PF-00835231

PF-00835231 is a protease inhibitor developed by Pfizer for coronavirus antiviral programs. Registered under CAS 870153-29-0, it is a ketone-based peptidomimetic aimed at the main protease that drives viral replication.

The molecular formula is C24H32N4O6 and the PF-00835231 molecular weight is 472.53 g/mol, with a cLogP near 0.73 that reflects a polar profile. The PF-00835231 chemical structure pairs an indole-2-carboxamide cap with a leucine-derived spacer and a glutamine-mimetic lactam, ending in a hydroxymethyl ketone. That ketone is the reactive center of the PF-00835231 molecular structure and sets how the inhibitor engages its target.

Pfizer PF-00835231 is often referenced alongside its phosphate prodrug PF-07304814. Catalogs and literature also list it as PF 00835231.

Application of PF-00835231

PF-00835231 is commonly used as a reference inhibitor in biochemical and cellular assays of coronavirus 3CLpro activity. Because its apparent antiviral potency depends on the cell model, assay conditions, and P-glycoprotein-mediated efflux, reference values should be interpreted against the corresponding experimental system rather than as a single universal EC50. 

That role plays out in a few scenarios. In screening campaigns for new 3CL protease inhibitors, it anchors the potency scale, letting teams place a hit against a molecule with known preclinical history. In mechanism-of-action work, its early point of intervention makes it a clean control for time-of-addition experiments: activity falls off once replication has progressed, which helps date each step of the cycle. In resistance studies, protease variants are profiled against it to see which mutations blunt binding.

PF-00835231 can also be used in mechanistic studies of 3CLpro-dependent polyprotein processing and downstream viral replication, including experiments designed to distinguish protease inhibition from effects on other stages of the viral life cycle. 

Two practical notes matter. PF-00835231 has limited aqueous solubility, which led to the development of the more soluble phosphate prodrug PF-07304814 for intravenous delivery. Researchers should therefore select solvents and dilution conditions according to the specific assay protocol and available batch documentation. The compound also ships as a dangerous good, affecting lead time and scheduling.

In Vitro

Against the USA-WA1/2020 isolate, PF-00835231 shows an EC50 near 0.22 µM at 24 hours and 0.16 µM at 48 hours. In VeroE6-enACE2 cells, PF-00835231 showed an EC50 of approximately 0.23 µM in the presence of the P-glycoprotein inhibitor CP-100356. Without efflux inhibition, its apparent potency was substantially lower because PF-00835231 is a P-glycoprotein substrate.  Antiviral activity has also been reported against the Omicron BA.2 variant, with potency values in the low-nanomolar range in the specific cellular assay used. Cytotoxicity stays low, with CC50 above 50 µM across several cell lines. Potency also holds in human polarized airway epithelium, closer to the real site of infection.

In Vivo

In a mouse-adapted SARS-CoV (MA15) BALB/c model, the inhibitor reduced multifocal lung lesions and lowered lung viral load. In the SARS-CoV MA15 model, treatment initiated at the time of infection produced the clearest antiviral effect. Delaying treatment by one day still resulted in a substantial numerical reduction in lung viral titers, whereas later initiation showed a weaker response. 

Biochemical and Physiological Actions

PF-00835231 blocks the SARS-CoV-2 cysteine 3C-like protease (3CLpro, or Mpro), which cuts viral polyproteins into functional units. The hydroxymethyl ketone warhead forms a covalent, reversible bond with the catalytic cysteine, halting cleavage. PF-00835231 inhibits SARS-CoV-2 3CLpro with a reported Ki of approximately 0.27 nM. Across a broader panel of coronavirus 3CL proteases, reported Ki values ranged from approximately 30 pM to 4 nM. PF-00835231 showed markedly weaker activity against non-coronavirus proteases and was inactive against the tested HIV protease and most human proteases at the evaluated concentrations. Detectable inhibition of human cathepsin B occurred only at micromolar concentrations, approximately 1000-fold above its potency against SARS-CoV-2 3CLpro. 

Features and Benefits of PF-00835231

The PF-00835231 structure earns its place through balance:

• Sub-nanomolar potency against the coronavirus main protease.
• Activity across SARS-CoV-2 isolates and variants, including Omicron BA.2.
• Wide selectivity window, backed by low cytotoxicity in common cell lines.
• Documented mouse-model efficacy, bridging cell and animal work.