimization into bioactive ligands targeting RNA. −

Supplementary Material

ja5c06802_si_001.pdf (10.4MB, pdf)
ja5c06802_si_002.xlsx (90.4KB, xlsx)
ja5c06802_si_003.xlsx (296KB, xlsx)

Acknowledgments

This work was funded by the National Institutes of Health (R35 NS116846 to M.D.D.), the Muscular Dystrophy Association (Grant ID 1069959 to M.D.D.), and the German Research Foundation (DFG) through a Walter Benjamin fellowship (#515396515 to P.R.A.Z.). This material is based upon work supported by the National Science Foundation Graduate Research Fellowship under Grant No. 2235200 (to N.A.S.). Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.5c06802.

  • Experimental methods; supplementary figures including results of electrophoretic mobility shift assays, MALDI-TOF analysis of a panel of nitrogen mustards, optimization of bead cleanup of RNA for MALDI-TOF analysis, structural analysis of the 2,000-member electrophile library, screening and hit validation of the 2,000-member electrophile library, SAR for a common binding scaffold which contains the 3-chloropivalamide electrophile, analysis of oxidized derivatives 10g and 10h that contribute to the activity of the originally identified hit 10, identification of the site of reactivity for the 3-chloropivalamide electrophile within the r­(CUG) repeat RNA, reactivity of electrophiles toward guanosine-like nucleophiles, rates of hydrolysis of 3-chloropivalamides, influence of the RNA-binding moiety of 10g on reactivity by NMR spectrometry and MALDI-TOF analysis, selectivity of 20 assessed by MALDI-TOF, electrophoretic mobility shift, and fluorescence-based binding assays, and molecular docking of 19 to DNA and RNA; a supplementary note, which describes the structural elucidation and activity data for 10 and 10a–h in more detail; additional information regarding the noncovalent derivatives of 10 (10i–m) and the analysis of their binding toward RNA1 by NMR are included; supplementary tables including rate constants for reactions involving eight 3-chloropivalamide electrophiles (11–18) and iodoacetamide reacting with three nucleophiles and sequences of oligonucleotides used in this study; synthetic procedures and compound characterization including synthetic schemes, NMR spectra, results from HR-MS, and analytical HPLC spectra (PDF)

  • Electrophile library of compounds (XLSX)

  • Hit validation of compounds (XLSX)

The authors declare no competing financial interest.

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