Using a function-first ‘scout fragment’-based approach to develop allosteric covalent inhibitors of conformationally dynamic helicase mechanoenzymes

Abstract

Helicases, classified into six superfamilies, are mechanoenzymes that utilize energy derived from ATP hydrolysis to remodel DNA and RNA substrates. These enzymes have key roles in diverse cellular processes, such as translation, ribosome assembly and genome maintenance. Helicases with essential functions in certain cancer cells have been identified and helicases expressed by many viruses are required for their pathogenicity. Therefore, helicases are important targets for chemical probes and therapeutics. However, it has been very challenging to develop chemical inhibitors for helicases, enzymes with high conformational dynamics. We envisioned that electrophilic “scout fragments”, which have been used in chemical proteomic studies, could be leveraged to develop covalent inhibitors of helicases. We adopted a function-first approach, combining enzymatic assays with enantiomeric probe pairs and mass spectrometry, to develop a covalent inhibitor that selectively targets an allosteric site in SARS-CoV-2 nsp13, a superfamily-1 helicase. Further, we demonstrate that scout fragments inhibit the activity of two human superfamily-2 helicases, BLM and WRN, involved in genome maintenance. Together, our findings suggest an approach to discover covalent inhibitor starting points and druggable allosteric sites in conformationally dynamic mechanoenzymes.

Developing chemical inhibitors for helicases, which are important targets for anti-viral and anti-cancer drugs, has been notoriously difficult1,2. There are at least two reasons why targeting helicases has been challenging. First, while high-throughput activity-based screens have yielded several hits for helicases, the vast majority of these compounds were subsequently found to be nonselective, false positives or indirect inhibitors (e.g., DNA intercalators).1 Second, these enzymes undergo substantial conformational changes during their ATP hydrolysis cycle, a property that poses major difficulties for structure-guided inhibitor design.² Of the six helicase superfamilies (SFs), the mechanochemical cycle of SF1 and SF2 helicases are well studied.³ For both helicase superfamilies, two RecA-like domains transition between “open” and “closed” conformations during the ATP hydrolysis cycle,² with changes in interdomain spacing reaching ~15 Å.⁴

We envisioned that a covalent inhibitor discovery approach for helicases could address both challenges, as these compounds would remain bound to the targets throughout the conformational changes linked to the enzymatic cycle (Figure 1A), and direct target engagement could be readily assessed using mass spectrometry (MS) techniques. The use of covalent probes to discover and target allosteric sites has been shown to be an effective strategy for other protein superfamilies that have been difficult to selectively inhibit,^5,6 such as Ras GTPases.⁷

Figure 1.

A “scout fragment”-based function-first approach to discover covalent inhibitors of helicases. (a) Schematic of irreversible versus reversible inhibition. (b) Domain organization of nsp13. ZBD: zinc binding domain; S: stalk; 1B: β-barrel domain. (c) Chemical structure of “scout fragments”, 1 (KB02) and 2 (KB05). (d) Dose-dependent inhibition of nsp13 helicase activity by 1 and 2 (IC₅₀: 1 = 198 ± 60 μM, 2 = 357 ± 139 μM; 8 h incubation; 4 °C). (e) nMS analysis of nsp13 liganding by 1 (200 μM, 4 °C; number of adducts: gray).

Our efforts were inspired by the use of electrophilic “scout fragments” in large-scale chemical proteomic workflows profiling ligandability across native proteomes.^8–11 Importantly, these chemical proteomic analyses have identified ligandable sites in helicases.^10,11 However, it is unclear if any of these liganding events inhibit helicase activity and if these fragments can be progressed into site-specific inhibitors. We also noted that, methods to assess if ligand engagement modulates protein function are now emerging.^12,13

Here, we combine the use of electrophilic “scout fragments” with biochemical assays, enantiomeric probe pairs,¹⁴ and mass spectrometry to identify starting points for inhibitors and targetable allosteric sites in helicases. To develop our approach, we first focused on nsp13, the SARS-CoV-2 helicase, a member of the SF1 helicases that is required for SARS-CoV-2 replication.^15,16 Nsp13 has been proposed to be an important target of antiviral therapies due to the high degree of conservation across coronaviruses of potentially druggable pockets in this enzyme.¹⁷ Nsp13 contains five domains (Figure 1B) and can unwind DNA or RNA substrates.^18–20 Efforts to identify inhibitors of nsp13 using crystallographic fragment screens have been reported, but it is unclear if the fragments inhibit helicase function¹⁹. In addition, target specificity has not been firmly established for inhibitors from high-throughput screening and drug repurposing efforts.^21–23

To identify covalent inhibitors for nsp13 we selected two previously characterized “scout fragments” (compounds 1 and 2; Figure 1C).^8–11 We generated recombinant nsp13 and adapted a fluorescence-based helicase assay,²⁰ and observed dose-dependent inhibition of nsp13 by compounds 1 and 2 (Figure 1D). We selected the more potent compound 1 for subsequent studies.

High-resolution native mass spectrometry (nMS) revealed predominantly three covalent adducts on nsp13 in the presence of compound 1 (Figure 1E). Nsp13 contains 26 cysteine residues,²⁴ and these data indicate that only a subset are liganded by compound 1. Site-mapping MS indicated that compound 1 modifies C441 and C444, residues in a loop within nsp13’s ATP-binding pocket (Figure S1, Table S1). We next generated a construct with two mutations (C441S and C444S, hereafter nsp13^{C441S C444S}), determined it to be enzymatically active (Figure S2, Table S2), and found that it remained sensitive to compound 1 (Figure S3A).

We performed nMS analysis on nsp13^{C441S C444S}, and interestingly, only a single adduct was detected after incubation with compound 1 (Figure S3B). This is consistent with the covalent adduction of three cysteine residues (C441, C444, and another) in nsp13^wt (Figure 1E). Site-mapping MS experiments indicated that C556 in nsp13^{C441S C444S} is the predominant site of modification (Figure S4, Table S3). We next generated a construct with three mutations (C441S C444S C556S), determined it to be enzymatically active (Figure S2, Table S2), and found that compound 1 does not substantially inhibit its helicase activity (Figure S5).

Encouraged by these data, we synthesized and tested four analogs of compound 1 with simple modifications, such as methyl substitutions (Figure S6). We identified a compound S1, a racemic mixture that was more than compound 1, and found that its purified enantiomers (compounds S1a and S1b) display different potencies (Figure S7). Covalent docking studies with S1b (Figure S8) guided the synthesis and testing of additional analogs that identified compound 3 (Figure S9). Enantiomers of compound 3 were isolated to obtain compounds 3a and 3b (Figure 2A) and again, we observed differences in the potency of nsp13 inhibition based on the methyl group stereochemistry (Figure 2B). Interestingly, the stereochemistry is different in 3b relative to S1b. Nonetheless, the observed potency differences between 3a and 3b suggest that non-covalent contacts play a role in the ligand-enzyme interaction. We also found similar potencies when compound activity was tested at RT (Figure S10). nMS analyses indicated that compound 3b, the more potent enantiomer of compound 3, liganded nsp13 predominantly at a single residue (Figure 2C).

Figure 2.

Characterizing analogs of 1. (a) Chemical structures of 3, 3a and 3b and potency rank ordering (indicated by chevrons). (b) Dose-dependent inhibition of nsp13 helicase activity by 3, 3a and 3b (IC₅₀: 3a > 50 μM; 3 = 8.60 μM ± 0.02; 3b = 5.04 ± 0.52 μM; 4 h incubation, 4 °C). (c) nMS analysis of nsp13^wt liganding by 3b (20 μM, 4 °C).

Site-mapping MS analyses revealed C556 is the primary site of nsp13^wt liganding by compound 3b (Figure S11, Table S4). We generated a construct with a point mutation at C556 (nsp13^C556S), determined it to be enzymatically active (Figure S2, Table S2), and gratifyingly, inhibition of this mutant construct by compound 3b was not detected (Figure 3A). We also performed nMS experiments and did not observe modification of nsp13^C556S by compound 3b (Figure 3B), indicating that 3b acts by selective covalent modification of the C556 residue in nsp13. We also generated a nsp13 construct with a C556W (nsp13^C556W) point mutation, as tryptophan mutations can mimic liganding by small molecules.²⁵ We found that in helicase assays, the tryptophan mutant was ~20 fold less active than nsp13^wt (Figure 3C). Taken together, these data suggest that covalent modification of C556, suppresses helicase activity.

Figure 3.

Liganding of C556 leads to nsp13 inhibition. (a) Dose-dependent inhibition of nsp13^wt and nsp13^C556S by 3b (data from Figure 2b provided for reference (dashed line); 4 h incubation, 4 °C). (b) nMS analysis of nsp13^C556S liganding by 3b (20 μM, 4 °C). (c) Enzyme velocity versus concentration of nsp13^wt and nsp13^C556W.

To profile selectivity, we tested inhibition of two mammalian helicases by compound 3b. We selected the superfamily-2 RecQ helicases Bloom syndrome (BLM) and Werner syndrome (WRN), enzymes involved in maintaining genome stability.²⁶ Importantly, WRN helicase has been identified as a unique vulnerability in certain cancer cell lines.^27–30 Guided by literature precedent, constructs for BLM and WRN (BLM^636–1298, WRN^515–1233, and WRN^1–1235 (Figure 4A); generating a near-full length BLM construct was not successful) were expressed in recombinant form and fluorescence-based activity assays were adapted (Figure S12).^31,32 We used BLM^636–1298 and WRN^1–1235 to profile specificity and found that nsp13 is inhibited by compound 3b more potently than WRN or BLM (Figure 4B). Together, these data indicate that compound 3b is a selective, site-specific, allosteric inhibitor of nsp13.

Figure 4.

Characterizing inhibition of the helicases BLM and WRN by scout fragments and their analogs. (a) Domain organization of BLM and WRN constructs used. RQC: recQ c-terminal domain; HRDC: helicase and RNAaseD c-terminal domain) domain; Exo: exonuclease domain. (b) Dose-dependent inhibition of nsp13, BLM and WRN helicase activity by 3b (IC50: BLM > 100 μM, WRN ~ 50 μM; nsp13: data from Figure 3d provided for reference (dashed line), 4 h incubation, 4 °C). (c) Dose-dependent inhibition of BLM and WRN helicase activity by 1 (IC₅₀: BLM = 243 ± 41 μM; WRN = 114 ± 22 μM; 8 h incubation, 4 °C). (d) Dose-dependent inhibition of BLM and WRN helicase activity by 2 (IC₅₀: BLM = 47.2 ± 20.5 μM; WRN = 24.9 ± 3.8 μM; 8h incubation, 4 °C). (e) Chemical structures of 4 and 5. (f) Dose-dependent inhibition of WRN by 4 and 5 (IC₅₀: 4 = 38.3 ± 7.6 μM, 5 = 17.9 ± 6.6 μM; 8 h incubation, 4 °C). Data for 2 from panel d is provided for reference (dashed line).

We next examined if a function-first “scout-fragment”-based approach can be used to identify inhibitors of mammalian RecQ helicases. Chemical proteomic studies have suggested that both BLM and WRN can be partially liganded by compounds 1 and 2.^10,11 For these experiments, we tested scout fragments against BLM^636–1298 and the equivalent WRN^515–1233 construct. We found that both BLM and WRN are inhibited by compounds 1 and 2, but more potently by compound 2 (Figure 4C, 4D). We synthesized and tested two analogs of compound 2, yielding compounds 4 and 5 (Figure 4E), and found that compound 6 is a more potent inhibitor of WRN than compound 2 (Figure 4F). These data suggest that compounds 2 and 5 could be useful starting points to develop covalent inhibitors for RecQ helicases.

Together, our findings suggest a function-first approach, based on biochemical testing of electrophilic scout fragments combined with the use of enantiomeric probe pairs¹⁴ and mass spectrometry, to identify starting points for inhibitors and druggable allosteric sites in helicase mechanoenzymes. In the case of nsp13, of the 26 cysteines, we identified a single residue in an allosteric site that can be liganded by compound 3b to inhibit helicase activity. Structural models reveal that C556 is not likely to be directly involved in ATP or RNA binding.³³ Further studies will be required to understand how liganding C556 in nsp13 allosterically inhibits helicase activity. Our finding that nsp13 activity is reduced by mutating this cysteine to serine (Figure S2, Table S2), a conservative amino acid substitution, suggests that potential inhibitor-resistance-conferring mutations would have an associated fitness cost for the virus. Consistent with this hypothesis, sequences of SARS-CoV-2 in the GISAID database indicate a low frequency of C556 mutations (132 of >15 million sequences available).³⁴ While electrophilic warheads such as acrylamides are found in clinically approved drugs,^35–37 the chloroacetamide warhead in 3b is more chemically reactive and likely to have additional cellular targets. Encouragingly, there are examples of chloroacetamide-containing selective probes that have cellular activity.¹¹ It is noteworthy that less reactive warheads, which retain the overall nucleophilic cysteine attack vector of chloroacetamides, have been developed and could be incorporated in 3b analogs,³⁸ as needed.

For the few helicases for which selective chemical inhibitors have been reported,^39–43 targeting allosteric sites has been an effective strategy. In the case of the hepatitis C NS3 helicase integrative approaches, which included high-throughput screens, fragment screens and structural analysis, were employed to identify druggable allosteric sites.^39,40 Both allosteric and orthosteric inhibitors of the Brr2 helicase, an enzyme involved in spliceosome function, were identified and the allosteric binders exhibited better specificity.⁴¹ Characterizing the mechanisms of action of natural products (e.g., rocaglamids) or screening hits has also serendipitously identified druggable allosteric sites in EIF4A⁴² and BLM helicases,⁴³ respectively. Our data suggest that characterizing scout fragment-based inhibition may present a useful approach to identify starting points for chemical inhibitors that selectively target allosteric sites in helicases and other conformationally dynamic mechanoenzymes.