How to Perform a Tracer Displacement BRET Assay for the TRPML1 Ion Channel

Background

The transient receptor mucolipin subtype 1 (TRPML1) is a lysosomal ion channel expressed in every cell type [1]. Its deregulation has been implicated in many diseases, including genetic diseases (e.g., Mucolipidosis type IV [2], Niemann–Pick [3], and Duchenne muscular dystrophy [4]), bacterial [5] and viral infections [6], and cancer [7–14]. Recent pharmaceutical interest in developing agonist ligands has emerged due to beneficial effects in neurodegenerative diseases [15]. The identification of cell-permeable modulators for TRPML1 has been challenging due to the dynamic nature of these proteins and the inability to express and purify them in bacteria because of their complex structure. As a result, in vitro biochemical assays are largely unavailable. To overcome these issues, researchers have essentially relied on compound screening in cells overexpressing a cytoplasmic version of TRPML1 rather than the endolysosomal form [16–19]. This allows the apical application of compounds and the direct measurement of channel activity by calcium (Ca²⁺⁾ imaging assay or by electrophysiology. However, these assays represent a non-physiological condition for drug screening because the compounds do not target the receptor in its native location [20,21]. More recently, the use of genetically encoded calcium indicators (GECI, such as GCaMP’s), specific intracellular calcium-sensitive dyes (such as Fura-2 and Fluo-4), and lysosomal preparations for patch-clamping has changed how we investigate compound binding to wild-type TRPML1 [22,23]. Despite these advancements, drug-screening campaigns remain extremely work-intensive due to successive washing steps and special handling of cells, and, more importantly, the use of expensive equipment for the functional assays.

Target engagement (TE) assays have emerged as an essential confirmatory assay for on-target activity in live cells, which accounts for compound permeability, biological target localization, and competition with intracellular constituents [24]. Among the different assay modalities, the bioluminescent resonance energy transfer (BRET) technology has been widely used to investigate compound binding to important targets such as kinases, GPCRs, and others [25]. This assay depends on the intracellular tracer that emits fluorescence once bound to the target protein fused to a luciferase [26]. The tracer (also called a BRET probe) is composed of a small molecule with experimental data on potency and mode of action in the target of interest, by either cryo-electron microscopy (cryo-EM), X-ray diffraction, or other structural biology techniques. This compound is rationally modified to covalently incorporate a fluorescent moiety in a position that would not interfere with binding to the protein target. This compound must also be cell-permeable in live cells and must bind to the target in a pocket that is placed at an optimal distance from the luciferase [27]. Once these parameters are achieved, one can perform the competition assay between a known concentration of the tracer and non-fluorescent test compounds, referred to as the tracer displacement (TD) assay. It is important to note that if the parent molecule that originated the tracer is promiscuous within a protein family, it allows the researchers to use the same tracer to investigate many proteins of the family [28]. Finally, it is worth mentioning that the BRET assay will only give data for compounds that bind to the same region of the tracer. Therefore, one of the major challenges to establishing a functional BRET assay is to develop a validated BRET tracer for the intended target.

Given the need for new screening assays for the TRPML1 ion channel and the ability of TE assays to investigate targets in their native environment, we set out to develop a BRET assay for TRPML1 using a new validated tracer called MRC087. The tracer was designed using a molecular docking approach to incorporate a fluorescent BODIPY-based compound into the mucolipin synthetic agonist-1 (referred to as ML-SA1). ML-SA1 was chosen due to its wide use in the scientific community as a TRPML1 agonist with a defined mode of action by cryo-EM [1,29,30] and reported structure-activity relationships [16].