Researchers in the United States have described a novel antiviral agent against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) currently being evaluated in clinical trials as a treatment for coronavirus disease 2019 (COVID-19).

“Alongside vaccines, antiviral therapeutics are an important part of the healthcare response to counter the ongoing threat presented by COVID-19,” says Dafydd Owen from Pfizer Worldwide Research in Cambridge, Massachusetts, and colleagues.

The team has now described an orally bioavailable inhibitor of the SARS-CoV-2 main protease (Mpro) that cleaves viral polyproteins into the shorter proteins needed for viral replication.

The inhibitor – called  PF-07321332 – also exhibits in vitro pan-human coronavirus antiviral activity and excellent in vivo safety profiles.

A pre-print version of the research paper is available on the medRxiv* server, while the article undergoes peer review.

Novel human coronaviruses pose a significant threat to global public health

Over the last two decades, the emergence of novel human coronaviruses, including SARS-CoV-1, Middle East Respiratory Syndrome (MERS), and SARS-CoV-2, has highlighted the significant potential threat that this class of viruses poses to global public health.

Since the COVID-19 outbreak began in late December 2019, SARS-CoV-2 has infected almost 200 million people and caused more than 4.2 million deaths globally.

The virus infects host cells when the receptor-binding domain of its surface spike protein binds to the human receptor angiotensin-converting enzyme 2 (ACE2).

While effective COVID-19 vaccines targeting this spike-ACE2 interaction have been developed and rolled out within record time, a substantial proportion of the population is either unwilling to be vaccinated or unable to due to existing medical conditions.

“Oral SARS-CoV-2 specific therapeutics that are applicable for treatment of the broad population upon COVID-19 diagnosis are urgently needed,” writes Owen and colleagues. “Such a treatment approach may prevent more severe disease, hospitalizations and deaths. Indirectly, it may also reduce further transmission from infected individuals.”

More about the SARS-CoV-2 main protease

The SARS-CoV-2 genome encodes two polyproteins (pp1a and pp1ab) that are cleaved by Mpro to yield shorter proteins that are crucial for viral replication.

The coronavirus Mpro is a three-domain cysteine protease with substrates that share common features, including the presence of a P1 glutamine (Gln) residue.

“No known human equivalent cysteine protease exploits a P1 Gln as the cleavage site prompt, thus offering an intriguing selectivity hypothesis for this viral target over the human proteome,” say the researchers.

Furthermore, since the SARS-CoV-2 Mpro and the spike protein are distinct entities, the antiviral efficacy of a small-molecule Mpro inhibitor is not expected to be affected by the spike mutations that variants of concern have evolved.  

SARS-CoV-2 Mpro structural biology. (A) Key H-bond interactions of PF-00835231 (1). (B) Modelled overlap of dimethyl-bicyclo[3.1.0] proline from compound 3 (blue) as a mimic of P2 leucine residue (cyan) found in the viral polyprotein substrate and 1. This tolerated P2 change eliminates an H-bond donor from resulting inhibitors. (C) Compound 3 effectively fills the lipophilic S2 pocket formed by Met49, Met165, and His41 but productive hydrogen bonding to Gln189 is no longer possible. (D) Compound 4 with optimized acyclic P3 group and restored Gln189 interaction. (E) Binding mode of clinical candidate PF-07321332 (6). A reversible covalent Cys145 adduct is formed with the reactive nitrile in compound 6.

SARS-CoV-2 Mpro structural biology. (A) Key H-bond interactions of PF-00835231 (1). (B) Modeled overlap of dimethyl-bicyclo[3.1.0] proline from compound 3 (blue) as a mimic of P2 leucine residue (cyan) found in the viral polyprotein substrate and 1. This tolerated P2 change eliminates an H-bond donor from the resulting inhibitors. (C) Compound 3 effectively fills the lipophilic S2 pocket formed by Met49, Met165, and His41 but productive hydrogen bonding to Gln189 is no longer possible. (D) Compound 4 with optimized acyclic P3 group and restored Gln189 interaction. (E) Binding mode of clinical candidate PF-07321332 (6). A reversible covalent Cys145 adduct is formed with the reactive nitrile in compound 6.

“Given the pivotal role of SARS-CoV-2 Mpro in viral replication, its potential for mechanistic safety, and expected lack of spike protein variant resistance challenges, SARS-CoV-2 Mpro inhibition represents an attractive small molecule approach for an oral antiviral therapy to treat COVID-19,” writes the team.

An inhibitor of SARS-CoV-1 Mpro was previously identified

As part of the response to the SARS-CoV-1 outbreak in 2002, researchers attempted to identify inhibitors of the SARS-CoV-1 Mpro.

This led to the discovery of PF-00835231 (referred to hereafter as 1) as a potent inhibitor of recombinant SARS-CoV-1 Mpro in a fluorescence resonance energy transfer (FRET)-based cleavage assay.

Furthermore, PF-00835231 has also been shown to potently inhibit recombinant SARS-CoV-2 Mpro, which shares 100% sequence homology with the binding site of SARS-CoV-1 Mpro.

The phosphate prodrug form of PF-00835231 is now currently being investigated as an intravenous treatment for patients hospitalized with COVID-19.

What did the current study involve?

As part of the researchers’ oral SARS-CoV-2 Mpro inhibitor program, they pursued two functional groups that precedented as covalent warheads for cysteine proteases.

These nitriles and benzothiazol-2-yl ketones replace the hydrogen bond donor of the P1’ α-hydroxymethyl ketone moiety in 1 to improve its absorptive permeability and oral absorption in animals.

The addition of the P1’ nitrile 6 led to the selection of what would ultimately become the clinical candidate PF07321332.

Testing the antiviral activity of PF-07321332 against coronaviruses

PF-07321332 demonstrated potent inhibition in FRET Mpro assays representing all coronaviruses known to infect humans – SARS-CoV-2, SARS-CoV-1, MERS, HKU1, OC43, 229E and NL63.

In vitro analysis of the antiviral activity in human adenocarcinoma-derived alveolar basal epithelial (A549) cells expressing ACE2 revealed that PF-07321332 inhibited SARS-CoV-2 replication with a half-maximal effective concentration (EC50) value and an EC90 value of 77.9nM and 215nM, respectively.

No cytotoxicity was detected at concentrations as high as 3µM.

Furthermore, PF-07321332 demonstrated potent antiviral activity against SARS-CoV-1 (EC90 = 317nM), MERS (EC90 = 351nM) and 229E (EC90 = 620nM) in cytopathic effect assays.

Next, the team evaluated the in vivo antiviral activity of PF-07321332 using a mouse-adapted model of SARS-CoV-2 infection. Following intranasal infection, twice daily (BID) oral administration of PF-07321332 protected BALB/c mice from weight loss, compared with mice that received a placebo.

Discovering a pharmacokinetic enhancer of PF-07321332

The researchers also performed in vitro studies of the enzymes involved in PF-07321332 metabolism, which established a predominant role for CYP3A4.

A potent inactivator of CYP3A4 called ritonavir (RTV) is already used as a pharmacokinetic enhancer of several marketed protease inhibitors that are subject to metabolic clearance via CYP3A4.

Owen and colleagues say the safety, tolerability, and pharmacokinetics of PF-07321332 as a single agent and in combination with RTV are currently under investigation in a randomized, double-blind, placebo-controlled trial.

The administration of two oral PF-07321332 doses has already been shown to be safe, well-tolerated, and to exhibit a significant boost in plasma concentrations when co-administered with RTV.

Next, “the efficacy of PF-07321332 in COVID-19 patients will be assessed with a BID dosing paradigm with the potential to increase the dose of PF-07321332 as a single agent and/or co-administration with RTV,” says the team.

*Important Notice

medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

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