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Kinetics Layout 1 23/06/2011 13:26 Page 39 Kinetics_Layout 1 23/06/2011 13:26 Page 39 Drug Discovery The need for high throughput kinetics early in the drug discovery process Generally kinetics are only investigated late in a programme by which time many compounds, slightly less potent but with a much better kinetic profile, have already been discarded. This paper argues that having kinetic information at the early stages of drug discovery can bring numerous benefits to the process. he majority of drug discovery projects rely is not possible to predict these from the KD as Dr Wilma Keighley upon estimates of compound affinity to a shown in Table 1. Ttarget protein to guide lead seeking medic- So, are on and off-rates critical information or inal chemistry in early stages when high compound merely nice to know? The overwhelming answer is numbers are being screened. At this stage it is now that they are critical. Andersson et al1 recognise quite common to test at IC50 level not only against kinetic binding data as a decisive element in drug the primary target but also against several selectiv- discovery, while the importance of drug-target res- ity and toxicity targets, so choosing compounds for idence time (as quantified by the dissociative half progression against multiple criteria. Nevertheless life of the drug-target binary complex) is empha- any efforts to measure the on and off-rates that sised for its potential impact on duration of effect underlie the affinity (to either the primary target or and target selectivity by Copeland et al2 and selectivity target) are left to much later stages of the Zhang and Monsma3. Hopkins (www.hop- campaign when fewer compounds are tested. This kinslab.org) puts this firmly into the perspective of is simply due to the high cost and low throughput the patient ‘understanding the binding kinetics can of the specialist biosensor instruments typically help shape the clinical profile of a drug that are used to determine binding kinetics. important to patients: efficacy, safety, duration of However, evidence is plentiful that compounds action, greater tolerability, indication and thera- with the same affinity but different on and off-rates peutic differentiation’. The following examples can have a very different biological activity profile. show why this is so. The affinity-driven triage strategy currently used therefore results in superior compounds being Slow off-rates rejected early in the discovery process. Improved Increased residence time gives methods and technologies enabling cost-effective, better duration of action convenient and high throughput access to kinetic A survey of the literature identifies a number of data would create opportunities for a much drugs, across a variety of target families, having improved early drug discovery paradigm. The exceptionally long duration of action, which can attributes of an ideal technology are discussed. be ascribed to very slow off-rates when compared Affinity (KD) for a target protein, derived from to other drugs of the same class. Examples include 4 5 6 IC50, is used regularly to rank compound perform- Aprepitant , Amlodipine , Maraviroc , 7 8 9 ance. KD is dependent on both the association (on) Candesartan , Saquinavir and Tiotropium . and dissociation (off) rates of the compound and it In all of these cases, affinity would have been no Drug Discovery World Summer 2011 39 Kinetics_Layout 1 23/06/2011 13:26 Page 40 Drug Discovery Table 1: For the same affinity, on and off-rates can vary enormously and cannot be predicted apparent for this drug over other mixed M1/M2/ (illustrated here using mock data) M3 antagonists. Changes in off-rate leading to large differences AFFINITY K-ON M-1 K-OFF MIN-1 SPEED in duration of action are most obvious where a -1 (KD) MIN compound series is subject to rapid clearance. Slowly dissociating antagonists, where the half life 1 x 10-9 105 0.0001 (6900 min) Exceptionally slow of the antagonist-receptor complex exceeds that of on/off the free antagonist are likely to maintain a longer receptor protection in vivo than fast dissociating 1 x 10-9 106 0.001 (690 min) Slow on/off antagonists. Where the half life of the free antago- nist is longer than the half life of the complex, 1 x 10-9 107 0.01 (69 min) Fast on/slow off longer occupation by the slowly dissociating antag- onist is only relevant if the receptor is subject to large fluctuations in free agonist concentration1. 1 x 10-9 108 0.1 (6.9 min) Very fast on/fast off Improved selectivity due to 1 x 10-9 109 1 (0.69 min) Exceptionally fast differential off-rates on/off 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 guide to efficacy duration and it is a general find- not only the dissociation rate of the compound ing that drugs with slow-off-rates are found by from its intended target but also from other poten- serendipity. We can see the impact of slow-off tial selectivity or ADME targets, it should be pos- clearly in Table 2 by comparing data from some sible to identify compounds with preferred charac- M3 antagonists. teristics where a faster off-rate from the unintend- In the case of the Tiotropium, the molecule ed targets is observed in the presence of slow off- exhibits very long lasting effects with very little rate for the intended target. Table 3 gives some variation in bronchiodilatation between peak and examples of these findings. trough (the time between administration and next administration) which rendered it the first truly Fast on rates once-a-day bronchodilator10. Plasma levels of Improved in vivo efficacy where Tiotropium at trough are in the low pg/ml range agonist concentrations are high and hence very unlikely to explain the sustained Despite a 50,000-fold difference in in vitro Ki val- effectiveness of the drug, which is instead ascribed ues for two thrombin inhibitors, hirudin and mala- to its slow dissociation from the M3 receptors in gatran, comparable plasma concentrations resulted the lung9. In addition to long duration of action, in comparable antithrombotic effects in vivo. In the ability to retain effectiveness at lower blood contrast, comparable in vitro Ki values and in vivo levels endows a large therapeutic window. EC50 scores were obtained when malagatran was Additionally, since Tiotropium has a faster dissoci- compared with inogatran. These differing profiles ation rate from M1 and M2 muscarinic receptors have been explained by the concentration of than from M3 receptors, an improved selectivity is thrombin in the thrombus and the rate of attaining an effective inhibitory concentration of antagonist drug. For the inhibitors tested (eight in total) there Table 2: Muscarinic M3 antagonists with similar affinities but very different off-rates9,10 was an inverse relationship between k-on values in KD/KI ON RATE OFF-RATE vitro and the slope of the dose response curves in X109M-1 MIN-1 MIN-1 vivo with inhibitors with k-on values of <1x107 M- 1 s-1 having steep slopes with Hill coefficients >1 Atropium 0.2 nM 1.5 0.27 (2 mins) and better therapeutic index due to rapidly reach- ing critical concentration for efficacy. Contrarily, Ipatropium 0.2 nM 0.5 0.07 (10 mins) the association time for inhibition of thrombin by Clidinium 0.3 nM 10 0.02 (30 mins) slow binding inhibitors is too long to give effective antithrombotic effects at low plasma concentra- Tiotropium 8 pM 0.16 0.0015 (34.7 hrs) tions. Where higher plasma concentrations are achieved, the association time decreases resulting 40 Drug Discovery World Summer 2011 Kinetics_Layout 1 23/06/2011 13:26 Page 41 Drug Discovery in a steeper dose response curve but at the price of laboratory species. If on/off-rates vary in human a narrowed therapeutic interval11. In examples versus animal receptors, an expected margin of such as this, because the need to achieve critical safety observed in one species could disappear due concentration in a short time is the crucial factor in to different kinetic profiles in the human at the rel- determining efficacy, on-rate is a much better pre- evant receptors – or vice versa: a compound reject- dictor of efficacy than Ki. ed due to poor margin of safety or selectivity in an animal species could be, in fact, adequately selec- How could we apply on and off-rate tive for the desired over the unwanted effects in properties to enable better drug human. Such variations would be particularly discovery? important where efficacy and safety studies are In vitro screening conducted in different species. Understanding of kinetics at early stages of Drug Is a fast Koff of use? It is possible to envisage that Discovery, even at the screening phase, ie before iv dosage forms of a drug with fast off-rate would many compounds have been rejected, gives access enable a fast wash out of drug – short acting anaes- to more diverse chemical space, more scope for thetic perhaps, or other use where tight control on intellectual property (IP) and patents, and better the duration of effect would be valuable. defined biology. Receptor selectivity can be driven by off-rate and not necessarily affinity and affinity Manipulation of kinetic profiles and off-rate SAR may diverge with large differ- An understanding of kinetics, at an early stage in a ences in dissociation rates within a chemical series lead seeking programme, could give real assistance being not uncommon. In addition to kinetics at the by knowing in which areas to focus where a com- primary target, this principle can apply equally to pound falls short of required effectiveness.
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