Off-rate screening (ORS)

Molecules that dissociate slowly from their target proteins are potentially useful because they can have a long-lasting effect even if they are rapidly cleared from circulation. However, it is next to impossible to predict whether a molecule will dissociate slowly or not. Moreover, the correlation with binding affinity is poor: weak binders generally don’t stay bound to their target for long, but even tight binders often rapidly dissociate. In the early stages of lead discovery most folk are focused on affinity, and it is usually only much later that kinetics enters in. In a new paper in J. Med. Chem., James Murray, Paul Brough, and colleagues at Vernalis introduce a technique that moves kinetics to the front of the line.

The technique, off-rate screening (ORS), relies on surface plasmon resonance (SPR), which is already commonly used to study binding kinetics. The trick here is using SPR to screen products in unpurified reaction mixtures. An initial fragment with known affinity is modified, and products screened for slower dissociation. Of course, the concentration of desired compound is likely to vary from mixture to mixture, but the great thing about looking at compound dissociation is that it is a zero order reaction: it does not depend on concentration. The researchers use mathematical simulations to show that even if the yield is only 5%, a product with a 10-fold slower dissociation rate constant could still be detected. Since off-rates can vary by orders of magnitude, this is not such a high bar.

Of course, simulations are one thing, but how does the technique actually work in practice? The researchers show examples on two targets, one using some of the early compounds for their HSP90 program, the other some of their PIN1 inhibitors. For PIN1, the researchers resynthesized some of the molecules in plastic tubes, which caused leaching of plastic into the reaction mixtures. Nonetheless, for both proteins the dissociation rate constants measured for unpurified reactions were very close to purified molecules, generally differing by less than 30%.

The researchers also tried subjecting compounds to eleven reaction conditions typically used in medicinal chemistry, evaporating the solvent, and testing the products; the idea was to see if the reagents or other components in the reaction mixture would interfere with the assay. Happily in all cases the dissociation rate constants differed by less than 20%, again pointing to the robustness of ORS.

Of course, as with any technique, there are limitations. Since the screening compounds are not purified from their starting materials, the desired products must dissociate sufficiently slowly from the protein to be distinguishable from other components in the reaction mixture; dissociation rate constants greater than about 1.2 s^-1 appear to be challenging. Also, if the starting material itself has a slow dissociation rate from the protein, it may be difficult to differentiate this from a low yield of slowly dissociating product. The researchers note that both cases could be addressed by changing the temperature, either lowering it to slow the dissociation rate constant or raising it to increase it.

All in all this is a nice approach, and it will be interesting to see how widely it catches on.