Assay Development and Screening

Measuring cell-free, label-free drug binding kinetics to native integral membrane protein targets in the membrane.

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  Jaime Arenas, PhD

  Sr. Director, Science & Technology
  Nanotech Biomachines

Numerous valuable therapeutic targets are integral membrane proteins (IMP) such as GPCRs, ion channels, and transporters. However, IMPs are unstable when purified away from the membrane and cannot be isolated in their native state to evaluate the binding of drug candidates. This limitation forces lead development programs to introduce mutations and use detergent-based solubilization and reconstitution techniques to stabilize purified IMPs for ligand binding characterization. This expensive and time-consuming approach has limited success and introduces a significant risk to yield inaccurate kinetic binding information for lead selection. To address this challenge, we have developed a graphene bio-electronic sensor technology (GBEST) specifically designed to measure label-free kinetic binding of drug candidates to native IMP targets without removing them from the cell membrane.

A simple cell membrane preparation, expressing the target IMP, is used to cover the GBEST sensor surface with a flat membrane. A small voltage across the sensor creates a current that is highly sensitive to electrostatic changes in the immediate vicinity of the sensor surface. Thus, electrostatic changes caused by the binding of drugs to target IMPs in the membrane can be monitored in real time to generate label-free kinetic binding data in a cell-free system that includes all the natural components of the cell membrane.

The fluidity of the membrane is critical to support the IMP’s native conformation and function. We have successfully demonstrated that cell membranes deposited on the GBEST sensors remain fluid and show lateral movement of target IMPs in the plane of the membrane. Furthermore, direct demonstration of IMP fluidity confirms that its native conformation has not been altered by adsorption to the sensor surface.

We show that the GBEST biosensor detects real-time kinetic binding of small molecule and biotherapeutic drugs to IMPs, and we show that the KD value obtained with GBEST correlates well with the expected benchmark value.

We will present recent data sets and discuss the capabilities of the GBEST biosensor to enable lead characterization with native IMPs. The use of GBEST in lead discovery will enable lead characterization directly on native membranes to generate biologically relevant data for lead selection.