Reversible inhibitors slow onset

To distinguish between simple, reversible slow binding (scheme B) and an enzyme isomerization mechanism (scheme C), one can examine the dependence of kobs on inhibitor concentration. If the slow onset of inhibition merely reflects inherently slow binding and/or dissociation, then the term kobs in Equations (6.1) and (6.2) will depend only on the association and dissociation rate constants k3 and k4 as follows ... 

In an attempt to avoid problems with the phosphate groups from the CoA moiety, the penultimate intermediate in the synthesis of the bisubstrate inhibitor, A-bromoacetyltryptamine, was tested as a possible affinity label inhibitor or as a substrate for the in situ enzymatic synthesis of the bisubstrate inhibitor through an acylation mechanism. N-Bromoacetyltryptamine did act as an inhibitor of the enzyme, but inhibition could be reversed by dialysis suggesting that the inhibition was not due to a covalent adduct. It was shown that the enzyme catalyzed the acylation of A-bromoacetyltryptamine by CoASH to form compound 6a with a rate enhancement of 3.3 x 10 relative to the uncatalyzed reaction. Ultimately, it was shown that the acylation reaction occurs at the same active site as the acetylation activity. A closer inspection of the kinetics of inhibition by tbe bisubstrate analog 6a resulted in the observation of slow-onset inhibition over the first few minutes of the reaction with a A) value of 84nmol 1 . Owing to its neutrality, A-bromoacetyltryptamine was tested as an inhibitor in vivo. The analog precursor was shown to inhibit melatonin production in norepinephrine-stimulated pinealocytes in a concentration-dependent manner and with low cytotoxicity. 

Finasteride was subsequentiy shown to be a slow-onset inhibitor of type 1 5AR by researchers at Glaxo Research Institute. Attempts to dialyze and remove the inhibitor from a saturated enzyme—inhibitor complex again failed to display any reactivation of the enzyme even after 3 days. This led to the conclusion that the complex is either irreversible or has a half-life of greater than 10 days. The authors of the second study also demonstrated that the presence of a double bond in ring A of 5AR inhibitors is responsible for the appearance of the slow-onset kinetics. This, combined with the inability to demonstrate reversibility, was cited as further evidence for a covalent modification of the enzyme active site. 

Time-dependent inhibition defined mainly by mechanism-based inhibition (MBI), which includes CYP suicide inactivation (irreversible inhibition, the more widely studied process) and metabolite-intermediate (MI) complex formation (quasi-irreversible inhibition), is responsible for most clinically significant DDIs (Silverman, 1995 Waley, 1980 Zhou et al., 2005). Suicide inactivation involves the formation of a reactive intermediate that irreversibly inactivates the CYP in the process of catalytic turnover. Quasi-irreversible inhibition occurs when the CYP produces a metabolite (e.g., nitroso intermediate) with the capacity to bind tightly to the CYP heme. TDI (time-dependent inhibition) can be characterized (1) to be dose dependent, (2) to be preincubation time dependent, (3) to have bioactivation of the inhibitor that is required for inactivation of the target enzyme, (4) to have de novo protein synthesis that is required to recover metabolic capacity, and (5) to have potentially slow onset of the effects but be more profound than reversible inhibition. If present, then TDI is the major component of overall enzyme inhibition and frequently leads to clinically relevant DDIs. Table 4.5 contains a list of inhibitors of TDI observed in vitro and in vivo. 

This inhibition was ascribed originally to a possible resemblance between this inhibitor and a possible carbonium ion intermediate in substrate transformation. Although inhibition was reversible and competitive as expected, it was later shown to be measurably slow in its onset, and both the rate of onset and the rate of release of inhibitor could be determined by measurement of residual catalytic activity using ordinary steady-state methods. Rates of onset and release were in reasonable agreement with the measured Ki value of galactal, but the released inhibitor was found to consist entirely of 2-deoxygalactose. This could be explained if the enzyme reacted covalently with the inhibitor to yield a 2-deoxygalactosyl-enzyme derivative, which is hydrolyzed slowly with recovery of activity. 

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