Although many of the speakers focused upon binding parameters, Robert Copeland, Ph.D., vp enzymology and mechanistic pharmacology, at GlaxoSmithKline (www.gsk.com), advocated a new approach to compound optimization based on residence time. The premise is that residence time is more important than binding parameters like affinity or target inhibition and can provide benefits in terms of target selectivity and duration of the pharmacologic effect.
“Residence time isn’t the only factor to look at,” but is proving to be an epiphany for many, Dr. Copeland said. Although “the concept is well-grounded in the literature, there have been no robust, systematic studies. Consequently, translating the concept to management and medicinal chemists is new, but it resonates with medicinal chemists.”
“When optimizing compounds, we often think of protein-drug interactions in terms of equilibrium constructs or equilibrium between bound and unbound states in small molecules,” but the most important feature, he insisted, is the time a drug spends on the molecule.
Usually, we think in terms of closed systems, in which the drug and target come into contact over time, or in terms of open systems, in which the drug flows in and is removed. But, Dr. Copeland said, “These are unrealistic ways to think about pharmacology.
“Measuring equilibrium in a closed system is a good way to screen or optimize drugs, but it doesn’t account for kinetics,” Dr. Copeland said. Likewise, two-step binding with target isomerization is more common, he noted, but causes researchers to “grossly underestimate affinity.”
“Additionally, there are a number of situations where one can have molecules with identical affinities but very different offerings. So, is it affinity of residence time that makes an effective drug?” he asked.
“You don’t need much residence time,” Dr. Copeland said. “A 30-minute half-life can have a great effect on the dynamics of a drug.” A large local concentration of drug in the viscous cytosol provides rapid rebinding, which leads to a long duration of molecular activity of the target. An HIV study showing viral replication and dissociation half life, he said, showed a “very strong correlation between dissociation half life and target inhibition (IC50) for viral replication. In terms of cellular activity, it is the dissociation half life that is most important.”
Another study, this time of HER2 receptors, showed a slight correlation between cell proliferation and affinity. The same cell-proliferation inhibition data, when plotted against dissociation half life showed “a great correlation.” It is also important, though, to view the inhibition curve over time, as inhibition wanes.
So, he asked, “Why do some drugs have long residence time and some do not?” A study of Tykerb (in Phase III/IV trials) showed conformational adjustments. Specifically, the ATP binding site closed down because the ATP cleft activity loop, the activation loop, folded over the binding site, Dr. Copeland explained.
Biacore’s Markku Hamalainen, senior scientist, chemometrics, also de-emphasizes the importance of binding affinity. In fragment screening, “affinity isn’t the most important issue. Ligand efficiency is key,” Dr. Hamalainen said. Rather than using structural analysis, which he terms “demanding and expensive,” he advocated an initial screening using SPR, followed by in silico screening with validation using x-ray crystallography or NMR.
One project he described, conducted by AstraZeneca (www.astrazeneca.com), used sensograms for data quality control, assaying four substances, including wild type thorombin, mutant thorombin, and GST. Affinities could be measured for 62 of the 80 fragments. Results showed that in-line mutant and reference proteins give invaluable information on binding specificities, but that secondary reactions seem slow.
“With thorombin, the P2/P3 pocket is shallow, so fragment binding is hard to identify,” Dr. Hamalainen said. Therefore, he used a concentration series of assays for a high confidence rate and ran 40 compounds in eight hours. He found an “excellent correlation between enzyme inhibition” and direct binding data, and found that SPR “is more sensitive for analysis of low affinity fragments.” Using Biacore’s A100 SPR analysis system, he said, can run about 1,400 fragments at 120 kDa per day with parallel analysis of multiple targets.