Rapid Screening of Allosteric Effectors Using Two-Dimensional Infrared Spectroscopy

Summary There is an unmet need for an experimental approach to screen small molecules as potential allosteric inhibitors to support drug discovery. Allostery involves binding an effector at a remote site on an enzyme that causes changes in the properties of the active site. Discovering drug candidate molecules that act

allosterically is difficult, however, because the screen should identify molecules that bind to the enzyme but only those that do so specifically, at sites other than the active site, and result in a change in the catalytic properties. Although screening methods can readily identify binding events, determining the

mode of binding or the effect on activity requires additional assessment. The central objective of this proposal is to evaluate the appropriateness and performance of two-dimensional infrared (2D IR) spectroscopy as a tool for screening for allosteric effectors of enzymes. 2D IR measures the conformational dynamics and heterogeneity of the environment around a vibrational chromophore with

femtosecond time resolution, and previous applications to proteins show that it is sensitive to subtle changes. The following two aims will achieve the goals of this project: Aim 1. Accelerate and automate 2D IR data collection for rapid screening; and Aim 2. Assess the sensitivity and specificity of 2D IR as a

probe of allosteric binding. The first aim will be accomplished by implementing a dual-beam approach for active background subtraction in 2D IR, introducing fitting methods to to extract the dynamic observables from a small number of measurements, and developing a multi-well sample holder and automating the

process of serially measuring the 2D IR data for a large set of samples in a rapid screen. In the second aim, which is entirely independent of the first, three model systems with known allosteric inhibitors will be studied to determine the effects of the binding of the allosteric effector on the dynamics measured at

the active site by 2D IR. These models are a diverse group of well-studied enzymes, HIV-1 reverse transcriptase, b-lactamase, and p38 mitogen-activated protein kinase. The probe, a cyanophenylalanine, will be mutated into each in place of an existing active-site phenylalanine residue for the 2D IR experiments. The approach is innovative because it develops a new and substantively different way of

discovering allosteric effectors of enzymes. If successful, 2D IR would be a new and enabling technology for drug discovery. The proposed studies are significant because they will provide the critical proof-of- concept for the idea and advance the technical approach to 2D IR to make it suitable for rapid screening.

Ultimately, 2D IR has the potential to be a powerful tool for drug discovery.