The effect on the efficacy and potency of a non-competetive antagonist binding to the active site of the receptor (dose-response curve)

Question

According to the book "Principles of Pharmacology: The Pathophysiologic Basis of Drug Therapy" by Golan et al, non-competetive antagonists can bind to both the allosteric site and the active site.

I know that a non-competetive antagonist that binds to the allosteric site causes a decrease in efficacy, but no decrease in potency (see figure below). Figure A shows the agonist alone and the agonist together with a competetive antagonist. This causes the dose response curve to shift to the right, thus increasing the EC50 and decreasing the potency of the agonist. Figure B shows the dose-response curve for the agonist alone and agonist together with a non-competetive antagonist that binds to an allosteric site. As you see this type of antagonist causes a decrease in efficacy, but no decrease in potency is observed.

But how does a non-competetive antagonist that binds to the active site affect the dose-response curve? Does it lead to both a decrease in efficacy and potency? Unfortunately, there is no figure in the book to answer my question.

Answer 1

Short anwer

'Non-competitive active site–binding inhibitors' are called mixed-type inhibitors. These inhibitors exhibit features of both competitive and non-competitive inhibitors, as they increase K_m (like a competitive inhibitor) and decrease V_max (like a non-competitive inhibitor).


Background

What an interesting question!

In theory, a reversible inhibitor binding to the active site of an enzyme is per definition competitive. In a review by Blat (2010) the author mentions that active site–binding inhibitors that display non-competitive inhibition are indeed unusual. Such inhibitors are referred to as mixed-type inhibitors. In many cases the unusual behavior is observed with (1) enzymes utilizing an exosite for substrate binding, or (2) isomechanism enzymes, (3) enzymes with multiple substrates/products and⁄or (4) products and two-step binding inhibitors.

(1) Enzymes with an exosite have a substrate recognition site that is different from the active site. For example, some proteases bind their target on the exosite and then catalyze proteolysis in the active site. Mixed-type inhibitors then bind to the exosite, thereby inducing non-competitive behavior, as the active site is not bound.

(2) isomechanism enzymes are enzymes that undergo several structural transitional changes during catalysis. When one of these conformations is rate limiting and binds the mixed-type inhibitor, it may non-competitively inhibit the enzyme when another conformational form binds the substrate.

(3) Enzymes with multiple substrates or products sometimes follow a sequential binding and release of two substrates (or products). Suppose substrate A and cofactor B (e.g. NADPH). The enzyme converts substrate A into A' and cofactor B to B'. Now suppose that the enzyme obligatory has to release A' before B' can be released and that the B' bound state is unable to bind substrate A in the active site, but is able to bind the mixed-type inhibitor. In this case, again, a non-competitive inhibition pattern is observed.

(4) Two-step binding inhibitors refer to inhibitors that cause a conformational change of the enzyme after binding its inhibitor, that very slowly reverts back. In the conformationally changed state, the enzyme cannot bind the substrate and competition with substrate is lost. The most extreme case are inhibitors that covalently bind to the active site (note this is the mechanism addressed by @RoverEye).

You ask what the dose-response graphs are of mixed-type inhibitors. A more common way of displaying inhibitor behavior is using Lineweaver-Burk plots. Many enzymes follow Michaelis-Menten kinetics (Berg et al., 2002) and by plotting the enzyme kinetics as Lineweaver-Burk plots more insight is gained into the affinity (K_m, you call this 'potency') and maximum rate of the enzyme (V_max, you call this 'efficacy') compared to dose-response graphs. In Lineweaver-Burk plots the reciprocal velocity of the enzyme is plotted against the reciprocal of the substrate concentration, resulting in a straight line out of which affinity an maximum velocity can be easily obtained via linear regression.

The following graphs obtained from Illinois Institute of Technology show the three different modes of inhibition discussed, starting with the two common types:

^{Competitive inhibitors increase K_m (i.e., decrease substrate affinity)}.

^{Non-competitive inhibitors decrease Vmax (i.e., decrease substrate turnover)}

^{Mixed-type inhibitors increase Km and decrease Vmax}


References
- Berg et al. (2002). Biochemistry, 5^th edition
- Blat, Chem Biol Drug Des 2010;75(6):535-40

Answer 2

This is a tough question. I was reading this paper Patrono, C., et al. "Clinical pharmacology of platelet cyclooxygenase inhibition." Circulation 72.6 (1985): 1177-1184. and they seemed t mention this paragraph in the introduction.

Platelet Cycloxygenase or prostaglandin (PG) H synthase (i.e., the enzyme that converts arachidonate released from membrane phospholipids into PG endoperoxides) is the target of several antiplatelet agents that can reversibly or irreversibly block the activity of the enzyme by competing with the substrate or permanently altering the active site, respectively.Such drugs belong to the class of so-called nonsteroidal anti-inflammatory agents (e.g., indomethacin, aspirin), but also include a uricosuric agent, i.e., sulfinpyrazone.

So we have to look for DRC of a NSAID to say COX. Now I was unable to find a good DRC for aspirin (If you find it, let me know in the comments below. I would love to take a look at it). But I did find DRC for other drugs that inhibit COX.

It led me to this paper Walker, M., et al. "A three-step kinetic mechanism for selective inhibition of cyclo-oxygenase-2 by diarylheterocyclic inhibitors." Biochem. J 357 (2001): 709-718. which described the inhibiton of COX2 by a NSAID called celecoxib.

The inhibition of COX isoenzymes by NSAIDs generally conforms to one of three inhibitory mechanisms: simple reversible inhibition, as demonstrated by ibuprofen ; time-dependent reversible inhibition, which includes both weaker binding inhibitors, such as naproxen , and tight binding inhibitors, such as indomethacin and meclofenamic acid ; and irreversible covalent inhibition, as demonstrated by aspirin and o-(acetoxy-phenyl)hept-2-ynyl sulphide ('APHS') . Most of the traditional NSAIDs display similar inhibitory mechanisms against both COX-1 and COX-2 , and are relatively non-selective. However, COX-2-selective diarylheterocyclic inhibitors demonstrate distinct inhibitory mechanisms for the two isoforms . For example, celecoxib has been reported to be a reversible competitive inhibitor of COX-1 while demonstrating time-dependent irreversible inhibition of COX-2.

Considering the highlighted statement, I think you can consider the COX1 curve as a standard competitive inhibition and COX2 as non competitive inhibition. The resulting change is essentially the curve shape itself (being shifted to the right).

The DRCs were reported here: Tacconelli, Stefania, et al. "The biochemical selectivity of novel COX-2 inhibitors in whole blood assays of COX-isozyme activity." Current Medical Research and Opinion® 18.8 (2002): 503-511.

Interestingly the second paper (By Walker) also described how (the authors proposed) the binding occurs. A very interesting read.