Pfizer moves towards an oral anti-COVID-19 therapy

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.

Journal reference:
Sally Robertson

Written by

Sally Robertson

Sally first developed an interest in medical communications when she took on the role of Journal Development Editor for BioMed Central (BMC), after having graduated with a degree in biomedical science from Greenwich University.

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Comments

  1. Jim Harrison Jim Harrison United States says:

    Pfizer and Merck are late to the game.  Oramed Pharmaceuticals (NASDAQ:ORMP). Has been working on an Oral pill for some time. The first trial involved 24 unvaccinated volunteers, each taking one or two pills, before moving on to larger Phase 3 trials. The vaccine should be "much more resistant to COVID-19 variants," according to Oramed CEO Nadav Kidron, since it trains the immune system against three viral proteins instead of the single protein targeted by Pfizer and Moderna's (NASDAQ:MRNA) shots.

The opinions expressed here are the views of the writer and do not necessarily reflect the views and opinions of News Medical.
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