**1. Introduction**

The recent outbreak of coronavirus disease 2019 (COVID-19) caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) affected more than 192 million people and killed 4.1 million across the globe (as of 23 July 2021) [1,2]. The genome sequence of SARS-CoV-2 is closely related to SARS-CoV and β-coronavirus, sharing 79.5% and 96.2% sequence identity, respectively [3–5]. Coronaviruses belong to the Coronaviridae family and are found in birds and mammals [6]. The genomic organizations of SARS-CoV and MERS-CoV are similar [7] and both viruses comprise two polypeptides, pp1a and pp1ab. These polypeptides are processed into nonstructural proteins that play a fundamental part in the replication of these viruses, and the whole process is mediated by two kinds of main proteases: 3-chymotrypsin like protease (3CLpro) and papain-like proteases [8,9].

3CLpro, also called the main protease, consists of 306 amino acid residues, and is known to cleave at 11 sites in the polyproteins. The cleaving phenomenon leads to the formation of a helicase, single-stranded-RNA–binding protein, RNA-dependent RNA polymerase, 2-*O*-ribose methyltransferase, endoribonuclease, and exoribonuclease [10,11]. 3CLpro forms a homodimer, and each subunit consists of three domains. The substratebinding site of 3CLpro contains four subsites, S1, S1, S2, and S4, and is highly conserved

**Citation:** Ahmad, B.; Batool, M.; Ain, Q.u.; Kim, M.S.; Choi, S. Exploring the Binding Mechanism of PF-07321332 SARS-CoV-2 Protease Inhibitor through Molecular Dynamics and Binding Free Energy Simulations. *Int. J. Mol. Sci.* **2021**, *22*, 9124. https://doi.org/10.3390/ ijms22179124

Academic Editor: Małgorzata Borówko

Received: 31 July 2021 Accepted: 21 August 2021 Published: 24 August 2021

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among all coronaviruses [3]. The active site of 3CLpro is located in a cleft between domains I and II, while domain III helps with the formation of the dimer and is connected to domain II via a long loop (residues 184–199) [3,8]. A 3CLpro antagonist will be highly specific to SARS-CoV-2 and will have minimal side effects because 3CLpro shares no homology with human proteases [3,12]. Owing to the essential role of 3CLpro in the transcription and replication of the viral genome and strong conservation of its binding-pocket residues, it is considered an ideal drug target in SARS-CoV-2 and other coronaviruses.

To date, more than 26% of world population has received at least one dose of COVID-19 vaccine [13]. The COVID-19 pandemic requires not only prevention via vaccine but also drug treatment. Antiretroviral drugs have been tested in past human coronavirus infections and against SARS-CoV-2, but a recent clinical trial of lopinavir and ritonavir failed to show any clinical benefit in COVID-19 disease. Protease inhibitors are used to treat HIV/AIDS and hepatitis C, but PF-07321332 (Figure 1A) is the first oral protease inhibitor to target the SARS-CoV-2 virus. In addition to PF-07321332, α-ketoamide (Figure 1B) has been reported to show in vitro inhibition of 3CLpro [14]. The bespoke clinical candidate PF-07321332 (NCT04756531) is a potent protease inhibitor with potent antiviral activity against SARS-CoV-2.

The development of new drugs or vaccines is time-consuming; therefore, a drugrepurposing strategy was approved for the treatment of COVID-19 at this critical time [15]. The use of FDA-approved anti–HIV-I drugs lopinavir and ritonavir (Figure 1C,D) in combination against MERS-CoV and SARS-CoV has been reported earlier [16–19]. This combination of lopinavir and ritonavir is currently in a phase II trial along with interferon β-1b against MERS-CoV [16]. In contrast, for the treatment of MERS, this regimen is in phase IV of clinical trials in combination with Arbidol® hydrochloride and oseltamivir (NCT04255017).

**Figure 1.** 2D structure representation of ( **A**) PF-07321332, (**B**) α-ketoamide, ( **C**) lopinavir, and (**D**) ritonavir.

PF-07321332 is a 3CLpro inhibitor with potent in vitro antiviral activity against SARS-CoV-2 and other coronaviruses (NCT04756531). Protease inhibitors interfere with the cutting step of the protease enzyme reaction. These drugs interact with the protease and block its polypeptide cutting ability. Lopinavir is used as a retroviral-protease inhibitor for the treatment of HIV-I infection and is usually co-administered with ritonavir, which increases the half-life of lopinavir and inactivates cytochrome P450 3A4 [20,21]. PF-07321332

is administered in combination with low doses of ritonavir as a booster to increase the bloodstream levels of PF-07321332. In the present study, the binding mechanisms of PF-07321332, α-ketoamide, lopinavir, and ritonavir were explored through molecular dynamics (MD) simulations and binding energy calculations. This study offers a deeper understanding of the mechanism of binding of these drugs to 3CLpro and will be helpful to identify potent protease inhibitor candidates for SARS-CoV-2 and other coronaviruses. In future, our data can be used to identify novel drug candidates using supervised machine learning methods [22].
