We have developed a fluorescence-based assay to determine whether small amphiphiles, such as drugs and drug leads, alter membrane protein function through drug-induced changes in membrane properties. The assay exploits the power of gramicidin channels as probes for changes in bilayer properties,11
as sensed by a bilayer-spanning channel (the energetic cost of a channel-bilayer hydrophobic mismatch). The results obtained using the fluorescence-based assay are in good agreement with results from single-channel gA experiments ( and ), indicating that this method can be used for mechanistic studies as well as for screening compound libraries. Using the present configuration of the assay, we can test dozens of samples a day; which is 1 to 2 orders of magnitude higher throughput than possible using the single-channel approach. There are no fundamental rate-limiting steps in the fluorescence-based assay, meaning that it can be extended to run in true high-throughput mode. Given the time course of fluorescence quenching (10–100
ms), due to the vesicles' small volume and the fast quencher influx through the gA channels, such implementations of the assay will require a rapid mixing system. This may be difficult to implement in a conventional 96- or 384-well plate format, but can be achieved by automating the loading of the stopped-flow system.
It remains unclear whether there is a causal relationship between increasing lipophilicity of drug leads and increasing attrition in drug development2–4
and, if so, what are the underlying mechanism(s)? Nevertheless, because membrane proteins tend to be regulated by changes in their membrane environment,10
it would be prudent to test whether amphiphilic drugs and drug leads alter pertinent lipid bilayer properties and, if so, at what concentrations? Because amphiphile partitioning into the lipid bilayer will deplete the aqueous phase; the relevant concentration is the free concentration in the aqueous phase—which may be orders of magnitude less than the nominal concentration in the system, for example.16,17,33
Determining drug concentrations in the aqueous and lipid phases may be problematical due to lipid composition-dependent partitioning and drug absorption to the experimental setup. When comparing to in vivo
plasma concentrations, it further becomes important to consider drug binding to plasma proteins. It is therefore important that one can vary the vesicle lipid composition, lipid:water ratio, and presence of plasma proteins. Addition of 600
μM serum albumin, for example, right-shifts the dose-response curve for Cap 3- to 5-fold (results not shown), presumably reflecting Cap binding to albumin.35
If a molecule's desired (biological) effects occur at concentrations where it alters bilayer properties, it becomes important to distinguish between the “non-specific,” bilayer-mediated changes in membrane protein function, as opposed to direct effects due to (high-affinity) binding to one or more target proteins. Knowing the bilayer-modifying propensity of a drug lead, discovered in conventional high-throughput screening, therefore is likely to be important for decisions regarding its further development.
Any amphiphile—at some concentration—will alter some bilayer property. Key concerns therefore become: at what concentration and are the changes in bilayer properties sensed by (bilayer-spanning) membrane proteins? Here, we exploit the ability of gramicidin to form channels by transbilayer dimerization.36
This makes them useful probes for the energetic coupling between lipid bilayers and bilayer-embedded proteins, and for exploring whether small molecules alter bilayer properties that are sensed by membrane proteins. The assay is fast, reliable, and scalable, and therefore suitable for both biophysical studies and for the screening compound libraries for drugs with potential bilayer-perturbing effects.