Safety, Tolerability, and Pharmacokinetics of Intravenous Doses of PF-07304814, a Phosphate Prodrug Protease Inhibitor for the Treatment of SARS-CoV-2, in Healthy Adult Participants.

Studies on targeted antivirals for treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of the ongoing pandemic, are limited. PF-07304814 (lufotrelvir) is the phosphate prodrug of PF-00835231, a protease inhibitor targeting the 3C-like protease of SARS-CoV-2. This phase 1 study evaluated the safety, tolerability, and pharmacokinetics (PK) of single ascending intravenous doses of lufotrelvir (continuous 24-hour infusion of 50, 150, 500, or 700 mg) versus placebo in healthy volunteers (2 interleaving cohorts: 1, n = 8; 2, n = 7). Each dosing period was separated by a washout interval (≥5 days). Treatment-emergent adverse events, PK, and biomarker concentrations were estimated from plasma/urine samples. Lufotrelvir was administered to 15 volunteers (mean [SD] age 39.7 [11.8] years). No serious adverse events, discontinuations, or deaths were reported. Mean maximum observed concentration of PF-00835231 (active moiety; 97.0 ng/mL to 1288 ng/mL) were observed between median time to maximum concentration of 14 to 16 hours after the start of the lufotrelvir infusion. Near-maximum plasma concentrations of PF-00835231 were observed ≈6 hours after infusion start and sustained until infusion end. PF-00835231 plasma concentrations declined rapidly after infusion end (mean terminal half-life: 500 mg, 2.0 hours; 700 mg, 1.7 hours). Approximately 9%-11% of the dose was recovered in urine as PF-00835231 across doses. A continuous, single-dose, 24-hour infusion of lufotrelvir (50-700 mg) was rapidly converted to PF-00835231 (active moiety), with dose-proportional PK exposures and no significant safety concerns. A daily, 24-hour continuous infusion of 270 to 350 mg is expected to maintain PF-00835231 concentration at steady state/above effective antiviral concentrations. Further studies exploring lufotrelvir efficacy in patients with coronavirus disease 2019 are ongoing.

Structural Basis of Main Proteases of Coronavirus Bound to Drug Candidate PF-07304814.

New variants of the severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) emerged and spread rapidly all over the world, which strongly supports the need for pharmacological options to complement vaccine strategies. Main protease (Mpro or 3CLpro) is a critical enzyme in the life cycle of SARS-CoV-2 and appears to be highly conserved among different genera of coronaviruses, making it an ideal target for the development of drugs with broad-spectrum property. PF-07304814 developed by Pfizer is an intravenously administered inhibitor targeting SARS-CoV-2 Mpro. Here we showed that PF-07304814 displays broad-spectrum inhibitory activity against Mpros from multiple coronaviruses. Crystal structures of Mpros of SARS-CoV-2, SARS-CoV, MERS-CoV, and HCoV-NL63 bound to the inhibitor PF-07304814 revealed a conserved ligand-binding site, providing new insights into the mechanism of inhibition of viral replication. A detailed analysis of these crystal structures complemented by comprehensive comparison defined the key structural determinants essential for inhibition and illustrated the binding mode of action of Mpros from different coronaviruses. In view of the importance of Mpro for the medications of SARS-CoV-2 infection, insights derived from the present study should accelerate the design of pan-coronaviral main protease inhibitors that are safer and more effective.

A VSV-based assay quantifies coronavirus Mpro/3CLpro/Nsp5 main protease activity and chemical inhibition.

Protease inhibitors are among the most powerful antiviral drugs. However, for SARS-CoV-2 only a small number of protease inhibitors have been identified thus far and there is still a great need for assays that efficiently report protease activity and inhibition in living cells. Here, we engineer a safe VSV-based system to report both gain- and loss-of-function of coronavirus main protease (Mpro/3CLpro/Nsp5) activity in living cells. We use SARS-CoV-2 3CLpro in this system to confirm susceptibility to known inhibitors (boceprevir, GC376, PF-00835231, and PF-07321332/nirmatrelvir) and reevaluate other reported inhibitors (baicalein, ebselen, carmofur, ethacridine, ivermectin, masitinib, darunavir, and atazanavir). Moreover, we show that the system can be adapted to report both the function and the chemical inhibition of proteases from different coronavirus species as well as from distantly related viruses. Together with the fact that live cell assays also reflect compound permeability and toxicity, we anticipate that this system will be useful for both identification and optimization of additional coronavirus protease inhibitors.

[Nirmatrelvir plus ritonavir (Paxlovid) a potent SARS-CoV-2 3CLpro protease inhibitor combination]. / Nirmatrelvir más ritonavir (Paxlovid) una potente combinación inhibidora de la proteasa 3CLpro del SARS-CoV-2.

All coronavirus, including SARS-CoV-2, encode two proteases needed for the processing of PP1A and PP1AB polyproteins. The main protease 3CL (chemotripsine-like) gives rise to the formation of NSP11/16 proteins. The 3CL protease has been constituted as one of the possible therapeutic targets for the development of antiviral drugs against SARS-COV-2 due to its highly conserved sequence and structure among all coronaviruses. During the SARS-COV-1 pandemic, a hydroxymethyl ketone derivative (PF-00835231) was identified with an intense inhibitory activity against the 3CL protease. Subsequent chemical modifications gave rise to derivative PF-07321332 (nirmatrelvir) which has shown a high antiviral efficacy against SARS-COV-2. The company's data indicate that it is capable of reducing 89% the risk of hospitalization and death of patients infected with hardly adverse effects. Its effectiveness improves if it is administered orally in the first 24-48 hours and the duration of treatment has been established between 3-5 days. The commercial form has been associated with the antiviral ritonavir that has shown the metabolism of nirmatrelvir, lengthening its average life. This antiviral would be effective against current and future viral variants, since 3CL is not modified in them. The FDA approved this antiviral in November 2021 and EMA is in the final evaluation phase.

Biosynthesis of Calipyridone A Represents a Fungal 2-Pyridone Formation without Ring Expansion in Aspergillus californicus.

A chemical investigation of the filamentous fungus Aspergillus californicus led to the isolation of a polyketide-nonribosomal peptide hybrid, calipyridone A (1). A putative biosynthetic gene cluster cpd for production of 1 was next identified by genome mining. The role of the cpd cluster in the production of 1 was confirmed by multiple gene deletion experiments in the host strain as well as by heterologous expression of the hybrid gene cpdA inAspergillus oryzae. Moreover, chemical analyses of the mutant strains allowed the biosynthesis of 1 to be elucidated. The results indicate that the generation of the 2-pyridone moiety of 1 via nucleophilic attack of the iminol nitrogen to the carbonyl carbon is different from the biosynthesis of other fungal 2-pyridone products through P450-catalyzed tetramic acid ring expansions. In addition, two biogenetic intermediates, calipyridones B and C, showed modest inhibition effects on the plaque-forming ability of SARS-CoV-2.

Preclinical characterization of an intravenous coronavirus 3CL protease inhibitor for the potential treatment of COVID19.

COVID-19 caused by the SARS-CoV-2 virus has become a global pandemic. 3CL protease is a virally encoded protein that is essential across a broad spectrum of coronaviruses with no close human analogs. PF-00835231, a 3CL protease inhibitor, has exhibited potent in vitro antiviral activity against SARS-CoV-2 as a single agent. Here we report, the design and characterization of a phosphate prodrug PF-07304814 to enable the delivery and projected sustained systemic exposure in human of PF-00835231 to inhibit coronavirus family 3CL protease activity with selectivity over human host protease targets. Furthermore, we show that PF-00835231 has additive/synergistic activity in combination with remdesivir. We present the ADME, safety, in vitro, and in vivo antiviral activity data that supports the clinical evaluation of PF-07304814 as a potential COVID-19 treatment.

Pan-3C Protease Inhibitor Rupintrivir Binds SARS-CoV-2 Main Protease in a Unique Binding Mode.

Rupintrivir targets the 3C cysteine proteases of the picornaviridae family, which includes rhinoviruses and enteroviruses that cause a range of human diseases. Despite being a pan-3C protease inhibitor, rupintrivir activity is extremely weak against the homologous 3C-like protease of SARS-CoV-2. In this study, the crystal structures of rupintrivir were determined bound to enterovirus 68 (EV68) 3C protease and the 3C-like main protease (Mpro) from SARS-CoV-2. While the EV68 3C protease-rupintrivir structure was similar to previously determined complexes with other picornavirus 3C proteases, rupintrivir bound in a unique conformation to the active site of SARS-CoV-2 Mpro splitting the catalytic cysteine and histidine residues. This bifurcation of the catalytic dyad may provide a novel approach for inhibiting cysteine proteases.

Is PF-00835231 a Pan-SARS-CoV-2 Mpro Inhibitor? A Comparative Study.

The COVID-19 outbreak continues to spread worldwide at a rapid rate. Currently, the absence of any effective antiviral treatment is the major concern for the global population. The reports of the occurrence of various point mutations within the important therapeutic target protein of SARS-CoV-2 has elevated the problem. The SARS-CoV-2 main protease (Mpro) is a major therapeutic target for new antiviral designs. In this study, the efficacy of PF-00835231 was investigated (a Mpro inhibitor under clinical trials) against the Mpro and their reported mutants. Various in silico approaches were used to investigate and compare the efficacy of PF-00835231 and five drugs previously documented to inhibit the Mpro. Our study shows that PF-00835231 is not only effective against the wild type but demonstrates a high affinity against the studied mutants as well.

A Quick Route to Multiple Highly Potent SARS-CoV-2 Main Protease Inhibitors*.

The COVID-19 pathogen, SARS-CoV-2, requires its main protease (SC2MPro ) to digest two of its translated long polypeptides to form a number of mature proteins that are essential for viral replication and pathogenesis. Inhibition of this vital proteolytic process is effective in preventing the virus from replicating in infected cells and therefore provides a potential COVID-19 treatment option. Guided by previous medicinal chemistry studies about SARS-CoV-1 main protease (SC1MPro ), we have designed and synthesized a series of SC2MPro inhibitors that contain ß-(S-2-oxopyrrolidin-3-yl)-alaninal (Opal) for the formation of a reversible covalent bond with the SC2MPro active-site cysteine C145. All inhibitors display high potency with Ki values at or below 100 nM. The most potent compound, MPI3, has as a Ki value of 8.3 nM. Crystallographic analyses of SC2MPro bound to seven inhibitors indicated both formation of a covalent bond with C145 and structural rearrangement from the apoenzyme to accommodate the inhibitors. Virus inhibition assays revealed that several inhibitors have high potency in inhibiting the SARS-CoV-2-induced cytopathogenic effect in both Vero E6 and A549/ACE2 cells. Two inhibitors, MPI5 and MPI8, completely prevented the SARS-CoV-2-induced cytopathogenic effect in Vero E6 cells at 2.5-5 µM and A549/ACE2 cells at 0.16-0.31 µM. Their virus inhibition potency is much higher than that of some existing molecules that are under preclinical and clinical investigations for the treatment of COVID-19. Our study indicates that there is a large chemical space that needs to be explored for the development of SC2MPro inhibitors with ultra-high antiviral potency.

Balancing the decomposable behavior and wet tensile mechanical property of cellulose-based wet wipe substrates by the aqueous adhesive.

With the current global outbreak of novel coronaviruses, the fabrication of decomposable wet wipe with sufficient wet strength to meet daily use is promising but still challenging, especially when renewable cellulose was employed. In this work, a decomposable cellulose-based wet wipe substrate is demonstrated by introducing a synthetic N-vinyl pyrrolidone-glycidyl methacrylate (NVP-GMA) adhesive on the cellulose surface. Experimental results reveal that the NVP-GMA adhesive not only significantly facilitates the chemical bonding between cellulose fibers in the wet state, but also increase the surface wettability and water retention. The as-fabricated cellulose-based wet wipe substrate displays a superb water retention capacity of 1.9 times, an excellent water absorption capacity (completely wetted with 0° water contact angle), and a perfect wet tensile index of 3.32 N.m.g-1. It is far better than state-of-the-art wet toilet wipe on the market (non-woven). The prepared renewable and degradable cellulose-based substrate with excellent mechanical strength has potential application prospects in diverse commercially available products such as sanitary and medical wet wipes.