INDEX protein adducts

The partitioning of the activated inhibitor between direct covalent inactivation of the enzyme and release into solution is an important issue for mechanism-based inactivators. The partition ratio is of value as a quantitative measure of inactivation efficiency, as described above. This value is also important in assessing the suitability of a compound as a drug for clinical use. If the partition ratio is high, this means that a significant proportion of the activated inhibitor molecules is not sequestered as a covalent adduct with the target enzyme but instead is released into solution. Once released, the compound can diffuse away to covalently modify other proteins within the cell, tissue, or systemic circulation. This could then lead to the same types of potential clinical liabilities that were discussed earlier in this chapter in the context of affinity labels, and would therefore erode the potential therapeutic index for such a compound. 

The amount of AG that is covalently bound to protein is dependent both on the concentration of AG and the degree of reactivity to form protein and peptide adducts. Historically, AG reactivity has been expressed as the percentage of total AG that is involved in the reaction with protein [26,27]. For this technique, the AG reactivity is calculated as reactivity index, C%, which is the ratio of ion current peak areas of AG peptide adducts (peak area a + b in Figure 10.15 A) to those that correspond to AGs from the same sample (peak area a -F b -F c in Figure 10.14A) multiplied by 100. The C% for DCL-AG was determined to be 0.88%. The AG reactivities of the seven drugs were ranked based on their C% (Table 10.3). 

The formation of adducts through a Schiff base mechanism was the basis to assess the reactivity of the seven model compounds with this new technique. The reactivity index generated with our method was consistent with those reported by Benet et al. [26] and Bolze et al. [27] (Table 10.3), which validated this technique to evaluate AG reactivity. Schiff base adducts of AGs and proteins were obtained from the literature for TOL, ZOM, and DCL... 

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