PF-4981517

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]
.

使用来自无功能 CYP3A5 供体的人肝微粒体 (CYP3A5 3/3)，在检查 CYP3cide 的抑制特性时，观察到非凡的代谢失活效率 (kinact/KI) 为 3300 至 3800 ml • min-1 • μmol-1 。该效率被发现对应于 420-480 nM 的表观 KI 和 1.6 min-1 的最大失活率 (kinact)。当在基因型多态性 CYP3A5 微粒体文库中以完全抑制 CYP3A4 的浓度和预孵育时间评估 CYP3cide 时，CYP3A5 丰度与剩余咪达唑仑 1'-羟化酶活性之间的联系很强[1]。

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1. 对重组人CYP3A4活性的抑制：将PF-4981517与重组人CYP3A4、NADPH生成系统及特异性底物咪达唑仑共同孵育时，其以浓度依赖性方式抑制咪达唑仑的1'-羟化反应。浓度为0.1 μM时，CYP3A4活性抑制率达>90%，IC50经测定为0.023 μM。通过超滤去除未结合药物后，抑制活性无显著丢失，证实其对CYP3A4的灭活具有不可逆性 [1]
2. 对人肝微粒体（HLMs）中CYP3A4的抑制：在混合人肝微粒体中，PF-4981517抑制咪达唑仑1'-羟化反应（CYP3A4的经典活性指标）的IC50为0.031 μM。在CYP3A5高表达供体（CYP3A51/1基因型）的肝微粒体中，PF-4981517（0.1 μM）可抑制>90%的CYP3A4介导咪达唑仑代谢，而剩余的代谢活性（归因于CYP3A5）不受影响；浓度高达10 μM时，其对CYP3A5介导咪达唑仑代谢的抑制率<10% [1]
3. 对其他CYP亚型的选择性：PF-4981517对其他主要人CYP亚型的抑制活性极低。对CYP1A2（底物：非那西丁）、CYP2C9（底物：甲苯磺丁脲）、CYP2C19（底物：奥美拉唑）、CYP2D6（底物：右美沙芬）、CYP2E1（底物：氯唑沙宗）的IC50均>10 μM；浓度为10 μM时，对这些CYP亚型的抑制率均<15% [1]
4. 对CYP3A4介导其他药物代谢的抑制：PF-4981517可有效抑制CYP3A4依赖的其他底物代谢。对人肝微粒体中睾酮6β-羟化反应（另一CYP3A4活性指标）的IC50为0.028 μM，对辛伐他汀内酯水解反应（CYP3A4介导）的IC50为0.035 μM；浓度为0.1 μM时，PF-4981517对这些代谢反应的抑制率均>85% [1]
。

1. 重组人CYP3A4抑制实验：将重组人CYP3A4、NADPH生成系统（葡萄糖-6-磷酸、葡萄糖-6-磷酸脱氢酶、NADP+）及特异性底物咪达唑仑（终浓度10 μM）在0.1 M磷酸钾缓冲液（pH 7.4）中混合，加入不同浓度的PF-4981517（0.001、0.005、0.01、0.025、0.05、0.1、0.5、1 μM）或溶媒对照（DMSO，终浓度≤0.1%）。加入NADPH生成系统启动反应，37℃孵育30分钟后，加入2倍体积含内标（如地西泮）的冰乙腈终止反应。10,000×g离心10分钟后，取上清液通过液相色谱-串联质谱（LC-MS/MS）定量咪达唑仑的主要代谢产物1'-羟基咪达唑仑。抑制率按[1 -（药物组代谢物浓度/对照组代谢物浓度）]×100%计算，采用非线性回归拟合浓度-抑制曲线获得IC50 [1]
2. 人肝微粒体CYP亚型选择性实验：使用混合人肝微粒体（来自>10名供体）或CYP3A5高表达人肝微粒体（CYP3A51/1基因型）。为每个CYP亚型加入特异性底物：非那西丁（CYP1A2）、甲苯磺丁脲（CYP2C9）、奥美拉唑（CYP2C19）、右美沙芬（CYP2D6）、氯唑沙宗（CYP2E1）或咪达唑仑（CYP3A4/CYP3A5）。实验体系包含肝微粒体、NADPH生成系统、底物及PF-4981517（0.001-10 μM）或溶媒对照，孵育及终止条件与重组CYP3A4实验一致。通过LC-MS/MS定量各底物的代谢产物（如非那西丁的代谢产物对乙酰氨基酚、甲苯磺丁脲的代谢产物4-羟基甲苯磺丁脲），计算每个CYP亚型的IC50 [1]
3. CYP3A4机制性灭活（kinact/Km）实验：将重组人CYP3A4与NADPH生成系统在缓冲液中与不同浓度的PF-4981517（0.01、0.025、0.05、0.1、0.2 μM）混合。在预设时间点（0、2、5、10、15、20、30分钟）取出部分混合液，稀释20倍后加入含咪达唑仑和新鲜NADPH生成系统的“反应体系”中（用于测定剩余酶活性）。稀释后的样品在37℃孵育15分钟，用冰乙腈终止反应，通过LC-MS/MS定量1'-羟基咪达唑仑。以剩余酶活性（相对于0时刻）的自然对数对孵育时间作图，计算各PF-4981517浓度下的灭活速率（kobs）；通过1/kobs对1/[药物浓度]的双倒数图，测定灭活速率常数（kinact）和灭活米氏常数（Km），灭活效率以kinact/Km比值表示 [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 acts as a mechanism-based (suicide) inhibitor of CYP3A4. Upon metabolism by CYP3A4, it forms a reactive intermediate that covalently binds to the heme group or apoprotein of CYP3A4, leading to irreversible inactivation of the enzyme. This mechanism distinguishes it from reversible CYP3A inhibitors (e.g., ketoconazole) and ensures prolonged inhibition of CYP3A4 in vitro [1]
2. Utility as an in vitro research tool: PF-4981517 is specifically designed for in vitro studies to delineate the relative contributions of CYP3A4 and CYP3A5 to the metabolism of drugs. Since it selectively inactivates CYP3A4 without affecting CYP3A5, treating HLM or recombinant CYP3A4/CYP3A5 mixtures with PF-4981517 (at concentrations ≥0.1 μM) allows quantification of CYP3A5-mediated metabolism (as the remaining activity after CYP3A4 is fully inhibited). This is critical for understanding interindividual variability in drug metabolism due to CYP3A5 genetic 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 exhibits >400-fold selectivity for CYP3A4 over CYP3A5 (based on IC50 ratios). This high selectivity avoids confounding results when studying CYP3A5-mediated metabolism, making it a superior tool for CYP3A subtype-specific research [1]
4. Limitations: PF-4981517 is intended for in vitro use only and has not been evaluated for in vivo applications (e.g., as a therapeutic agent). Its inhibitory activity is dependent on NADPH (required for CYP3A4-mediated activation of the drug to its reactive intermediate), so 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

67.9

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)