key: cord-0306512-pm9utuxk
authors: Johansen-Leete, Jason; Ullrich, Sven; Fry, Sarah E.; Frkic, Rebecca; Bedding, Max J.; Aggarwal, Anupriya; Ashhurst, Anneliese S.; Ekanayake, Kasuni B.; Mahawaththa, Mithun C.; Sasi, Vishnu M.; Passioura, Toby; Larance, Mark; Otting, Gottfried; Turville, Stuart; Jackson, Colin J.; Nitsche, Christoph; Payne, Richard J.
title: Discovery of Antiviral Cyclic Peptides Targeting the Main Protease of SARS-CoV-2 via mRNA Display
date: 2021-08-24
journal: bioRxiv
DOI: 10.1101/2021.08.23.457419
sha: 712df5a6f3ebc959744ff64dcce7aea0c9d7ea0a
doc_id: 306512
cord_uid: pm9utuxk

Antivirals that specifically target SARS-CoV-2 are needed to control the COVID-19 pandemic. The main protease (Mpro) is essential for SARS-CoV-2 replication and is an attractive target for antiviral development. Here we report the use of the Random nonstandard Peptide Integrated Discovery (RaPID) mRNA display on a chemically cross-linked SARS-CoV-2 Mpro dimer, which yielded several high-affinity thioether-linked cyclic peptide inhibitors of the protease. Structural analysis of Mpro complexed with a selenoether analogue of the highest-affinity peptide revealed key binding interactions, including glutamine and leucine residues in sites S1 and S2, respectively, and a binding epitope straddling both protein chains in the physiological dimer. Several of these Mpro peptide inhibitors possessed antiviral activity against SARS-CoV-2 in vitro with EC50 values in the low micromolar range. These cyclic peptides serve as a foundation for the development of much needed antivirals that specifically target SARS-CoV-2.

The COVID-19 pandemic, caused by infection with severe acute respiratory syndrome 38 coronavirus 2 (SARS-CoV-2) has caused widespread morbidity and mortality as well across the world. Whilst vaccines will enable protective immunity in most, there will be 46 populations where vaccine-based immunity may fail, and these individuals will be 47 vulnerable to SARS-CoV-2 infection in the future. It is furthermore unclear what 48 changes will appear in the virus in contemporary SARS-CoV-2 viral variants 49 (highlighted by the recent emergence of the delta variant B.1.617.2) 3 , and how those 50 variants will navigate both convalescent and vaccine immune responses. Given that 51 this is the third coronavirus that has crossed via zoonoses, antiviral development 52 against SARS-CoV-2, and future coronaviruses with pandemic potential, are 53 desperately needed in addition to prophylactic vaccines. 54 55 While there have been significant efforts toward the discovery of effective antivirals for 56 SARS-CoV-2, the vast majority of molecules that have completed (or are currently 57 I interferon treatments 9,10 , and the antimalarial hydroxychloroquine 11-13 ; however, 66 these have not demonstrated improvement in disease progression over standard care. 67

In fact, it has recently been suggested that many repurposing efforts may be 68 compromised by experimental artefacts reflecting the physicochemical properties of 69 certain drugs rather than specific target-based activities 14 . To date, the most effective 70 therapeutic intervention for improving COVID-19 patient outcomes in a hospital setting 71 is the use of the corticosteroid dexamethasone, which reduces inflammation-mediated 72 lung injury associated with SARS-CoV-2 infection in patients with elevated levels of C-73 reactive protein 15 16 . Based on the above, there is an urgent need for the discovery of 74 effective antivirals for COVID-19, ideally with mechanisms of action that specifically 75 target proteins critical in the SARS-CoV-2 lifecycle. 76 77

receptor binding domain of the trimeric viral spike protein (S) with the host cell-surface 79 receptor angiotensin converting enzyme 2 (ACE2) (Figure 1 ). Following receptor 80 binding of the virus, the spike protein is activated by cleavage between the S1 and S2 81 domains leading to host cell entry via two distinct pathways: 1) an endocytic pathway 82 through endosomal-lysosomal compartments with spike cleavage facilitated by 83 lysosomal cathepsins, or 2) a cell surface pathway following activation by a serine 84 protease such as TMPRSS2 [17] [18] [19] . Following proteolysis, the N-terminus of the cleaved 85 S2 domain is embedded into the cell membrane and leads to fusion of the membranes 86 of the virus and the host cell, followed by transfer of viral RNA into the cytoplasm 20 . polymerase (RdRp, nsp12) and helicase (nsp13) 20 . These proteins become functional 99 only after proteolytic release by two viral proteases. The first of these is a domain of 100 nsp3 called the papain-like protease (PL pro ) which cleaves the pp1a and pp1ab at three 101 sites, releasing nsp1, nsp2 and nsp3 20,21 . The second is the SARS-CoV-2 main 102 protease (M pro ), also called nsp5 or the chymotrypsin-like protease (3CL pro ), which 103 cleaves pp1a and pp1ab at a minimum of 11 distinct cleavage sites to release nsp4-104 16 (Figure 1 ) 20 . Interestingly, M pro has also been found to aid in immune evasion by 105 inhibiting type I IFN production, contributing to the impaired type I IFN response that 106 has become a hallmark of severe SARS-CoV-2 infection, with persistent viral load and 107 poor patient outcomes 22-25 . M pro forms a catalytically active homodimer which cleaves 108 with high specificity at Leu-Gln¯Xaa (where ¯ represents the cleavage site and Xaa 109 can be Ser, Ala or Asn) 26-28 . Such sequence specificity has not been observed for any 110 human proteases and therefore peptide or peptidomimetic based inhibitors are 111 predicted to inhibit SARS-CoV-2 M pro with high selectivity and with minimal off-target 112 effects in humans 20,29 . 113

The key role of M pro for the replication and viability of SARS-CoV-2 has naturally led 115 to the search for novel inhibitors of the protease. Perhaps the most promising of these 116 are the peptidomimetic compounds developed by Pfizer, inspired by PF-00835231 117 (IC50 = 4-8 nM against SARS-CoV-2 M pro ) that was originally developed against SARS-118

CoV-1 M pro , which possesses high homology to the SARS-CoV-2 protease 30-32 . 119

Specifically, a phosphate prodrug of this inhibitor (PF07304814) has recently 120 completed a phase 1b trial (clinical trials identifier NCT04535167). A second-121 generation orally available peptidomimetic M pro inhibitor developed by Pfizer (PF-122 07321332, Figure 1 ) has also recently entered phase 2 clinical trials for treatment of Macrocyclic peptides are attractive chemotypes for medicinal chemistry efforts due to 136 their ability to bind targets with high affinity and selectivity, whilst exhibiting greater 137 proteolytic stability and membrane permeability than their linear counterparts 40-42 . In 138 this work we describe several potent cyclic peptide inhibitors of the SARS-CoV-2 M pro , 139

identified through the use of the Random nonstandard Peptide Integrated Discovery 140 (RaPID) technology, which couples mRNA display with flexizyme-mediated genetic 141 code reprogramming 42-44 . Importantly, we also report a crystal structure of the SARS-142

CoV-2 M pro dimer bound to our most potent cyclic peptide inhibitor that highlights the 143 residues important for binding both at the catalytic site and across the dimer interface. 144

Finally, we demonstrate that three of the cyclic peptides identified exhibited antiviral 145 activity against SARS-CoV-2 in vitro, with an additional peptide gaining antiviral activity 146 upon conjugation to a cell penetrating peptide. 147 148

Selection against chemically cross-linked SARS-CoV-2 M pro homodimer. In order 150 to identify cyclic peptide inhibitors of SARS-CoV-2 M pro , we sought to utilize RaPID, 151 which allows the screening of >10 12 cyclic peptides for affinity against a protein target 152 of interest immobilized on magnetic beads. However, functional SARS-CoV-2 M pro is 153 a homodimer with relatively weak affinity between the monomers (KD = 2.5 µM), and 154

we hypothesized that the protein may exist in a predominantly monomeric (i.e. 155

inactive) form when immobilized on magnetic beads 27 . Consistent with this, we found 156 that the catalytic activity (measured by fluorescent substrate cleavage) of C-terminally 157

His6-tagged M pro was significantly diminished (ca. 30%) when immobilized on His Pull-158 down Dynabeads TM compared to that of the wild-type protein in solution 159

( Supplementary Fig. 1a) ; this indicated that a significant proportion of the immobilized 160 M pro was unable to form an active homodimer. We therefore investigated the use of a within the same monomer unit; however, in this case we could not differentiate 172 between intermolecular or intramolecular cross-links by mass spectrometry 173 (Supplementary Fig. 1b) . Importantly, cross-linked M pro exhibited catalytic activity 174 comparable to that of wild-type M pro (in solution), following immobilization on magnetic 175 beads (Supplementary Fig. 1a ). Based on these data, we moved on to RaPID 176 selections against the cross-linked SARS-CoV-2 M pro with a view to discovering novel 177 cyclic peptide inhibitors. corroborate this data, we used 3D NMR spectroscopy (specifically TROSY-HNCO 258 spectra) to analyze SARS-CoV-2 M pro after titration with peptide 1 (Figure 4b ). This 259 revealed shifts of backbone NMR resonances of residues near the active site, 260 consistent with binding of peptide 1 at this location. However, shifts were also 261 observed for residues located far from the active site ( Supplementary Fig. 3) , 262

suggesting that the protein responds to the binding of 1 with global allosteric changes. It was therefore hypothesized that this Leu-Gln motif embedded within 1 mimics the 303 natural substrate and binds to the catalytic site of the protease. Importantly, this 304 proposal is supported by the purely competitive inhibition mode observed for 1 305 (Supplementary Fig. 5) . However, this also raises the possibility that the protease 306 may be able to cleave 1 next to the Gln-Leu recognition sequence. To test this, we 307 Fig. 6a and 6b) . We also 314 synthesized an authentic standard of the resulting cleavage product, which was 315 verified to be identical to M pro -cleaved 1 by LC-MS/MS (Supplementary Fig. 6b) . 316

Finally, we assessed whether the linear peptide product resulting from M pro cleavage 317 of 1 possessed inhibitory activity against SARS-CoV-2 M pro . Interestingly, the peptide 318 exhibited an IC50 of 23.2 ± 5 µM, ~330-fold higher than the IC50 of 70 nM for 1 319 (Supplementary Fig. 6c) . This two-orders of magnitude loss in activity upon 320 linearization suggests that the conformation of cyclic peptide 1 is pre-organised for 321 optimal interaction with the protease. 322

In order to assess the importance of each residue in 1 for M pro inhibitory activity, we 324 systematically replaced all residues in the peptide with alanine (except alkyl side 325 chain-containing amino acids Ala5, Val6 and Leu7) and determined the inhibitory 326 activity of the resulting mutants against M pro (Figure 4d, Supplementary Fig. 7) . 327

Consistent with the known recognition sequence for SARS-CoV-2 M pro and supported 328 by the mass spectrometry results described above, mutation of either Leu2 or Gln3 to 329 Ala (that would be predicted to bind in the S2 and S1 recognition sites, respectively) 330 led to more than two orders of magnitude reduction in inhibitory activity. Remarkably, 331 mutation of Tyr4 (which would be predicted at P1') also led to a significant loss in 332 inhibitor activity (IC50 = 1.9 µM); while this is consistent with the established importance 333 of aromaticity in P1', small residues such as alanine are often found in substrates at 334 this position, and the dramatic reduction in inhibitory potency was therefore 335 unexpected 27,28 . Interestingly, mutation of Arg11, which is distal from the most 336 prominent recognition residues of the cyclic peptide, also led to a significant reduction structures, with a notably long b axis. There were four protein subunits within the 360 asymmetric unit comprising two physiological dimers (Figure 5a) . 361

Although the peptide is not fully resolved in the structure, 9-13 residues were 363 observable in various chains with good electron density; the selenoether linkage and 364 the first 5 residues (D-Tyr1, Leu2, Gln3, Tyr4, Ala5) were very stable, with the 365 remainder of the peptide (7-12) appearing somewhat disordered (Figure 5b) . The 366 peptide displayed a consensus binding pose across three subunits of the crystal 367 structure. The fourth subunit displayed an alternative binding pose in which Leu2 and 368

Gln3 were present in identical positions, but the flanking D-Tyr1 and Tyr4 residues 369 adopted alternative conformations (Supplementary Fig. 9a and 9b) . Closer 370 inspection revealed a non-Pro cis-peptide bond between Tyr4 and Ala3 in chains A, 371 C, D, which results in a tight kink in the helix, while in chain B this bond remains in the 372 Fig. 9c and 9d) . The Gln3 of the peptide 373 occupies the canonical S1 subsite of the protease (following the numbering by Lee et 374 al.) 50 in every peptide:protein complex, facilitated by interactions with His163, Glu166, 375 and Asn142 (Figure 5b) . Likewise, Leu2 is always bound in the S2 subsite comprised 376 of His41, His164 and Gln189. D-Tyr1 is in the solvent-exposed S3 site bordered by 377

Gln189, Ala191 and Pro16, while the peptide twists at the selenoether/D-Tyr1 linkage, 378 turning away from the canonical S4 site to be positioned adjacent to Glu166. Tyr4 is 379 therefore positioned in the S1' (P1') position in ¾ chains, fully consistent with the slow 380 proteolysis between Gln3 and Tyr4 observed by mass spectrometry (Figure 4c) . In 381 chain B, the peptide backbone and Tyr4 have swapped positions (Supplementary 382 Fig. 9a and 9b) . in activity when these positions are mutated to Ala, while D-Tyr1 and Tyr4 also form 399 significant interactions with the protease on either side of these residues. Other 400 positions (8, 9, 10, 12 and 13) that were observed to have little influence on inhibitory 401 activity are either disordered or solvent-exposed. MD simulations suggest that Arg11, 402 the mutation of which to Ala had a significant effect on inhibition (Figure 4d) , makes 403 contacts across the dimer interface (Supplementary Fig. 9e) , interacting with the side 404 chain and main chain carbonyl of Q256*. This is consistent with electron density 405

showing Arg11 within hydrogen bonding distance of the neighbouring chain B in one 406 dimer (Supplementary Fig. 9f) . These observations are consistent with the SEC-407

MALLS and NMR data that shows the peptide binds the dimeric form of SARS-CoV-2 408 M pro exclusively for its mode of inhibition (Figure 4a and 4b) . Antennapedia homeodomain 52 (to afford pen-1). It should be noted that during the 441 selection process the C-terminus of the peptides is conjugated to a large mRNA:cDNA 442 tag, and therefore addition of a CPP to the C-terminus of the peptide (to afford pen-1) 443 was deemed unlikely to affect binding and inhibition of M pro . Pleasingly, pen-1 had 444 equivalent in vitro inhibitory activity against SARS-CoV-2 M pro to peptide 1 (Ki = 9 nM 445 for pen-1 vs 14 nM for 1, Supplementary Fig. 11) indicating that addition of a CPP to 446 the C-terminus of the peptide did not affect binding and inhibition of the protease. We . 12) . Pleasingly, we found that pen-1 and pen-6 both exhibited significantly 454 improved antiviral activity, with EC50 values of 15.2 µM and 6.6 µM, respectively 455 ( Figure 6 ). As expected, the marked improvement in antiviral activity of peptide 1 by 456 conjugation to the penetratin CPP correlated with enhanced cellular uptake, whereby 457 LC-MS/MS analysis showed a 5.5-fold increase in levels of pen-1 compared to 1 in 458 cell lysates (Supplementary Fig. 13) . selections; in this case by covalently cross-linking the M pro homodimer. Co-crystal 479 structures of M pro and 1 and the selenocysteine derivative Sec-1 solved by X-ray 480 crystallography revealed the canonical interaction between a Gln residue and subsite 481 S1, flanked by hydrophobic residues. This half of the peptide was relatively stable, 482 while the hydrophilic face (8-12) was relatively mobile, interacting with solvent and 483 across the dimer interface to facilitate an exclusive dimer binding mode for the inhibitor. After induction, the culture was grown at 18 °C overnight for protein expression. Cells 517 were harvested by centrifugation at 5,000 g for 15 minutes and lysed by passing twice 518 through a Emulsiflex-C5 homogenizer (Avestin, Canada). The lysate was centrifuged 519 at 13,000 g for 60 minutes and the filtered supernatant was loaded onto a 5 mL Ni-520 NTA column (GE Healthcare, USA) equilibrated with binding buffer (50 mM Tris-HCl 521 pH 7.5, 300 mM NaCl, 5 % glycerol). The protein was eluted with elution buffer (binding 522 buffer containing, in addition, 300 mM imidazole) and the fractions were analyzed by 523 Crosslinked peptides were identified with a 2% false-discovery rate using the Byonic 543 search engine (Protein Metrics) and a custom database containing the 3CL protein 544 sequence and common proteomics contaminants (e.g. trypsin, albumin, keratins, etc.). 545 DSG crosslinks and hydrolysis products were allowed for the 3CL protein only. 546

Carbamidomethylation was specified as a fixed modification on C, while oxidation of 547 methionine, deamidation of N/Q and pyro-Glu for N-terminal Q/E were variable 548 modifications. Plots of crosslinked residues were generated using UCSF Chimera 549 1.15rc with the 3CL dimer structure (PDB: 6Y2E). using an excitation wavelength of 340 nm. Initial velocities were derived from the linear 601 range of the enzymatic reaction. For IC50 determination, 100% enzymatic activity was 602 defined as the initial velocity of control triplicates containing no inhibitor and the 603 percentage of inhibition was calculated in relation to 100% enzymatic activity. An 604 EDANS standard curve generated as described by Ma et al. 35 To obtain a crystallographic complex of SARS-CoV-2 M pro and 1, the peptide was 613 dissolved in DMSO to a stock concentration of 50 mM. Protein, prepared as described 614

above, was diluted to 4 mg/mL in TBS (50 mM Tris-HCl pH 7.5, 300 mM NaCl) and 615 incubated with 2.5-fold molar excess (~300 µM) 1 for 2 hours on ice to saturate the 616 binding sites of the protease. The complex was briefly buffer exchanged to remove 617 unbound peptide using an Amicon Ultra 0.5 mL centrifugal filter with a 10 kDa MWCO 618 to a final protein concentration of 4 mg/mL. This complex was used for high-throughput 619 crystallography trials at 18 °C using drop sizes of 0.5 µL protein and 0.5 µL reservoir, 620 which yielded a hit in the Index sparse matrix screen (Hampton Research). This hit 621 was optimized but did not yield crystals of suitable diffraction quality; however, the 622 crystals were able to be crushed and used to seed crystals with better morphology. 623

Serial seeding was performed using a protein concentration of 1 mg/mL for several 624 rounds until crystals of suitable diffracting quality were obtained, which were formed 625 at 18 °C in a drop size of 1 µL reservoir, 1.5 µL protein, and 0.5 µL seed stock against 626 a reservoir solution of 25% PEG 3350, 0.1 M Bis-Tris pH 6.5, 0.3 M NaCl. Crystals 627 were flash frozen without cryoprotecting and diffraction data were collected at 100 K 628 using the MX2 beamline at the Australian Synchrotron. As the diffraction was not 629 adequate to unambiguously model the inhibitor into the crystal structure, we solved 630 the co-crystal structure of the SARS-CoV-2 Mpro-Se-1 complex. The complex was 631 prepared the same as with 1 but with overnight incubation at 4 °C. The complex was 632 diluted to a protein concentration of 1 mg/mL and crystallized at 18 °C in a drop size 633 of 1 µL reservoir, 1.5 µL protein and 0.5 µL seed stock against a reservoir solution of 634 22% PEG 3350, 0.1 M Bis-Tris pH 6.0, 0.3 M NaCl. Crystals were seeded using crystal 635 seeds of the SARS-CoV-2 Mpro-1 crystals. Crystals formed as thin plates after ~4-5 636 days and were flash frozen without cryoprotection. CO2. 20 µL of virus solution at 8x10 3 TCID50/mL 67 was then added to the wells and 666 plates were incubated for a further 24 hours at 37 °C, 5% CO2. Stained cells were then 667 imaged using the InCell 2500 (Cytiva) high throughput microscope, with a 10× 0.45 668 NA CFI Plan Apo Lambda air objective. Acquired nuclei were counted using InCarta 669 high-content image analysis software (Cytiva) to give a quantitative measure of CPE. 670

Virus inhibition/neutralization was calculated as %N= (D-(1-Q))x100/D, where; "Q" is 671 the value of nuclei in test well divided by the average number of nuclei in untreated 672 uninfected controls, and "D"=1-Q for the wells infected with virus but untreated with 673 inhibitors. Thus, the average nuclear counts for the infected and uninfected cell 674 controls get defined as 0% and 100% neutralization respectively. To account for cell 675 death due to drug toxicity, cells treated with a given compound alone and without virus 676 were included in each assay. The % neutralization for each compound concentration 677 in infected wells was normalized to % neutralization in wells with equivalent amount of 678 compound but without the virus to yield the final neutralization values for each 679 condition. Inhibition curves and 50% (EC50) effective concentrations were determined 680 tryptic peptides were plotted using Xcalibur Qual Browser (Thermo Scientific). The 708 area under the curve for each peak was used for quantification. 709

The X-Ray co-crystal structure of SARS-CoV-2 M pro -Se-1 has been deposited to the 711 PDB (PDB ID: 7RNW). All other data supporting the findings reported in this study are 712 available in the article and its Supplementary Information. 713

GraphPad software, USA)

Cytotoxicity and targeted proteomics on HEK293-ACE2-TMPRSS2 cell

HEK293-ACE2-TMPRSS2 cells were maintained in Dulbecco's Modified Eagle 687

Cells were sub-cultured 689 between 70-90% confluency and tested regularly to ensure free of mycoplasma 690 contamination. Cytotoxicity of 1, pen-1, 6 and pen-6

Invitrogen) cell viability assay as per manufacturer's instructions

5x10 4 cells were seeded into wells of a 96-well flat bottom culture plate (Corning) and 693 once adhered, compounds or vehicle control (DMSO) were added at varying 694 concentrations and incubated for 24 hours (37 o C, 5% CO2)

Relative fluorescent units (RFU) were determined per well at ex/em 560/590 nm 697 (Tecan Infinite M1000 pro plate reader). Increasing RFU is proportional to cell viability

For targeted proteomics, cells in 6-well plates at 70% confluency were treated with 700 vehicle control (DMSO) or inhibitors at 10 µM. At various time points

M Tris-HCl pH 8.0) then immediately 703 heated to 95 o C for 10 mins before freezing at -30 o C prior to processing for targeted 704

Cell lysates were digested to peptides as described previously. Peptides 705 were analyzed by LC-MS/MS using a data-independent acquisition method as 706 described previously. 69 Extracted ion chromatograms for fragment

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Flexizymes for genetic code reprogramming

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We acknowledge the ARC Centre of Excellence for Innovations in Peptide and Protein 888

Lamberton Research Scholarship (to J.J-L) and Research Training Program from the 890

B) for PhD scholarships

The authors declare no competing interests. 914