Irreversible (covalent) enzyme inhibitors cannot be unambiguously ranked for biochemical potency by using IC₅₀ values determined at a single point in time, because the same IC₅₀ value could originate either from relatively low initial binding affinity accompanied by high chemical reactivity, or the other way around. To disambiguate the potency ranking of covalent inhibitors, here we describe a data-analytic procedure relying on two separate IC₅₀ values, determined at two different reaction times. In the case of covalent inhibitors following the two-step kinetic mechanism E + I ⇌ E·I → EI, the two IC₅₀ values alone can be used to estimate both the inhibition constant (K_i) as a measure of binding affinity and the inactivation rate constant (k_inact) as a measure of chemical reactivity. In the case of covalent inhibitors following the one-step kinetic mechanism E + I → EI, a simple algebraic formula can be used to estimate the covalent efficiency constant (k_inact/K_i) from a single experimental value of IC₅₀. The two simplifying assumptions underlying the method are (1) zero inhibitor depletion, which implies that the inhibitor concentrations are always significantly higher than the enzyme concentration; and (2) constant reaction rate in the uninhibited control assay. The newly proposed method is validated by using a simulation study involving 64 irreversible inhibitors with covalent efficiency constants spanning seven orders of magnitude.

中文翻译：

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用于评估不可逆酶抑制剂的生化效力的两点 IC50 方法

不能通过使用在单个时间点确定的IC ₅₀值对不可逆（共价）酶抑制剂的生化效力进行明确排序，因为相同的 IC ₅₀值可能源自伴随高化学反应性的相对较低的初始结合亲和力，或其他绕路。为了消除共价抑制剂的效力等级的歧义，我们在此描述了依赖于在两个不同反应时间确定的两个独立 IC ₅₀值的数据分析程序。在共价抑制剂遵循两步动力学机制E + I ⇌ E · I → EI的情况下，两个 IC ₅₀单独的值可用于估计抑制常数 ( K _i ) 作为结合亲和力的量度和失活速率常数 ( k _inact ) 作为化学反应性的量度。在共价抑制剂遵循一步动力学机制E + I → EI的情况下，可以使用简单的代数公式从 IC ₅₀的单个实验值估算共价效率常数 ( k _inact / K _{i )}. 该方法的两个简化假设是（1）零抑制剂消耗，这意味着抑制剂浓度总是显着高于酶浓度；(2) 不受抑制的对照试验中的恒定反应速率。新提出的方法通过使用涉及 64 种不可逆抑制剂的模拟研究得到验证，共价效率常数跨越 7 个数量级。