The target of PF-4981517 (CYP3cide) is cytochrome P450 3A4 (CYP3A4), a mechanism-based (suicide) inhibitor with high selectivity for CYP3A4 over CYP3A5. Key parameters include:
- For recombinant human CYP3A4 (using midazolam 1'-hydroxylation as the activity marker): Half-maximal inhibitory concentration (IC50) = 0.023 μM [1]
- For CYP3A4 in pooled human liver microsomes (HLMs, using midazolam 1'-hydroxylation as the marker): IC50 = 0.031 μM [1]
- Mechanism-based inactivation parameters for recombinant CYP3A4: Inactivation rate constant (kinact) = 0.14 min⁻¹, Michaelis constant for inactivation (Km) = 0.056 μM, and the inactivation efficiency (kinact/Km) = 2.5 μM⁻¹min⁻¹ [1]
- For CYP3A5 (using CYP3A5-high expressor HLMs and midazolam as the substrate): IC50 > 10 μM (negligible inhibitory activity) [1]
.

Using human liver microsomes from donors with nonfunctioning CYP3A5 (CYP3A5 3/3), an extraordinary metabolic inactivation efficiency (kinact/KI) of 3300 to 3800 ml • min-1 • μmol-1 is observed when examining the inhibitory characteristics of CYP3cide. This efficiency was found to correspond to an apparent KI of 420–480 nM and a maximal inactivation rate (kinact) of 1.6 min-1. The connection between CYP3A5 abundance and the remaining midazolam 1'-hydroxylase activity is strong when CYP3cide is assessed in a library of genotyped polymorphic CYP3A5 microsomes at a concentration and preincubation period that totally inhibits CYP3A4[1].

---

1. Inhibition of recombinant human CYP3A4 activity: When PF-4981517 was incubated with recombinant CYP3A4, NADPH-generating system, and the specific substrate midazolam, it inhibited the 1'-hydroxylation of midazolam in a concentration-dependent manner. At a concentration of 0.1 μM, the inhibition rate of CYP3A4 activity reached >90%, and the IC50 was determined to be 0.023 μM. No significant loss of inhibitory activity was observed after removing unbound drug via ultrafiltration, confirming irreversible inactivation [1]
2. Inhibition of CYP3A4 in human liver microsomes (HLMs): In pooled HLMs, PF-4981517 inhibited midazolam 1'-hydroxylation (a canonical CYP3A4 activity) with an IC50 of 0.031 μM. In HLMs from donors with high CYP3A5 expression (CYP3A51/1 genotype), PF-4981517 (0.1 μM) inhibited >90% of CYP3A4-mediated midazolam metabolism, while the remaining metabolic activity (attributed to CYP3A5) was unaffected. At concentrations up to 10 μM, it inhibited <10% of CYP3A5-mediated midazolam metabolism [1]
3. Selectivity against other CYP isoforms: PF-4981517 showed minimal inhibitory activity against other major human CYP isoforms. For CYP1A2 (substrate: phenacetin O-deethylation), CYP2C9 (substrate: tolbutamide 4-hydroxylation), CYP2C19 (substrate: omeprazole 5-hydroxylation), CYP2D6 (substrate: dextromethorphan O-demethylation), and CYP2E1 (substrate: chlorzoxazone 6-hydroxylation), the IC50 values were all >10 μM. At a concentration of 10 μM, the inhibition rate of each of these CYP isoforms was <15% [1]
4. Inhibition of CYP3A4-mediated metabolism of other drugs: PF-4981517 effectively inhibited CYP3A4-dependent metabolism of additional substrates. For testosterone 6β-hydroxylation (another CYP3A4 marker reaction) in HLMs, the IC50 was 0.028 μM; for simvastatin lactone hydrolysis (CYP3A4-mediated), the IC50 was 0.035 μM. At 0.1 μM, PF-4981517 inhibited >85% of these metabolic reactions [1]
.

1. Recombinant human CYP3A4 inhibition assay: Recombinant human CYP3A4, NADPH-generating system (glucose-6-phosphate, glucose-6-phosphate dehydrogenase, NADP+), and the substrate midazolam (final concentration 10 μM, a selective CYP3A4 substrate) were mixed in 0.1 M potassium phosphate buffer (pH 7.4). Different concentrations of PF-4981517 (0.001, 0.005, 0.01, 0.025, 0.05, 0.1, 0.5, 1 μM) or vehicle control (DMSO, final concentration ≤0.1%) were added to the mixture. The reaction was initiated by adding the NADPH-generating system and incubated at 37°C for 30 minutes. The reaction was terminated by adding 2 volumes of ice-cold acetonitrile containing an internal standard (e.g., diazepam). After centrifugation at 10,000 × g for 10 minutes, the supernatant was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantify the formation of 1'-hydroxymidazolam (the major metabolite of midazolam). The inhibition rate was calculated as [1 - (metabolite concentration in drug group / metabolite concentration in control group)] × 100%, and the IC50 value was obtained by fitting the concentration-inhibition curve using non-linear regression [1]
2. Human liver microsomal (HLM) CYP isoform selectivity assay: Pooled HLMs (from >10 donors) or CYP3A5-high expressor HLMs (CYP3A51/1 genotype) were used. For each CYP isoform, a specific substrate was added: phenacetin (CYP1A2), tolbutamide (CYP2C9), omeprazole (CYP2C19), dextromethorphan (CYP2D6), chlorzoxazone (CYP2E1), or midazolam (CYP3A4/CYP3A5). The assay mixture contained HLMs, NADPH-generating system, substrate, and PF-4981517 (0.001-10 μM) or vehicle control. Incubation and termination conditions were the same as the recombinant CYP3A4 assay. Metabolites of each substrate (e.g., acetaminophen for phenacetin, 4-hydroxytolbutamide for tolbutamide) were quantified by LC-MS/MS, and IC50 values for each CYP isoform were calculated [1]
3. Mechanism-based inactivation (kinact/Km) assay for CYP3A4: Recombinant human CYP3A4 and NADPH-generating system were mixed with different concentrations of PF-4981517 (0.01, 0.025, 0.05, 0.1, 0.2 μM) in buffer. At predetermined time points (0, 2, 5, 10, 15, 20, 30 minutes), aliquots of the mixture were removed and diluted 20-fold into a "reaction mix" containing midazolam and fresh NADPH-generating system (to measure remaining enzyme activity). The diluted aliquots were incubated at 37°C for 15 minutes, terminated with ice-cold acetonitrile, and 1'-hydroxymidazolam was quantified by LC-MS/MS. The natural logarithm of remaining enzyme activity (relative to time 0) was plotted against incubation time to calculate the inactivation rate (kobs) for each PF-4981517 concentration. A double-reciprocal plot of 1/kobs versus 1/[drug concentration] was used to determine the inactivation rate constant (kinact) and Michaelis constant (Km) for inactivation. The inactivation efficiency was expressed as the ratio kinact/Km [1]
.

[1]. Selective mechanism-based inactivation of CYP3A4 by CYP3cide (PF-04981517) and its utility as an in vitro tool for delineating the relative roles of CYP3A4 versus CYP3A5 in the metabolism of drugs. Drug Metab Dispos. 2012 Sep;40(9):1686-97.

1. Mechanism of action: PF-4981517 is a mechanism-based (suicidal) CYP3A4 inhibitor. After being metabolized by CYP3A4, it forms an active intermediate that covalently binds to the heme group or apolipoprotein of CYP3A4, leading to irreversible inactivation of the enzyme. This mechanism distinguishes it from reversible CYP3A inhibitors (such as ketoconazole) and ensures sustained inhibition of CYP3A4 in vitro [1] 2. Use as an in vitro research tool: PF-4981517 is designed specifically for in vitro studies to elucidate the relative contributions of CYP3A4 and CYP3A5 in drug metabolism. Because it can selectively inactivate CYP3A4 without affecting CYP3A5, treatment of HLM or recombinant CYP3A4/CYP3A5 mixtures with PF-4981517 (concentration ≥0.1 μM) can quantify CYP3A5-mediated metabolism (due to the remaining activity after complete inhibition of CYP3A4). This is crucial for understanding inter-individual differences in drug metabolism due to CYP3A5 gene polymorphisms (e.g., CYP3A51/1 vs. CYP3A53/3) [1]
3. Comparison with other CYP3A inhibitors: Unlike non-selective CYP3A inhibitors such as ketoconazole (which inhibits both CYP3A4 and CYP3A5) or ritonavir (a potent but non-specific CYP3A inhibitor), PF-4981517 is more than 400 times more selective for CYP3A4 than for CYP3A5 (based on IC50 ratios). This high selectivity avoids confounding results when studying CYP3A5-mediated metabolism, making it an excellent tool for CYP3A subtype specificity studies [1]
4. Limitations: PF-4981517 is for in vitro use only and its in vivo applications (e.g., as a therapeutic) have not been evaluated. Its inhibitory activity depends on NADPH (a substance necessary for the drug to be activated into an active intermediate via CYP3A4), and therefore it is ineffective in systems lacking functional CYP3A4 or NADPH[1].

C26H32N8

456.59

456.274

1390637-82-7

1390637-82-7;

71315139

Off-white to light yellow solid powder

1.3±0.1 g/cm3

630.2±55.0 °C at 760 mmHg

334.9±31.5 °C

0.0±1.8 mmHg at 25°C

1.725

2.77

0

6

4

34

678

1

CC1=CC=C(C=C1)C2=C(C=NN2C)C3=NN(C4=C3C(=NC=N4)N5CC[C@@H](C5)N6CCCCC6)C

WDWIMDKOXZZYHH-FQEVSTJZSA-N

InChI=1S/C26H32N8/c1-18-7-9-19(10-8-18)24-21(15-29-31(24)2)23-22-25(32(3)30-23)27-17-28-26(22)34-14-11-20(16-34)33-12-5-4-6-13-33/h7-10,15,17,20H,4-6,11-14,16H2,1-3H3/t20-/m0/s1

1-methyl-3-[1-methyl-5-(4-methylphenyl)pyrazol-4-yl]-4-[(3S)-3-piperidin-1-ylpyrrolidin-1-yl]pyrazolo[3,4-d]pyrimidine

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)