key: cord-346546-yffwd0dc
authors: Douangamath, Alice; Fearon, Daren; Gehrtz, Paul; Krojer, Tobias; Lukacik, Petra; Owen, C. David; Resnick, Efrat; Strain-Damerell, Claire; Aimon, Anthony; Ábrányi-Balogh, Péter; Brandaõ-Neto, José; Carbery, Anna; Davison, Gemma; Dias, Alexandre; Downes, Thomas D; Dunnett, Louise; Fairhead, Michael; Firth, James D.; Jones, S. Paul; Keely, Aaron; Keserü, György M.; Klein, Hanna F; Martin, Mathew P.; Noble, Martin E. M.; O’Brien, Peter; Powell, Ailsa; Reddi, Rambabu; Skyner, Rachael; Snee, Matthew; Waring, Michael J.; Wild, Conor; London, Nir; von Delft, Frank; Walsh, Martin A.
title: Crystallographic and electrophilic fragment screening of the SARS-CoV-2 main protease
date: 2020-05-27
journal: bioRxiv
DOI: 10.1101/2020.05.27.118117
sha: 
doc_id: 346546
cord_uid: yffwd0dc

COVID-19, caused by SARS-CoV-2, lacks effective therapeutics. Additionally, no antiviral drugs or vaccines were developed against the closely related coronavirus, SARS-CoV-1 or MERS-CoV, despite previous zoonotic outbreaks. To identify starting points for such therapeutics, we performed a large-scale screen of electrophile and non-covalent fragments through a combined mass spectrometry and X-ray approach against the SARS-CoV-2 main protease, one of two cysteine viral proteases essential for viral replication. Our crystallographic screen identified 71 hits that span the entire active site, as well as 3 hits at the dimer interface. These structures reveal routes to rapidly develop more potent inhibitors through merging of covalent and non-covalent fragment hits; one series of low-reactivity, tractable covalent fragments was progressed to discover improved binders. These combined hits offer unprecedented structural and reactivity information for on-going structure-based drug design against SARS-CoV-2 main protease.

through ribosome frame-shifting, generates two polyproteins pp1a and pp1ab (Bredenbeek 33 et al., 1990) . These polyproteins produce most of the proteins of the replicase-transcriptase 34 complex (Thiel et al., 2003) . The polyproteins are processed by two viral cysteine proteases: 35 a Papain-like protease (PL pro ) which cleaves three sites, releasing non-structural proteins 36 nsp1-3 and a 3C-like protease, also referred to as the main protease (M pro ), that cleaves at 11 37 sites to release non-structural proteins (nsp4-16). These non-structural proteins form the 38 replicase complex responsible for replication and transcription of the viral genome and have 39 led to M pro and PL Pro being the primary targets for antiviral drug development (Hilgenfeld, 40 2014) . 41

Structural studies have played a key role in drug development and were quickly applied 42 during the first coronavirus outbreak. Early work by the Hilgenfeld group facilitated targeting 43 the M pro of coronarviruses (Hilgenfeld, 2014) , spectrum antivirals. The most successful have been peptidomimetic α-ketoamide inhibitors 50 (Zhang et al., 2020a) , which have been used to derive a potent α-ketoamide inhibitor that 51 may lead to a successful antiviral (Zhang et al., 2020b) . 52

To date, no drugs targeting SARS-CoV-2 have been verified by clinical trials and treatments 53 are limited to those targeting disease symptoms. To contribute to future therapeutic 54 possibilities, we approached the SARS-CoV-2 M pro as a target for high throughput drug 55 discovery using a fragment-based approach (Thomas et al., 2019) . We screened against over 56 1250 unique fragments leading to the identification of 74 high value fragment hits, including 57 23 non-covalent and 48 covalent hits in the active site, and 3 hits at the vital dimerization 58

interface. Here, these data are detailed along with potential ways forward for rapid follow-59 up design of improved, more potent, compounds. 60 61

We report the apo structure of SARS-CoV-2 M pro with data to 1.25 Å. The construct we 64 crystallised has native residues at both N-and C--terminals, without cloning truncations or 65 appendages which could otherwise interfere with fragment binding. Electron density is 66 present for all residues, including 26 alternate conformations, many of which were absent in 67 previous lower resolution crystal structures. The protein crystallised with a single protein 68 polypeptide in the asymmetric unit, and the catalytic dimer provided by a symmetry-related 69 molecule. The structure aligns closely with the M pro structures from SARS-CoV-1 and MERS 70 (rmsd of 0.52 Å and 0.97 Å respectively). The active site is sandwiched between two β-barrel 71 domains, I (residue 10-99) and II (residue 100-182) ( Figure 1A ). Domain III (residue 198-306), 72 forms a bundle of alpha helices and is proposed to regulate dimerization (Shi and Song, 2006) . 73

The C-terminal residues, Cys300-Gln306, wrap against Domain II. However, the C terminal 74 displays a degree of flexibility and wraps around domain III in the N3 inhibitor complex (Shi 75 and Song, 2006) (PDB ID 6LU7). His41 and Cys145 comprise the catalytic dyad and 76 dimerisation completes the active site by bringing Ser1 of the second dimer protomer into 77 proximity with Glu166 ( Figure 1B ). This aids formation of the substrate specificity pocket and 78 the oxyanion hole (Hilgenfeld, 2014 between hits. Screening at more stringent conditions (5 µM per electrophile; 1.5 hours; 25°C) 108 resulted in 8.5% of the library labelling above 30% of protein (Table S1a) . These hits revealed 109 common motifs, and we focused on compounds which offer promising starting points. 110

Compounds containing N-chloroacetyl-N'-sulfonamido-piperazine or N--chloroacetylaniline 111 motifs were frequent hitters. Such compounds can be highly reactive. Therefore, we chose 112 series members with relatively low reactivity for follow up crystallization attempts. For 113 another series of hit compounds, containing a N-chloroacetyl piperidinyl-4-carboxamide 114 motif (Table S2 ) which displays lower reactivity and were not frequent hitters in previous 115 screens, we attempted crystallization despite their absence of labelling in the stringent 116

conditions. 117

While mild electrophilic fragments are ideal for probing the binding properties around the 118 active site cysteine, their small size prevents extensive exploration of the substrate binding 119 pocket. We performed an additional crystallographic fragment screen to exhaustively probe 120 the M pro active site, and to find opportunities for fragment merging or growing. The 68 121 electrophile fragment hits were added to crystals along with a total of 1176 unique fragments 122 from 7 libraries (Table S3) . Non-covalent fragments were soaked (Collins et al., 2017) , 123 whereas electrophile fragments were both soaked and co-crystallized as previously described 124 (Resnick et al., 2019) , to ensure that as many of the mass spectrometry hits as possible were 125 structurally observed. A total of 1742 soaking and 1139 co-crystallization experiments 126 resulted in 1877 mounted crystals. While some fragments either destroyed the crystals or 127 their diffraction, 1638 datasets with a resolution better than 2.8 Å were collected. The best 128 crystals diffracted to better than 1.4 Å, but diffraction to 1.8 Å was more typical, and no 129 datasets worse than 2.8 Å were included in analysis ( Figure S2 ). We identified 96 fragment 130 hits using the PanDDA method (Pearce et al., 2017) , all of which were deposited in the Protein 131

Data Bank (Table S2) Active-site fragments 141

Eight fragments were identified that bind in the S1 subsite and frequently form interactions 142

with the side chains of the key residues His163, through a pyridine ring or similar nitrogen 143 containing heterocycle, and Glu166 through a carbonyl group in an amide or urea moiety 144 ( Figure 3 ). Several also reach across into the S2 subsite. 145 this location, which we termed the "aromatic wheel" because of a consistent motif of an 157 aromatic ring forming hydrophobic interactions with Met49 or π-π stacking with His41, with 158 groups variously placed in 4 axial directions. Particularly notable is the vector into the small 159 pocket between His164, Met165 and Asp187, exploited by three of the fragments 160 (Z1220452176 (x0104), Z219104216 (x0305) and Z509756472 (x1249)) with fluoro and cyano 161 substituents ( Figure 3 ). 162

Of the four fragments exploring subsite S3, three contain an aromatic ring with a 163 sulfonamide group forming hydrogen bonds with Gln189 and pointing out of the active site 164 towards the solvent interface ( Figure 3 ). These hits have expansion vectors suitable for 165 exploiting the same His164/Met165/Asp187 pocket mentioned above. 166

The experiment revealed one notable conformational variation, which was exploited by one 167 fragment only (Z369936976 (x0397); Figure 4 ): a change in the sidechains of the key catalytic 168

residues His41, Cys145 alters the size and shape of subsite S1ʹ and thus the link to subsite S1. 169

This allows the fragment to bind, uniquely, to both S1 and S1ʹ. In S1, the isoxazole nitrogen 170

hydrogen-bonds to His163, an interaction that features in several other hits; and in S1ʹ, the 171 cyclopropyl group occupies the region sampled by the covalent fragments. Notably, the N-172 methyl group offers a vector to access the S2 and S3 subsites. 173 174 Thus, compounds that interfere with dimerization might serve as quasi-allosteric inhibitors of 184 protease activity. In this study three compounds bound at the dimer interface. 185 Fragment Z1849009686 (x1086; Figure 5A ) binds in a hydrophobic pocket formed by the 186 sidechains of Met6, Phe8, Arg298 and Val303. It also mediates two hydrogen bonds to the 187 sidechain of Gln127 and the backbone of Met6. Its binding site is less than 7 Å away from 188

Ser139, whose mutation to alanine in SARS-CoV-1 protease reduced both dimerization and 189

protease activity by about 50% ( Ser123. Finally, POB0073 (x0887; York 3D library; Figure 5C ), binds only 4 Å from Gly2 at the 194 dimer interface and is encased between Lys137 and Val171 of one protomer and Gly2, Arg4, 195 Phe3, Lys5 and Leu282 of the second, including two hydrogen bonds with the backbone of 196

Phe3. 197 heterobenzyl-piperazine motif crystallized in one binding mode with respect to the 232 piperazinyl moiety ( Figure 6C ) (with one exception, PCM-0102287 (x0830)). Two structures 233

(PCM-0102277 (x1334), PCM-0102169 (x1385)) with a 5-halothiophen-2-ylmethylene moiety 234 exploit lipophilic parts of S2, which is also recapitulated by the thiophenyl moiety in an 235

analogous carboxamide (PCM-0102306 (x1412)). The other five structures point mainly to S2, 236

offering an accessible growth vector towards the nearby S3 pocket. 237 A series of compounds containing a N-chloroacetyl piperidinyl-4-carboxamide motif showed 238 promising binding modes. To follow up on these compounds we performed a rapid second-239 generation compound synthesis. Derivatives of this chemotype were accessible in mg-scale 240 by reaction of N-chloroacetyl piperidine-4-carbonyl chloride with various in-house amines, 241

preferably carrying a chromophore to ease purification. These new compounds were tested 242 by intact protein mass-spectrometry to assess protein labelling (5 uM compound; 1.5h 243 incubation, RT; Table S1b ). Amides derived from non-polar amines mostly outcompeted their 244 polar counterparts, hinting at a targetable lipophilic sub-region in this direction. The two 245

amides with the highest labelling PG-COV-35 and PG-COV-34 (figure 6G,H) highlight the 246 potential for further synthetic derivatization by amide N-alkylation or cross-coupling, 247

respectively. 248

The screen revealed unexpected covalent warheads from the series of 3-bromoprop-2-yn- Two PepLites, containing threonine (NCL-00025058 (x0978)) and asparagine (NCL00025412 257 (x0981)) bound covalently to the active site cysteine (Cys145), forming a thioenolether via C-258 2 addition with loss of bromine ( Figure 6E ,F). The covalent linkage was unexpected and 259 evidently the result of significant non-covalent interactions, specific to these two PepLites, 260 that position the electrophile group for nucleophilic attack. We note the side-chains make 261

hydrogen-bonding interactions with various backbone NH and O atoms of Thr26 and Thr24; 262

in the case of threonine, it was the minor 2R,3R diastereomer (corresponding to D-263 allothreonine) that bound. The only other PepLite observed (tyrosine, NCL-00024905 264 (x0967)) bound non-covalently to a different subsite. 265

The highlighted structure activity relationships is important for further optimisation. 266

Bromoalkynes have intrinsic thiol reactivity that is lower than that of established acrylamide- proteins. 284

The data presented herein provides many clear routes to developing potent inhibitors 286

against SARS-CoV-2. The bound fragments comprehensively sample all subsites of the active 287 site revealing diverse expansion vectors, and the electrophiles provide extensive, systematic 288 as well as serendipitous, data for designing covalent compounds. 289

It is widely accepted that new small molecule drugs cannot be developed fast enough to 290 help against COVID-19. Nevertheless, as the pandemic threatens to remain a long-term 291 problem and vaccine candidates do not promise complete and lasting protection, antiviral 292 molecules will remain an important line of defence. Such compounds will also be needed to 293

fight future pandemics (Hilgenfeld, 2014) . Our data will accelerate such efforts: 294 therapeutically, through design of new molecules and to inform ongoing efforts at 295 repurposing existing drugs; and for research, through development of probe molecules 296 (Arrowsmith et al., 2015) to understand viral biology. One example is the observation that 297 fragment Z1220452176 (x0104) is a close analogue of melatonin, although in this case, it is 298 unlikely that melatonin mediates direct antiviral activity through inhibition of M pro , given its 299 low molecular weight; nevertheless, melatonin is currently in clinical trials to assess its 300 immune-regulatory effects on COVID19 (Clinicaltrials.gov identifier NCT04353128). 301

In line with the urgency, results were made available online immediately for download. 302

Additionally, since exploring 3D data requires specialised tools ( figure 7 . These 308

can be expected to result in potent M pro binders and compound synthesis is ongoing. 309 

Collectively, the covalent hits provide rational routes to inhibitors of low reactivity and high 316 selectivity. Rationally designed covalent drugs are gaining traction, with many recent FDA 317 approvals (Singh et al., 2011 , Bauer, 2015 . Their design is based on very potent reversible 318 binding, that allows precise orientation of a low reactivity electrophile, so that formation of 319 the covalent bond is reliant on binding site specificity, with minimal off-targets. ( protease and other impurities were removed from the cleaved target protein by reverse 363

Nickel-NTA. The relevant fractions were concentrated and applied to an S200 16/60 gel 364 filtration column equilibrated in 20 mM Hepes pH 7.5, 50 mM NaCl buffer. The protein was 365 concentrated to 30 mg/ml using a 10 kDa MWCO centrifugal filter device. 366

Crystallisation and structure determination: Protein was thawed and diluted to 5 mg/ml 367 using 20 mM Hepes pH 7.5, 50 mM NaCl. The sample was centrifuged at 100 000 g for 15 368

minutes. Initial hits were found in well F2 of the Proplex crystallisation screen, 0.2 M LiCl, 369 0.1M Tris pH 8, 20% PEG 8K. These crystals were used to prepare a seed stock by crushing the 370 proteins with a pipette tip, suspending in reservoir solution and vortexing for 60 s in the 371 reservoir solution with approximately 10 glass beads (1.0mm diameter, BioSpec products). 372

Adding DMSO to the protein solution to a concentration of 5% and performing microseed 373 matrix screening, many new crystallisation hits were discovered in commercial crystallisation 374 screens. Following optimisation, the final crystallisation condition was 11% PEG 4K, 6% DMSO, 375 0.1M MES pH 6.7 with a seed stock dilution of 1/640. The seeds were prepared from crystals 376 grown in the final crystallisation condition. The drop ratios were 0.15 µl protein, 0.3 µl 377 reservoir solution, 0.05 µl seed stock. Crystals were grown using the sitting drop vapor 378 diffusion method at 20 °C and appeared within 24 hours. 379

Initial diffraction data was collected on beamline I04 at Diamond Light Source on a crystal 380 grown in 0.1 M MES pH 6.5, 5% PEG6K, cryoprotected using 30% PEG400. Data were 381 processed using Dials ( Light Source on crystals grown using the 0.1 M MES pH 6.5, 15% PEG4K, 5% DMSO condition. 385

To create a high-resolution dataset, datasets from 7 crystals were scaled and merged using 386

Aimless (Evans and Murshudov, 2013) . Waters ACUITY UPLC class H instrument, in positive ion mode using electrospray ionization. 395 UPLC separation used a C4 column (300 Å, 1.7 μm, 21 mm × 100 mm). The column was held 396

at 40 °C and the autosampler at 10 °C. Mobile solution A was 0.1% formic acid in water, and 397 mobile phase B was 0.1% formic acid in acetonitrile. The run flow was 0.4 mL/min with 398 gradient 20% B for 4 min, increasing linearly to 60% B for 2 min, holding at 60% B for 0.5 min, 399 changing to 0% B in 0.5 min, and holding at 0% for 1 min. The mass data were collected on a 400

Waters SQD2 detector with an m/z range of 2−3071.98 at a range of 1000−2000 m/z. Raw 401 data were processed using openLYNX and deconvoluted using MaxEnt. Labelling assignment 402 was performed as previously described (Resnick et al., 2019) . 403 Fragment Screening: Fragments were soaked into crystals as previously described (Collins 404 et al. Libraries.html). 414 Electrophile fragments identified by mass spectrometry were soaked by the same 415 procedure as the other libraries, but in addition, they were also co-crystallised in the same 416 crystallisation condition as for the apo structure. The protein was incubated with 10 to 20-417 fold excess compound (molar ratio) for approximately 1h prior to the addition of the seeds 418 and reservoir solution (following Resnick et al (Resnick et al., 2019) ). 419

Data were collected at the beamline I04-1 at 100K and processed to a resolution of 420 approximately 1.8 Å using XDS (Kabsch, 2010) The corresponding amides were prepared by addition of the acid chloride (1 equiv.) as a 449 DCM solution to the pertinent amines (1 equiv.) in presence of pyridine (1 equiv.) in DCM. 450

Heterogeneous reaction mixtures were treated with a minimal amount of dry DMF to achieve 451 full solubility. After stirring the reaction mixtures overnight, the solvents were removed in by 452 rotary evaporation, re-dissolved in 50% aq. MeCN (and a minimal amount of DMSO to achieve 453 higher solubility), followed by purification by (semi-)preparative RP-HPLC in mass-directed 454 automatic mode or manually. 455 Synthesis of PepLites HATU (1.5 eq.), DIPEA (3.0 eq.) and the acid starting material (1.5 eq.) 456 were dissolved in DMF (3 -6 mL) and stirred together at room temperature for 10 min. 3-457

Bromoprop-2-yn-1-amine hydrochloride was added and the reaction mixture was stirred at 458 40 °C overnight. The reaction mixture was allowed to cool to room temperature, diluted with 459

EtOAc or DCM and washed with saturated aqueous sodium bicarbonate solution, brine and 460

water. The organic layer was dried over MgSO4, filtered and evaporated to afford crude 461 product. The crude product was then purified by either normal or reverse phase 462 chromatography. 463 (3-bromoprop-2-yn-1-yl) N-(3-bromoprop-2-yn-1-yl)-3-(tert-butoxy) butanamide 485 (2S,3S)-2-Acetamido-N-(3-bromoprop-2-yn-1-yl)-3-(tert-butoxy)butanamide The reaction mixture was allowed to warm to room temperature and was stirred at room 503 temperature for 3 h then evaporated to dryness to afford crude product. The crude product 504 was purified by flash silica chromatography, elution gradient 0 -15% MeOH in DCM. Pure 505 fractions were evaporated to dryness to afford (2S,3S)-2-acetamido-N-(3-bromoprop-2-yn- (S)-2-Acetamido-N 1 -(3-bromoprop-2-yn-1-yl)succinimide (asparagine PepLite) 513 514 (S)-2-Acetamido-N 1 -(3-bromoprop-2-yn-1-yl)succinamide was synthesized according to 515

General procedure A using (s)-2-acetamido-5-amino-5-oxobutanoic acid (155 mg, 0.89 mmol) 516

and evaporating the reaction mixture to afford the crude product without aqueous work-up. 517

The crude product was purified by flash silica chromatography, elution gradients 0 -10% 518

MeOH in DCM. Pure fractions were evaporated to dryness to afford (S)-2-acetamido-N 1 - (3-519 bromoprop-2-yn-1-yl)succinamide (50 mg, 30%) as a white solid. 

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