Drug Discovery
Table 1: For the same affinity, on and off-rates can vary enormously and cannot be predicted (illustrated here using mock data)
AFFINITY (KD)
1 x 10-9 1 x 10-9 1 x 10-9 1 x 10-9 1 x 10-9 105 106 107 108 109
K-ON M-1 MIN-1
K-OFF MIN-1 0.0001 (6900 min) 0.001 (690 min) 0.01 (69 min) 0.1 (6.9 min) 1 (0.69 min) SPEED
Exceptionally slow on/off
Slow on/off Fast on/slow off Very fast on/fast off
Exceptionally fast on/off
Improved selectivity due to differential off-rates
guide to efficacy duration and it is a general find- ing that drugs with slow-off-rates are found by serendipity. We can see the impact of slow-off clearly in Table 2 by comparing data from some M3 antagonists.
In the case of the Tiotropium, the molecule exhibits very long lasting effects with very little variation in bronchiodilatation between peak and trough (the time between administration and next administration) which rendered it the first truly once-a-day bronchodilator10. Plasma levels of Tiotropium at trough are in the low pg/ml range and hence very unlikely to explain the sustained effectiveness of the drug, which is instead ascribed to its slow dissociation from the M3 receptors in the lung9. In addition to long duration of action, the ability to retain effectiveness at lower blood levels endows a large therapeutic window. Additionally, since Tiotropium has a faster dissoci- ation rate from M1 and M2 muscarinic receptors than from M3 receptors, an improved selectivity is
Table 2: Muscarinic M3 antagonists with similar affinities but very different off-rates9,10 KD/KI
ON RATE X109M-1 MIN-1 Atropium Ipatropium Clidinium Tiotropium 40 0.2 nM 0.2 nM 0.3 nM 8 pM 1.5 0.5 10 0.16 0.27 (2 mins) 0.07 (10 mins) 0.02 (30 mins) 0.0015 (34.7 hrs)
Since many compounds bind to a number of pro- teins in addition to their intended target, improved selectivity for the chosen target can be a strong dif- ferentiator across a class of drugs. By monitoring not only the dissociation rate of the compound from its intended target but also from other poten- tial selectivity or ADME targets, it should be pos- sible to identify compounds with preferred charac- teristics where a faster off-rate from the unintend- ed targets is observed in the presence of slow off- rate for the intended target. Table 3 gives some examples of these findings.
Fast on rates Improved in vivo efficacy where agonist concentrations are high Despite a 50,000-fold difference in in vitro Ki val- ues for two thrombin inhibitors, hirudin and mala- gatran, comparable plasma concentrations resulted in comparable antithrombotic effects in vivo. In contrast, comparable in vitro Ki values and in vivo
OFF-RATE MIN-1
EC50 scores were obtained when malagatran was compared with inogatran. These differing profiles have been explained by the concentration of thrombin in the thrombus and the rate of attaining an effective inhibitory concentration of antagonist drug. For the inhibitors tested (eight in total) there was an inverse relationship between k-on values in vitro and the slope of the dose response curves in
vivo with inhibitors with k-on values of <1x107M- 1 s-1 having steep slopes with Hill coefficients >1 and better therapeutic index due to rapidly reach- ing critical concentration for efficacy. Contrarily, the association time for inhibition of thrombin by slow binding inhibitors is too long to give effective antithrombotic effects at low plasma concentra- tions. Where higher plasma concentrations are achieved, the association time decreases resulting
Drug Discovery World Summer 2011
apparent for this drug over other mixed M1/M2/ M3 antagonists.
Changes in off-rate leading to large differences in duration of action are most obvious where a compound series is subject to rapid clearance. Slowly dissociating antagonists, where the half life of the antagonist-receptor complex exceeds that of the free antagonist are likely to maintain a longer receptor protection in vivo than fast dissociating antagonists. Where the half life of the free antago- nist is longer than the half life of the complex, longer occupation by the slowly dissociating antag- onist is only relevant if the receptor is subject to large fluctuations in free agonist concentration1.
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