key: cord-0731723-zmw6a1i2
authors: Qin, Bo; Craven, Gregory B.; Hou, Pengjiao; Lu, Xinran; Child, Emma S.; Morgan, Rhodri M. L.; Armstrong, Alan; Mann, David J.; Cui, Sheng
title: Acrylamide Fragment Inhibitors that Induce Unprecedented Conformational Distortions in Enterovirus 71 3C and SARS-CoV-2 Main Protease
date: 2020-11-06
journal: bioRxiv
DOI: 10.1101/2020.11.06.370916
sha: 62bc4fd781c91d228cbf4791b10d0221261d5e72
doc_id: 731723
cord_uid: zmw6a1i2

RNA viruses are critically dependent upon virally encoded proteases that cleave the viral polyproteins into functional mature proteins. Many of these proteases are structurally conserved with an essential catalytic cysteine and this offers the opportunity to irreversibly inhibit these enzymes with electrophilic small molecules. Here we describe the successful application of quantitative irreversible tethering (qIT) to identify acrylamide fragments that selectively target the active site cysteine of the 3C protease (3Cpro) of Enterovirus 71, the causative agent of hand, foot and mouth disease in humans, altering the substrate binding region. Further, we effectively re-purpose these hits towards the main protease (Mpro) of SARS-CoV-2 which shares the 3C-like fold as well as similar catalytic-triad. We demonstrate that the hit fragments covalently link to the catalytic cysteine of Mpro to inhibit its activity. In addition, we provide the first demonstration that targeting the active site cysteine of Mpro can also have profound allosteric effects, distorting secondary structures required for formation of the active dimeric unit of Mpro. These new data provide novel mechanistic insights into the design of EV71 3Cpro and SARS-CoV-2 Mpro inhibitors and identify acrylamide-tagged pharmacophores for elaboration into more selective agents of therapeutic potential.

RNA viruses cause significant morbidity and mortality in human and animal hosts. 1 , 2 For example, Enteroviruses (EV) include many important human pathogens with the best characterised being enterovirus 71 (EV71), rhinovirus (HRV), coxsackievirus B3 (CVB3), enterovirus D68 (EV-D68) and poliovirus (PV). EV71 is one cause of hand, foot and mouth disease (HFMD) in humans and is associated with severe neurological disease with considerable mortality. 3 Vaccines against EV71 have been developed and approved 4 but outbreaks persist and there are no antiviral drugs available for treating EV71. 5 , 6 Like many other RNA viruses, EV71 relies on proteases to cleave a polyprotein precursor into individual functional mature proteins. For enteroviruses, the majority of this proteolytic processing utilises the 3C protease (3C pro ). 7 Given the essential role of virally-encoded proteases in viral life-cycles, numerous protease inhibitors have been developed for potential clinical use. 8 These include a large collection of picornaviral 3C pro inhibitors, such as an HRV 3C pro inhibitor Rupintrivir (AG7088) 9 that failed to show patient benefit in phase II clinical trials. 10 To date, no 3C pro inhibitors have been approved for clinical use.

With the global rise of COVID-19, scientific attention has focussed on the causative agent, SARS-CoV-2. 11 This virus expresses two precursor polyproteins (pp1a and pp1ab) that are cleaved by both main protease (M pro ) 12 and papain-like protease (PL pro ). 13 Interestingly, M pro and 3C pro share a similar fold, active site architecture and catalytic triads, both proteases being absolutely reliant on the catalytic cysteine for activity. 14 Exploiting these structural similarities may enable identification of pharmacophores that target a wide variety of viral proteases.

Therapeutics with a covalent mechanism of action are becoming more widely accepted for a range of diseases. 15 Targeted covalent therapeutics such as Ibrutinib 16 and Osimertinib 17 use a Michael acceptor to react with the thiolate of cysteine, giving irreversible target engagement. 18 Identifying the starting points for such agents is often the bottleneck in development. Fragment-based approaches offer an efficient starting point and have already shown significant promise in targeting SARS-CoV-2 M pro . 19 We have recently developed quantitative irreversible tethering (qIT), a highthroughput method for identifying selective covalent fragments that bind to a desired cysteine on a target protein. 20 qIT enables hit prioritization and minimization of false positives and negatives through normalization of the rate of protein modification by compound intrinsic reactivity. 21 Here we employ qIT to identify inhibitory fragments that covalently target the active site cysteine of EV71 3C pro . Cocrystals of 3C-fragment complexes demonstrated the occupancy of a novel, cryptic pocket in 3C pro . Furthermore, when repurposed towards M pro , the covalent fragments also preferentially targeted the active site cysteine, inhibiting the enzyme activity and, in one case, additionally disrupting the quaternary structure of M pro .

Covalent fragment screening against EV71 3C pro by quantitative Irreversible Tethering (qIT).

To target cysteine C147 on EV71 3C pro , we constructed a 1040-member covalent fragment library using a combination of in-house parallel synthesis and commercial vendors ( Figure 1A and 1B) . Fragment-like core scaffolds were functionalized with cysteine-reactive chemical groups, with the majority (>95%) being acrylamides. Acrylamide "warheads" are featured in several clinically approved covalent drugs and are favoured for their mild electrophilic reactivity, minimizing potential non-specific reactivity and associated toxicity. 22 In line with the generally accepted FBDD guidelines, the library was designed to maximise scaffold diversity and to conform to the 'rule of 3': 23 MW < 300, clogP £ 3, H-bond donors/acceptors £ 3 ( Figure 1C ). We applied our fluorescence-based covalent fragment screening platform (qIT) to identify fragments which covalently bind to C147 on EV71 3C pro (Figure 2A ). To determine the rate of reaction between a cysteine thiol and an acrylamide fragment, the cysteine quantification probe CPM is employed to measure the degree of cysteine modification at a series of timepoints. The CPM probe competes with the acrylamide for modification of the cysteine residue such that the fluorescence signal is inversely proportional to the extent of acrylamide-cysteine labelling, allowing the rate of reaction (v) to be determined by exponential regression analysis in high-throughput. Our workflow uses GSH as a control cysteine-containing biomolecule and hit fragments are those that react significantly faster with 3C pro than with GSH ( Figure 2B ). The selectivity of the fragment towards the 3C pro is quantified by the rate enhancement factor (REF) which was used to identify and prioritise hit fragments ( Figure 2C ). The target thiol (5 μM), EV71 3C pro or glutathione, is reacted with acrylamide fragments (0.5 mM) under pseudo-first order conditions. Reaction progress is followed by discrete measurements of free target thiol concentration using the fluorogenic probe CPM and the rate of reaction (v) are derived from exponential regression analysis. (B) Fluorescence intensity is converted into percentage cysteine modification by normalizing to DMSO control = 0%, no thiol = 100%. Fragments are characterized as (i) non-reactive, (ii) reactive but non-selective or (iii) reactive and selective by comparing the reactivity profiles between EV71 3C pro and GSH. (C) Kinetic selectivity is quantified by the rate enhancement factor (REF) which is used to identify and prioritise hit compounds. The 1040-member acrylamide fragment library was screened at 500 µM against EV71 3C pro (5 µM) or GSH (5 µM) in parallel and the fluorescence intensity measured over 24 hours. The majority of the library (61%) displayed measurable reactivity with EV71 3C pro over the 24-hour time course, with roughly half of those fragments showing selectivity over GSH (REF > 1) ( Figure 3A ). There were 13 fragments which had a REF greater than three standard deviations (1SD = 2.5) over the geometric mean (geomean REF = 1.0), for example acrylamide 1, and these were taken forward for repeat qIT testing and mass spectrometry validation ( Figure S1 ). Pleasingly, four of those fragments both had reproducible qIT profiles and clearly mono-modified EV71 3C pro by intact protein mass spectrometry ( Figures 3B, 3C and S2). The glutathione selectivity of the four validated hit fragments ranged from REF = 8.5-24.3 and the compounds shared some common chemical features: Acrylamides 1 and 2 both contain the same 1,3-thiazole core and methylene linker while acrylamides 3 and 4 have similar isoxazole motifs with more extended linkers. Encouragingly, all 4 hits are alkyl acrylamides which typically are associated with low levels of off-target reactivity and this is supported by their slow reactivity with glutathione. 24 The hit fragments covalently bind to residue C147 of EV71 3C pro and accommodate a novel cryptic pocket.

To reveal the binding site of the cysteine-reactive fragments, we labelled recombinant EV71 3C pro with acrylamide fragments 1-4 and subjected the resulting complexes to crystallization trials. Unfortunately, the WT EV71 3C profragment complexes did not yield crystals so we employed a 3C pro mutant construct bearing the H133G to expedite the structural studies. The H133G mutant has WT-level protease activity, containing a WT-like catalytic triad, and harbors the H133G mutation at the hinge region of the β-ribbon, which improves the flexibility of the β-ribbon. 25 Using the H133G mutant, we were able to determine crystal structures of 3C pro -1 and 3C pro -2 complexes; however, the structure determination of the other complexes remained unsuccessful.

Crystal structures of 3C pro -1 and 3C pro -2 complexes were solved to the resolution of 1.2-1.3 Å respectively, which provided atomic details of the 3C pro -fragment interactions. We found that both fragments 1 and 2 bind to the same pocket on 3C pro , with the acrylamide functionalities forming covalent bonds to C147 (bond length C(acrylamide)-S(C147)= 1.8 Å). Unexpectedly, neither of the fragments occupy the central substrate pocket (S1-S4) of 3C pro , as is observed for AG7088 and other characterized 3C pro inhibitors. 9 Instead, the fragments accommodate a cryptic pocket on the other side of the catalytic cysteine, denoted the S' pocket. To obtain bias-free structural insight, we calculated the composite omit maps for fragments 1 and 2, which clearly show . Comparing the binding of fragments 1 and 2, we find that the trifluoromethyl and cyclohexane functionalities make less contact with protein and are associated with weaker electron density, hinting that the thiazole ring is the key pharmacophore. Conformational rearrangement of the active site induced by covalent fragment binding.

To our knowledge, the cryptic S' pocket identified in our 3C pro -fragment complexes has not been observed in the APO 3C pro structures or other 3C pro -inhibitor complex structures in the public database. By superimposing our structures with an EV71 3C pro -AG7088 complex (PDB ID; 3R0F), we observed a large conformational rearrangement of a catalytically important loop 141-147 aa. This loop harbors the catalytically critical residue C147 and constitutes the upper wall of the S1 pocket. While the 141-147 aa loop remains flexible in the absence of ligand, the binding of substrate (or inhibitor) can hold this loop in the catalytically active conformation, allowing residues G145 and C147 to form the oxyanion hole for the binding of tetrahedral intermediate anion. pro -1 complex. AG7088 is modeled to substrate binding pockets of 3C pro via structure superimposition. Due to 1 binding induced conformational changes, S1 pocket collapsed and it cannot accommodate the P1 residue. Substrate pockets S1-S5 are highlighted in yellow, the cryptic pocket on leaving group side S' is highlighted in cyan. (D) Molecular surface of 3C pro -AG7088 complex. AG7088 occupies substrate pockets S1-S5, highlighted in yellow; the residues forming the cryptic pocket on leaving group side S' are highlighted in cyan.

The unusual conformation of the 141-147 aa loop in 3C pro -1 and 3C pro -2 structures led to a series of conformational rearrangements: (1) Residue C147 side chain tilted towards the leaving group side of the active site. Comparing to EV71 3C pro -AG7088 complex, displacement of the nucleophilic Sγ atom in our structures was 6.4 Å, indicating the geometry of the Ser-His-Asp catalytic triad was disrupted. Displacement of the NH group of G145 was 4.6 Å and displacement of NH of C147 was 1.8 Å, indicating the oxyanion hole could not form. (2) The upper wall of the S1 pocket (the most important pocket for substrate recognition) collapsed, and the size of the pocket became too narrow to accommodate the P1 residue. (3) The leaving group side pockets S1' and S2' disappeared, and a previously unobserved cryptic S' pocket was generated. Residues constituting the cryptic S' pocket involve I104, T106, H108, M019, M112, V114, F140, T142, A144, G145 and Q146.

The hit acrylamide fragments inhibit EV71 3C pro and SARS-CoV-2 M pro activity in vitro.

To investigate which structural features of the thiazoleacrylamide fragments are key to binding, we tested analogues 5, 6 and 7 for their kinetic binding profiles against 3C pro using qIT (Table 1) . Benzothiazole 5 retained potency (REF = 13.5) with a similar kinetic profile to thiazole 1, further indicating that the thiazole motif drives the binding. Conversely, the N-H acrylamides 6 and 7 reacted with 3C pro >50 times more slowly than the parent acrylamide 1, indicating that alkyl functionalization of the amide nitrogen is required for efficient binding. Indeed, tolerance of the cyclopropane ring of acrylamide 5 indicates that larger substituents may be introduced here and based on the crystal structures of fragments 1 and 2, this represents a suitable vector for fragment growth towards the canonical binding groove. Table 1 . Biochemical characterization of acrylamide fragments.

Next we employed a fluorogenic peptide-based 3C pro protease activity assay to assess biochemical potency of the acrylamides. In accordance with the qIT data, acrylamides 1-5 all demonstrated concentration dependent inhibition of 3C pro with modest potency (IC50 = 30-230 µM) that is typical of unoptimized fragments.

With the emergence of SARS-CoV-2 as a threat to global public health, we sought to determine if our 3C pro -selective fragments could be re-purposed towards this second plus strand RNA virus. Given that 3C pro and M pro are both cysteine proteases that share similar chymotrypsin-folds, we hypothesised that our acrylamide fragments might also be effective against M pro . The first examples of irreversible SARS-CoV-2 M pro inhibitors have already emerged, 19, 26 , 27 but novel acrylamide-based M pro inhibitor scaffolds remain highly desirable. Accordingly, we incubated each fragment with SARS-CoV-2 M pro and used intact protein mass spectrometry to check for covalent modification (Table 1 and Figure S3 ). Encouragingly, acrylamides 1, 3 and 4 showed partial modification while fragments 2 and 5 both labelled M pro to completion. Using an M pro activity assay, we validated these results and found that the inhibitory potency against M pro is overall greater than 3C pro (IC50 = 10-60 µM), with acrylamides 2 and 5 being the most potent against either proteases. Although other covalent fragment inhibitors of M pro have recently been disclosed, 19 to our knowledge, our fragments represent the first examples of acrylamide-based fragment inhibitors of M pro . Acrylamidebased electrophiles offer low pharmacological risk as indicated by their widespread clinical use, emphasizing the development potential of fragments 2 and 5.

Mechanism of the fragment efficacy against M pro .

To reveal the inhibitory mechanism of these fragments against M pro , we determined the crystal structure of M pro with 2 and with 5 ( Figure 6 ). The crystals of M pro -5 complex diffracted to 2.3Å, had a P212121 space group and contained one M pro dimer in the asymmetric unit (ASU). Structural comparison of two monomers in ASU gave an r.m.s.d. of 0.73Å. The crystals of the M pro -2 complex diffracted to 1.8Å, had a C2 space group and contained a single M pro molecule in the ASU. It formed a typical M pro dimer with the symmetry mate (-x, y, -z), suggesting two M pro protomers have identical conformation.

We identified electron density of compound 5 connecting to the active site cysteine C145 in both M pro monomers in ASU. For the M pro -2 complex, we observed electron density of 2 connecting to the active site residue C145. We generated the polder maps for the above structures with the compound 2 or 5 omitted ( Figure S4 ). Positive densities clearly delineated the structure of compounds 2 and 5, confirming the presence of the fragments.

In the active site, the acrylamide moiety of 5 forms a covalent bond with C145 ( Figure 6 A&B) . While the R' group (benzothiazole) of 5 is accommodated in the deep S2 pocket of M pro , the cyclopropane group is exposed to solvent. The benzothiazole/S2 pocket interaction is mainly hydrophobic, involving residues H41, M49, Q189 and M165. The stacking of the H41 imidazole side chain with the benzothiazole moiety stabilizes the fragment. Similarly, the acrylamide moiety of 2 is covalently linked to residue C145 (Figure 6 C&D) . Owing to the high resolution of M pro -2 structure and unambiguous electron density for the fragment, we were able to build the fragment more accurately. We measured the length of the S-C bond between C145 and compound 2 to be 1.8 Å, very close to the average length of single S-C bond, 1.82Å. 28 The trifluoromethyl thiazole moiety of compound 2 is also accommodated by the S2 pocket. While the trifluoromethyl group touches the apex of the pocket and the thiazole ring π-stacks with the side chain of H41. Both 2 and 5 occupy only the S1' and S2 subsites, implying substantial opportunity to develop these fragments.

While picornavirus 3C pro functions as a monomer, coronavirus M pro is an obligate dimer. An additional C-terminal domain in M pro stabilizes dimerization and the dimerization interface is essential to maintain the active conformation. We next investigated the oligomerization state of the five M pro -fragment complexes using size-exclusion chromatography ( Figure S5 ). As expected, M pro -2, M pro -3 and M pro -4 eluted as dimers with calculated molecular masses of 45.7kDa, 45.7 kDa and 47.9 kDa, respectively. Interestingly, however, M pro -5 eluted as a monomer. The calculated molecular mass of M pro -5 is 25.3 kDa, whereas the theoretical molecular mass of M pro monomer is 33.7 kDa. The retention volume of M pro -1 lies between the monomeric and dimeric forms, with a calculated molecular mass of 37.6 kDa. This suggests the dimerization was partially impaired. To further validate the effects of 5 on M pro dimerization, we tested the oligomerization state of two M pro mutants, C145A and C156W in the absence and presence of ligand. As well as the active site cysteine (C145), C156 is also surface exposed and potentially reactive so the C156W mutant served as a control mutation. Indeed, mutant C156W behaved similarly to the wild-type enzyme: apo-M pro C156W eluted as dimers in size-exclusion chromatography, whilst labelling with 5 retarded M pro elution to that expected for a monomer ( Figure 7A ). By contrast, mutant C145A remained dimeric irrespective of the presence of 5. Given mutation C145A prevents the labelling of the active site cysteine, these results clearly indicate that the labelling of C145 by acrylamide 5 is solely responsible for dimer disruption.

Our crystallographic data provides further insights into the inhibition mechanism. Although M pro -5 forms dimers in crystal lattices, these are notably different from authentic M pro dimers: (1) Most published M pro dimers have 2-fold symmetry between two protomers, but M pro -5 protomers exhibit marked difference, r.m.s.d =0.73Å. The structure of each M pro -5 protomer is also notably different from the free enzyme (PDB id: 6Y2E), r.m.s.d= 0.71-0.78 Å. In this regard, M pro -2 is more similar to the free enzyme. The structure of M pro -2 dimer has 2-fold symmetry and each protomer is highly similar to the free enzyme, r.m.s.d =0.22 Å.

(2) The binding of 5 enlarged the substrate binding pockets and affected the nearby regions ( Figure 7B ). Comparing to the free enzyme, the loops surrounding 5 expanded to make room for benzothiazole motif. This induced conformational changes of several residues and regions at the dimerization interface. In the chain A of M pro -5 dimer, the extreme C-terminal region at the dimer interface went disordered, which was likely caused by the labelling of compound 5 on C145. The fragment induced conformational alterations may contribute to the destabilization of M pro -5 dimers. In summary, we found that 5 has at least two mechanisms of action to inhibit M pro : (1) covalently linking to the catalytically important cysteine and occupying the substrate binding pockets; (2) destabilizing the dimerization of M pro . In conclusion, we have identified acrylamide fragments that target both the EV71 3C pro and SARS-CoV-2 M pro , and inhibit their activity by covalently reacting with their catalytic cysteines. Importantly, some of these hit fragments cause profound structural rearrangements of each protease: in the case of EV71 3C pro a new subsite pocket is formed at the expense of the normal active site architecture whilst with M pro key structural features required for dimerization are distorted preventing formation of the active dimeric unit. The discovery of these conformational change-based mechanisms of action on covalent fragment binding demonstrates the utility of solution-based screening methodologies as an alternative to crystallographic fragment screening in which structural rearrangements are unlikely. These hit ligands also provide excellent candidates for development of potent protease inhibitors.

The Supporting Information is available free of charge on the ACS Publications website.

Experimental details, supporting Figures S1-5 and Table S1 (PDF)

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The manuscript was written through contributions of all authors. # These authors contributed equally.