Irreversible Inhibitors

Irreversible inhibitors are important for research that involves studying reaction mechanisms. Discover more about irreversible inhibitors including the types of irreversible inhibitors: suicide inhibitors, heavy metal inhibitors, and time-dependent inhibitors.

Read more about

• What Are Irreversible Inhibitors?
• Suicide Inhibitors
• Heavy Metal Inhibitors
• Time-Dependent Inhibitors
• Related Inhibitor Products

What Are Irreversible Inhibitors?

Irreversible inhibitors are inhibitors that bind to enzymes and inactivate them, making the substrate concentration irrelevant (i.e., adding more substrate does not reverse the enzyme inhibition). They are non-competitive in nature. Irreversible inhibitors include nonspecific protein denaturing agents, such as acids and alkalis, and specific agents, which attack a specific component of the holoenzyme system.

Specific inhibitors can be grouped as:

• Coenzyme inhibitors
• Inhibitors of a specific ion cofactor
• Prosthetic group inhibitors
• Apoenzyme inhibitors
• Physiological modulators of the reaction
  • For example, the pH and temperature that denature the enzyme catalytic site

Most irreversible inhibitors interact with functional groups on the enzyme and destroy enzyme activity. These interactions are covalent in nature. These inhibitors are highly useful in studying reaction mechanisms. Common types of irreversible inhibitors are suicide inhibitors, heavy metal inhibitors, and time-dependent inhibitors.

Suicide Inhibitors

Suicide inhibitors are a special group of irreversible inhibitors that are relatively unreactive until they bind to the active site of the enzyme. In the first few steps of the reaction, they function as a normal substrate, but then they are converted into a very reactive compound that combines with the enzyme to block its activity. Because they use the normal enzyme reaction mechanism to inactivate the enzyme, they are also known as mechanism-based inhibitors or transition state analogs. Suicide inhibitors that exploit the transition state-stabilizing effect of the enzyme result in higher enzyme binding affinity than do substrate-based inhibitor designs.

This approach is highly useful in developing pharmaceutical agents with minimal side effects. However, designing drugs that precisely mimic the transition state is a real challenge because of the unstable, poorly characterized structure of the transition state. Prodrugs undergo initial reaction(s) to form an overall electrostatic and three-dimensional intermediate transition state complex form with close similarity to that of the substrate. These prodrugs serve as guidelines to further develop transition state molecules with modifications.

The suicide inhibitor removes the enzyme and reduces the formation of the enzyme-substrate (ES) complex. The V_max value is reduced, and inhibition cannot be overcome by adding extra substrate. In this regard, suicide inhibition resembles noncompetitive inhibition.

Examples of Suicide Inhibitor Drugs

Allopurinol

A common example of a suicide inhibitor is allopurinol, the anti-gout drug that inhibits xanthine oxidase activity. The enzyme commits suicide by initially activating allopurinol into oxypurinol (a transition state analog) that binds very tightly to the molybdenum-sulfide (Mo-S) complex at the active site of xanthine oxidase.

Acyclovir

Acyclovir (acycloguanosine (2-amino-9-((2- hydroxyethoxy)methyl)-1H-purin-6(9H)-one) is one of the most commonly used antiviral agents with very low toxicity. It is selectively converted into acyclo-guanosine monophosphate (acyclo-GMP) by viral thymidine kinase. Acyclo-GMP is further phosphorylated into the active triphosphate form, acyclo-GTP, by cellular kinases. Acyclo-GTP is a very potent inhibitor of viral DNA polymerase with over 100-fold greater affinity for viral polymerase than cellular polymerase. It is incorporated into viral DNA, resulting in chain termination.

Heavy Metal Inhibitors

Heavy metal ions, such as mercury and lead, can bind tightly to enzymes and inhibit their activity. Heavy metal inhibitors exhibit a higher affinity for enzymes with sulfhydryl (–SH) groups. When they are present in larger quantities their action is rather nonspecific, and they can inhibit multiple enzymes. This can make it unclear which particular enzyme is most affected. Heavy metal inhibition of critical enzymes may result in poisoning, which can be treated by administering metal ion chelators.

Time-Dependent Inhibitors

Time-dependent inhibitors are inhibitors that exhibit slow binding to the enzyme. The observed onset of inhibition is slower. These inhibitors display nonlinear initial velocities and nonlinear recoveries of enzyme activity with slow k_off values (the rate constant of dissociation between enzyme and inhibitor). Time-dependent inhibition is a severe form of inhibition and overcoming this inhibition requires de novo enzyme synthesis.

Some time-dependent inhibitors covalently interact with enzymes. For these inhibitors, the k_off value approaches zero, and inhibition is irreversible. These molecules are less useful for most biological research unless the covalent species provides good information about the reaction mechanism.

Interestingly, many successful therapeutic drugs are time-dependent inhibitors. In these cases, with slow k_off values, the rate of release of the inhibitor from the enzyme-inhibitor complex proceeds independent of the substrate concentration, making them attractive for the drug discovery process.

Examples of Time-Dependent Inhibitor Drugs

One attractive therapeutic target of time-dependent inhibition is cytochrome P450 (CYP) 3A, which is responsible for the metabolism of about 60% of currently known drugs. Inhibition of CYP by interactions of co-administered drugs can lead to overexposure and has been attributed to the withdrawal of several drugs from the market. Time-dependent inhibition can increase the potency of drugs by blocking their degradation. This is due to either the formation of a tight-binding, quasi-irreversible inhibitory metabolite or by inactivation of CYP enzymes by covalent adduct formation. Some of the inhibitors of CYP include popular antibiotics like azithromycin and the antidepressant fluoxetine.

Explore more about inhibitors in our “How to Use Inhibitors” article.