First-in-class drug design series: A review of the development of Pfizer’s oral anti-coronavirus drug Paxlovid

Although vaccination is the most effective way to prevent and control the new crown epidemic, in the face of new Omicron mutant strains, the protective effects of most vaccines and neutralizing antibodies are significantly reduced
.
The development of new broad-spectrum antiviral therapies is still of great significance for the treatment of COVID-19
.
Viral proteases such as RNA-dependent RNA polymerase (RdRp), PLpro (papain-like protease), 3CLpro protease (main protease Mpro) and helicase of the new coronavirus are the main drug targets
.
Both the RdRp inhibitor Molnupiravir jointly developed by Merck and Ridgeback and Pfizer's main protease inhibitor Paxlovid (mainly PF-07321332 and Ritonavir) have passed the emergency use authorization of the FDA
.
Today we will review Paxlovid's discovery and development process
.
The SARS-CoV-2 genome encodes two polyproteins, pp1a and pp1ab, and four structural proteins
.
The main protease of SARS-CoV-2 can cleave polyproteins at 11 different sites to produce non-structural proteins that are essential for virus replication
.
The main protease of the coronavirus is a cysteine ​​protease with three domains, which is characterized by the Cys145-His41 catalytic dimer located between domains I and II
.
The common feature of the main protease substrate is the presence of a Gln residue at P1, and known human cysteine ​​proteases are not cleaved after Gln, so this provides potential selectivity for the main protease as an antiviral drug target
.
In view of the success of small-molecule oral inhibitors of viral protease in the treatment of HIV and HCV, and the key role of the SARS-CoV-2 main protease in viral replication, the main protease Mpro has become another research hotspot besides RdRp
.
The development of drugs targeting the main protease is mainly focused on the substrate site, allosteric site and dimerization interface
.
Since the main protease substrate binding sites of SARS-CoV-1 and SARS-CoV-2 have 100% sequence homology, Pfizer discovered the SARS-CoV-1 main protease inhibitor PF- in response to the SARS outbreak in 2002.
00835231 (Compound 1) also has a strong inhibitory activity on the SARS-CoV-2 main protease, and has a strong anti-SARS-CoV-2 phenotype at the cellular level
.
Compound 1 has poor membrane permeability and oral bioavailability.
In order to overcome this problem, the researchers tried to reduce the hydrogen bond donor at the P1' position and introduced two covalent covalents: cyano and benzothiazol-2-yl ketone, respectively.
Target head
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The oral absorption of cyano group-introduced compound 2 was significantly improved in rats and maintained good metabolic stability, but the inhibitory effect on the main protease in vitro and the anti-SARS-CoV-2 virus activity were reduced
.
The introduction of benzothiazol-2-yl ketone at position P1' and the introduction of cyclic leucine peptidomimetic analogues at position P2 to obtain compound 3 can significantly improve membrane permeability, and reduce the inhibitory effect on the main protease mainly due to the lack of inhibitors The hydrogen bond interaction with Gln189
.
In order to better occupy the S3 pocket, the researchers introduced branched acyclic groups at the P3 position
.
The compound 4 obtained by introducing methanesulfonamide end-capping stretches below Gln189, which enhances the binding to P3 pocket residues and improves the hydrogen bond interaction with the Glu166 skeleton
.
Compared with compound 3, compound 4 has improved in vitro inhibitory activity on main protease, antiviral activity on SARS-CoV-2 and oral absorption
.
The P3 end-capping was optimized to obtain the trifluoroacetamide-terminated compound 5, which maintained the inhibitory effect on the main protease and improved the anti-SARS-CoV-2 virus activity, membrane permeability, metabolic stability and oral bioavailability
.
After replacing the P1' position with a cyano group, compound 6 (PF-07321332) was obtained, which further improved the in vitro inhibitory activity of the main protease, maintained the anti-SARS-CoV-2 virus activity and metabolic stability, and improved the oral bioavailability of rats Spend
.
The dilution experiment proved that PF-07321332 is a reversible covalent inhibitor of SARS-CoV-2 main protease
.
Compared with compound 5, PF-07321332 is easier to synthesize and scale up, and its solubility is improved, and it is easy to be administered as a preparation, so it is developed as a clinical candidate drug
.
PF-07321332 has strong in vitro inhibitory activity against all major proteases of coronaviruses known to infect humans, but has no obvious inhibitory activity against mammalian cysteine, serine and aspartic proteases
.
PF-07321332 also has strong antiviral activity in physiologically relevant A549 and dNHBE cell models.
It has antiviral activity against SARS-CoV-1, SARS-CoV-2, MERS and human coronavirus 229E in cytopathic experiments (CPE).
Strong antiviral activity
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In mice, the plasma unbound drug concentration of PF-07321332 at a dose of 300 mg/kg can maintain EC90 above the in vitro cell experiment for a long time
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In the SARS-CoV-2 mouse model, PF-07321332 can protect mice from weight loss caused by virus infection, inhibit virus replication, significantly reduce the level of virus in the lungs of mice, and reduce lung pathological damage
.
The half-life of PF-07321332 after intravenous administration in rats and monkeys is 5h and <1h, respectively
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In the 2-week repeated dosing toxicity experiment in rats and monkeys, the in vivo exposure of PF-07321332 has a good dose correlation, is well tolerated, and has no obvious adverse reactions.

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Because CYP3A4 is the main metabolic enzyme of PF-07321332, and Ritonavir (RTV), a potent inhibitor of CYP3A4, can be used as a pharmacokinetic enhancer for multiple protease inhibitors metabolized by CYP3A4 (such as Darunavir, Lopinavir, etc.
) Therefore, in a randomized, double-blind, placebo-controlled single-dose ramp-climbing trial in healthy adult subjects, PF-07321332 was used alone and in combination with RTV (Paxlovid).
PF-07321332 The safety and tolerability of Paxlovid are good.
After combined with RTV, the oral blood concentration is significantly increased.
After 12 hours of administration, it still maintains an in vitro anti-SARS-CoV-2 virus EC90 or higher.
Therefore, Paxlovid is expected to be clinically strong The broad-spectrum anti-coronavirus activity
.
In summary, PF-07321332 can be administered orally, has good selectivity and safety, and can prevent infection in a mouse model
.
In a phase I clinical trial, the drug reached the expected concentration of viral suppression
.
Therefore, the combination of the main protease inhibitor Paxlovid and the RdRp inhibitor Molnupiravir and vaccine may further enhance the prevention and control of the new coronavirus epidemic
.
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Zhang, L.
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Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved α-ketoamide inhibitors.
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Discovery of Ketone-based Covalent Inhibitors of Coronavirus 3CL Proteases for the Potential Therapeutic Treatment of COVID-19.
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