This publication is Open Access under the license indicated. Learn More. ACS Editors' Choice® is a collection designed to feature scientific articles of broad public interest. Read the latest articles.
The development of therapies for the treatment of neurological cancer faces a number of major challenges including the synthesis of small molecule agents that can penetrate the blood-brain barrier (BBB). Given the likelihood that in many cases drug exposure will be lower in the CNS than in systemic circulation, it follows that strategies should be employed that can sustain target engagement at low drug concentration. Time dependent target occupancy is a function of both the drug and target concentration as well as the thermodynamic and kinetic parameters that describe the binding reaction coordinate, and sustained target occupancy can be achieved through structural modifications that increase target (re)binding and/or that decrease the rate of drug dissociation. The discovery and deployment of compounds with optimized kinetic effects requires information on the structure–kinetic relationships that modulate the kinetics of binding, and the molecular factors that control the translation of drug–target kinetics to time-dependent drug activity in the disease state. This Review first introduces the potential benefits of drug-target kinetics, such as the ability to delineate both thermodynamic and kinetic selectivity, and then describes factors, such as target vulnerability, that impact the utility of kinetic selectivity. The Review concludes with a description of a mechanistic PK/PD model that integrates drug–target kinetics into predictions of drug activity.
The treatment of primary infiltrative and secondary metastatic CNS tumors requires the development of drugs that can penetrate the blood-brain barrier (BBB), a selectively permeable barrier composed of epithelial cells held together by tight junctions that is rich in efflux transporter proteins such as P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp). (1) The BBB severely limits the ability of many drugs to penetrate into the brain and is a major impediment to the development of new CNS therapies. For instance, given the success at developing drugs that target kinases in peripheral tumors, it is salutatory that no kinase inhibitor has yet received approval for the treatment of primary CNS cancers. (2) In addition, brain metastasis is a common resistance mechanism during treatment of peripheral tumors due to the inability of drugs to penetrate the BBB. Thus, since drug exposure is likely to be lower in the brain than in systemic circulation, strategies should be adopted that involve the design and synthesis of compounds that remain bound to their targets even when drug concentration is low. However, the general reliance in drug discovery programs on in vitro assays performed at constant drug concentration limits the identification and progression of compounds that display kinetic effects. This knowledge gap will also impact the development of covalent inhibitors since the benefit gained from prolonged target engagement depends on factors such as target vulnerability that can only be assessed using time-dependent assays.
Drug discovery is predicated on the identification and optimization of drug leads through a series of in vitro experiments that are usually performed at constant concentration. The quantitative metrics that result, such as the IC50 values for engagement of the purified target or for activity in a cell-based assay, are used to select and prioritize lead compounds, make assessments about the possibility for off-target effects that impact the therapeutic index, and ultimately to predict drug activity. However, equilibrium parameters are not able to fully account for time-dependent changes in target engagement in the dynamic environment of the human body where drug (and target) concentrations fluctuate. Instead, both the thermodynamics and kinetics of drug-target interactions must be utilized to fully account for time-dependent changes in target engagement. The role of drug–target kinetics in drug discovery has been discussed in a number of reviews and opinions, (3-11) and key concepts include potential mechanisms that modulate the rates of drug–target complex formation (kon) and breakdown (koff), (12-16) including the development of covalent inhibitors, (17) and the role that drug–target residence time (1/koff), kon, and pharmacokinetics play in dictating target engagement. (18-26) In addition, the role of binding kinetics has been explored in forward-thinking programs, such as the K4DD Innovative Medicines Initiative. (27) The present Review reiterates some of the basic concepts that govern drug–target interactions and shows that access to the on and off rates for formation and breakdown of the drug–target complex provides an additional dimension of information that can be used to prioritize drug leads based on kinetic selectivity. Subsequently it is shown that the translation of kinetic effects to time-dependent changes in drug activity depends on target vulnerability which directly impacts kinetic selectivity. The review concludes with a discussion of pharmacokinetic/pharmacodynamic (PK/PD) models that integrate drug-target kinetics into predictions of drug activity in order to facilitate the prospective use of in vitro kinetic data.
Drug-target complex formation occurs because the complex is more thermodynamically stable than free unbound drug and target: thus, thermodynamics provides the driving force for drug binding. However, the value for the equilibrium constant that describes binding provides no information on the rate at which the complex forms and breaks down. Instead, the on (kon) and off (koff) rates for drug binding are controlled by the difference in free energy between the relevant ground and transition states on the binding reaction coordinate (Figure 1).
Since kon and koff depend on the difference in free energy between the ground and transition states on the binding reaction coordinate, efforts to improve drug potency may have unpredictable effects on the kinetics of drug-target complex formation and breakdown. Potency is normally used as a descriptor of affinity, and thus an increase in potency is associated with an increase in the affinity of the drug-target interaction quantified by a decrease in the Kd or IC50 value. However, the impact of stabilizing the E-I ground state on the kinetics for binding depends on whether the transition state is affected. (8) Several scenarios can be envisaged, the simplest of which is that stabilization of E-I has no effect on the transition state. In this case a decrease in Kd will lead to a decrease in koff with no change in kon. In other words, a more potent compound will have a slower off-rate. Alternatively, if the changes in compound structure that lead to an increase in affinity also result in equal stabilization of the transition state, then the increase in potency will have no effect on the rate at which the drug dissociates from the target. In addition, a third scenario can be envisaged where two molecules have identical affinities for the target (the same Kd or IC50 values) but different kon and koff values. Importantly, any differences in kon and koff values, either between two molecules binding to the same target or a molecule interacting with two different biological molecules (e.g., on and off-target proteins), will not be revealed by approaches that only evaluate compound affinity (potency).
Two important misconceptions abound. First, it is often assumed that an increase in potency will result in a decrease in koff. It may do, but it does not have to. Although there are many examples of long residence time compounds, kinetic data for structurally related analogs are often not available, preventing an analysis of whether or not residence time is driven by affinity. Examples where changes in structure within a compound series lead to a decrease in both IC50 and koff include inhibitors of Pseudomonas LpxC, (21) human protein methyltransferase DOT1L, (32) and CDK8/CycC. (33) In addition, if stabilization of E-I also results in stabilization of the transition state, then once the theoretical limit for kon is reached, which is the second order rate constant for encounter of drug and target (109 M–1 s–1), any further increase in affinity must lead to a reduction in the off-rate. A simple calculation reveals that the residence time of a 1 pM drug on the target must be at least 12 min whereas a 1 fM drug will have a residence time of at least 11.5 days. However, there are also examples where changes in affinity and off-rate are disconnected, which are particularly relevant where affinities are in the micromolar to nanomolar range: a 1 nM drug may only have a residence time of 1 s on the target. For example, the quinazoline-based inhibitors gefitinib and lapatinib have Kiapp values for EGFR of 0.4 and 3 nM, respectively, but while gefitinib has a residence time of <14 min, lapatinib has a residence time of 430 min. (34) Other examples include antagonists of the muscarinic M3 receptor, (35) antagonists of the chemoattractant receptor-homologous molecule CRTh2/DP2, (36) and inhibitors of p38α MAP kinase. (37) The second major misconception is that if a compound has similar IC50 values for two different proteins, the drug-target and an off-target protein associated with unwanted side-effects, then the compound has no selectivity. Indeed, the compound has no thermodynamic selectivity, but if the kon and koff values differ between the two targets, it can still have kinetic selectivity. (Text Box 1). This is very important given the implicit relationship between selectivity and therapeutic index.
The relative affinity of a compound for the target and for any kno
