key: cord-0755695-5kv3zdni
authors: Greasley, Samantha E.; Noell, Stephen; Plotnikova, Olga; Ferre, RoseAnn; Liu, Wei; Bolanos, Ben; Fennell, Kimberly; Nicki, Jennifer; Craig, Tim; Zhu, Yuao; Stewart, Al E.; Steppan, Claire M.
title: Structural basis for the in vitro efficacy of nirmatrelvir against SARS-CoV-2 variants
date: 2022-04-22
journal: J Biol Chem
DOI: 10.1016/j.jbc.2022.101972
sha: 3c9c9b24352b786dbee0384d8a554ec154a0460b
doc_id: 755695
cord_uid: 5kv3zdni

The COVID-19 pandemic continues to be a public health threat with emerging variants of SARS-CoV-2. Nirmatrelvir (PF-07321332) is a reversible, covalent inhibitor targeting the main protease (M(pro)) of SARS-CoV-2 and the active protease inhibitor in PAXLOVID™ (nirmatrelvir tablets and ritonavir tablets). However, the efficacy of nirmatrelvir is underdetermined against evolving SARS-CoV-2 variants. Here, we evaluated the in vitro catalytic activity and potency of nirmatrelvir against the M(pro) of prevalent variants of concern (VOC) or variants of interest (VOI): Alpha (α, B.1.1.7), Beta (β, B.1.351), Delta (δ, B1.617.2), Gamma (γ, P.1), Lambda (λ, B.1.1.1.37/C37), Omicron (ο, B.1.1.529) as well as the original Washington or wildtype strain. These VOC/VOI carry prevalent mutations at varying frequencies in the M(pro) specifically for: α, β, γ (K90R), λ (G15S) and ο (P132H). In vitro biochemical enzymatic assay characterization of the enzyme kinetics of the mutant M(pros) demonstrate that they are catalytically comparable to wildtype. We found that nirmatrelvir has similar potency against each mutant M(pro) including P132H that is observed in the Omicron variant with a Ki of 0.635 nM as compared to a Ki of 0.933 nM for wildtype. The molecular bases for these observations were provided by solution-phase structural dynamics and structural determination of nirmatrelvir bound to the ο, λ and β M(pro) at 1.63 - 2.09 Å resolution. These in vitro data suggest that PAXLOVID™ has the potential to maintain plasma concentrations of nirmatrelvir many-fold times higher than the amount required to stop the SARS-CoV-2 VOC/VOI, including Omicron, from replicating in cells.

. Later, WHO designated this virus as the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) owing to its similarity with the previous SARS-CoV (3) . Then at the end of January 2020, WHO declared this viral outbreak as a public health emergency of international concern (4), and subsequently characterized it as a pandemic.

Genetic lineages of SARS-CoV-2 have been emerging and circulating around the world since the beginning of the COVID-19 pandemic. Like all viruses, SARS-CoV-2 is constantly changing through mutation and each virus with a unique sequence is considered a new variant. The World

Health Organization, as well as other public health organizations, monitor all variants that cause COVID-19 for increased risk to global public health and classify as variants being monitored, variants of interest (VOI), variants of concern (VOC) and variants of high consequence. As of SARS-CoV-2 is a highly infectious beta coronavirus that can be life-threatening in serious cases.

While effective COVID-19 vaccines have been developed, for individuals who have yet to be J o u r n a l P r e -p r o o f vaccinated or cannot be vaccinated, such as due to pre-existing medical conditions, therapeutics will likely be needed to effectively combat coronavirus disease 2019 . (6) SARS-CoV-2 main protease (M pro , also referred to as 3CL protease) is a cysteine protease that is critical for the processing of the two polyproteins (pp1a and pp1ab) encoded by the SARS-CoV-2 genome. The protease cleaves these polyproteins into shorter, non-structural proteins that are essential for viral replication (6) . Owing to this key role in viral replication, small molecule inhibitors of SARS-CoV-2 M pro represent attractive therapeutics for the treatment of COVID-19.

We have previously reported on the discovery and antiviral efficacy of nirmatrelvir (PF-07321332), an orally bioavailable SARS-CoV-2-M pro inhibitor with in vitro pan-human coronavirus antiviral activity with excellent off-target selectivity and in vivo safety profiles (6) .

Nirmatrelvir has demonstrated oral activity in a mouse-adapted SARS-CoV-2 model and has achieved oral plasma concentrations that exceed the in vitro antiviral cell potency, in a phase I clinical trial in healthy subjects (6) . Here we report on the catalytic activity of frequently observed M pro mutations in SARS-CoV-2 VOC/VOI, in vitro efficacy of nirmatrelvir against these mutant M pros , the solution-phase structural dynamics and structure of nirmatrelvir bound to the M pros from three VOCs,   and .

Full-length wildtype M pro from the original Washington variant (USA-WA1/2020) and VOC/VOI SARS-CoV-2 M pro were expressed and purified to near homogeneity as demonstrated by a singular protein peak with a confirmed intact mass of 33.8 kDa for a fully authentic form of each protein ( Figure 1A) . A final and size exclusion chromatography step showed the wildtype, K90R, G15S and P132H M pro proteins to be nearly 100% pure by Western blot analysis ( Figure 1B ). An established M pro fluorescence resonance energy transfer (FRET)-based cleavage assay was used to determine enzyme catalytic activity by monitoring initial velocities of the proteolytic activities at varying substrate (SARS canonical peptide) concentrations (6,7,8) ( Figure 1C ). The turnover number (kcat) and Michaelis constants (Km) was determined for the wildtype, K90R, G15S and P132H M pro proteins, respectively ( Table 1) The crystal structures of nirmatrelvir bound to the three VOC were determined to 2.09 Å (K90R), 1.68 Å (G15S) and 1.63 Å (P132H) resolution ( Figure 2 ). Superposition of each of these mutant structures with the wildtype M pro , the structure of which was reported in detail previously (6, 9) , shows that the binding mode of nirmatrelvir is unperturbed by the mutations, with the ligand maintaining the protein interactions observed in the wildtype M pro , as shown in Figure 2 . Indeed, these mutations are distal to the PF-07321332 binding pocket, with Pro 132 located approximately 16 Å (C-Pro132 to C-Glu166) from the binding pocket, while K90R and G15S reside 19 Å and 17 Å respectively (C-R90/S15 to C-Cys145) from the PF-07321332 binding pocket ( Figure   2D ). The crystal structures also show that the mutations do not give rise to any signification changes of the protein around the binding pocket or the site of the mutation ( Figure 2D (10, 11) . Nirmatrelvir also appears to retain its in vitro antiviral efficacy against Omicron relative to wildtype (12) (13) (14) (15) .

The emergence of naturally occurring SARS-CoV-2 variants exemplify its ability to mutate and signify the continued potential for this pandemic to be problematic. It is important to continue monitoring emerging variants of concern to quickly understand potential challenges to the efficacy of current and future anti-viral therapies. More broadly, the availability of naturally occurring mutations could provide opportunities to more deeply understand additional aspects of protease structure-function and fitness. Studies described here demonstrate the in vitro inhibitory activity of nirmatrelvir against the Alpha, Beta, Gamma, Lambda and Omicron variants of M pro and indicate the structural basis for retention of in vitro potency against these mutant proteins. They also inform the methods for assessing activity against subsequent variants possessing mutations in the M pro protein.

Protein production and purification.

Briefly, the wild type SARS-Cov2-M pro construct was designed based on Su et al 2020 (16) . An additional N-terminal PreScission protease cleavage site was inserted between GST and the selfcleavage site. Site directed mutagenesis was performed to make each of the variants (K90R, G15S or P132H). The resulting plasmid was then transformed into BL21 (DE3) cells for protein expression. One liter of LB media was inoculated with 30 mL of overnight culture and grown at 37°C until an OD600 of 0.6 was reached. The culture was induced using a final concentration of 0.2 mM IPTG and harvested 1 hour post induction. Cells were lysed in 20 mM Tris pH 8.0 buffer containing 500 mM NaCl, 10% glycerol, 0.2 mM TCEP with a microfluidizer, and the mixture was clarified by centrifugation at 15000 x g. The resulting supernatant was purified by a Niaffinity column using a step gradient, followed by C-terminal His-tag cleavage with PreScission protease, and a secondary Ni-affinity purification to remove non-cleaved M pro and PreScission protease.

The enzymatic activity of the main protease, M pro of SARS CoV-2 wildtype and variants was monitored using a continuous fluorescence resonance energy-transfer (FRET) assay (6, 7, 8) . The X-ray diffraction data were collected at -173°C at IMCA-CAT 17-ID beamline (17, 18) of the Advanced Photon Source (APS) at Argonne National Labs (19) using the Eiger 2 x 9M detector (Dectris). The structures were determined by difference Fourier and refined using the anisotropically scaled data as described previously for wildtype SARS-CoV-2-M pro in complex with PF-07321332 (6) . Diffraction data processing and model refinement statistics for each of the mutant M pro are given in Table 3 .

Native protein was deuterium exchanged at four time points (15s, 1m, 6m, 1h) , and subsequently digested with two protease columns (Protease XIII/Pepsin (NovaBioAssays) and Nepenthesin-1 (AffiPro) to generate deuterated peptides for LC-MS analysis. A total of 405 WT peptides were identified (Table 4) J o u r n a l P r e -p r o o f J o u r n a l P r e -p r o o f 

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Use of the IMCA-CAT beamline 17-ID (or 17-BM) at the Advanced Photon Source was supported by the companies of the Industrial Macromolecular Crystallography Association through a contract with Hauptman

This research used resources at the Industrial Macromolecular Crystallography Association Collaborative Access Team (IMCA-CAT) beamline 17-ID, supported by the companies of the Industrial Macromolecular Crystallography Association through a contract with Hauptman

This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No

On the use of the merging R factor as a quality indicator for X-ray data

Linking crystallographic model and data quality

We thank Mark Noe, Gretchen Dean, Annaliesa Anderson and Charlotte Allerton for critical reading of the manuscript and leadership.

J o u r n a l P r e -p r o o f Rfree is the same as Rcryst, but for 5% of the data randomly omitted from refinement. (22) J o u r n a l P r e -p r o o f