Theoretical validation of the use of the Golden Approach for Open Channel Blocking Drugs

GE Jarvis1, AJ Thompson2. 1Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UK, 2Department of Biochemistry, University of Cambridge, Cambridge, UK

We recently reported a simple method for investigating the mechanism of the action of ion channel blocking drugs (1). It addresses the question: “Do two channel-blocking drugs bind at the same site?” and works by determining the extent of channel blockade in the presence of two channel-blocking drugs using concentrations that each elicit inhibition equal to the Golden Ratio (~61.8%) when acting alone.

There are two key mechanistic conditions that underlie the logic of the Golden Approach: (i) the channel-blocking drugs are not state selective; (ii) each drug does not exert an allosteric effect that modifies the binding affinity of the other. It is widely accepted that many channel-blocking drugs do not conform to this restrictive profile. To validate the Golden Approach for open channel blockers, we have conducted theoretical modelling using the following model of agonist-induced channel opening:

A + R ⇔ AR ⇔ AR*

A = agonist; R = channel in closed conformation

R* = channel in open conformation

Kd = dissociation constant (Kd = [A][R]/[AR])

E = agonist efficacy (E = [AR*]/[AR])

We modelled simultaneous binding of two channel-blocking drugs that act either at the same site (syntopically) or at separate sites (allotopically) under the following conditions:

1. Non-selective Channel Blockade (NCB): the affinity of the blockers is the same for R, AR and AR*

2. Absolutely-selective Open Channel Blockade (OCB): the blockers only bind to AR*

Using the model, we identified conditions that maximised the predicted difference between pairs of channel blockers acting either syntopically or allotopically. Supra-maximal agonist concentrations ([A] = 1,000 × Kd) are used to ensure full occupancy. For NCB, maximisation occurs, irrespective of the value of E, when the concentrations of channel blockers used cause inhibition equal to ~61.8% when used alone. For OCB, the conditions for maximisation are dependent on E. However, Fig. 1 shows that when using high efficacy agonists (E>10, 91% channels open) the Golden Approach remains applicable for OCB.

When two channel-blocking drugs bind allotopically, variance between experimentally observed and theoretically predicted results may arise as a consequence of allosteric effects. The difference between the predicted and observed inhibitions is therefore a measure of this allosterism. Further modelling will enable this effect to be quantified for both open and non-selective channel blockers.

(1) Jarvis GE & Thompson AJ (2013) Trends in Pharm Sci 34: 481-488.