Focal adhesion kinase (FAK) is a promising cancer drug target due to its massive overexpression in multiple solid tumors and its critical role in the integration of signals that control proliferation, invasion, apoptosis, and metastasis. Previous FAK drug discovery and high-throughput screening have exclusively focused on the identification of inhibitors that target the kinase domain of FAK. Because FAK is both a kinase and scaffolding protein, the development of novel screening assays that detect inhibitors of FAK protein–protein interactions remains a critical need. In this report, we describe the development of a high-throughput fluorescence polarization (FP) screening assay that measures the interactions between FAK and paxillin, a focal adhesion–associated protein. We designed a tetramethylrhodamine (TAMRA)-tagged paxillin peptide based on the paxillin LD2 motif that binds to the focal adhesion targeting (FAT) domain with significant dynamic range, specificity, variability, stability, and a Z’-factor suitable for high-throughput screening. In addition, we performed a pilot screen of 1593 compounds using this FP assay, showing its feasibility for high-throughput drug screening. Finally, we identified three compounds that show dose-dependent competition of FAT–paxillin binding. This assay represents the first described high-throughput screening assay for FAK scaffold inhibitors and can accelerate drug discovery efforts for this promising drug target.

The Gamma-aminobutyric acid (GABA) system is the main inhibitory neurotransmitter system in the central nervous system (CNS) of vertebrates and is involved in critical cellular communication and brain function. The endocannabioid system (ECS) was only recenty discovered and quickly recognized to be abundantly expressed in GABA-rich areas of the brain. The strong relationship between the GABA system and ECS is supported both by studies of the neuraoanatomy of mammalian nervous systems and the chemical messaging between neurons. The ECS is currently known to consist of two endocannabinoids, Anandamide (AEA) and 2-Arachidonyl Glycerol (2-AG), that function as chemical messengers between neurons, at least two cannabinoid receptors (CB1 and CB2), and complex synthetic and degradative metabolic systems. The ECS differs from the GABA system and other neurotransmitter systems in multiple ways including retrograde communication from the activated post-synaptic neuron to the presynaptic cell. Together, this molecular conversation between the ECS and GABA systems regulate the homeostasis and the chemical messaging essential for higher cortical functions such as learning and memory and may play a role in several human pathologies. Phytocannabinoids are synthesized in the plant Cannabis sativa (C. sativa). Within the family of phytocannabinoids at least 100 different cannabinoid molecules or derivatives have been identified and share the properties of binding to the endogenous cannabinoid receptors CB1 and CB2. The well-known psychoactive phytocannabinoid Δ9-tetrahydrocannabinol (THC) and the non-psychoactive cannabidiol (CBD) are just two of the many substances synthesized within C. sativa that act on the body. Although the phytocannabinoids THC and CBD bind to these endogenous receptors in the mammalian CNS, these plant derived molecules have little in common with the endocannabinoids in structure, distribution and metabolism. This overlap in receptor binding is likely coincidental since phytocannabinoids evolved within the plant kingdom and the ECS including the endocannabinoids developed within animals. The GABA and ECS networks communicate through carefully orchestrated activities at localized synaptic level. When phytocannabinoids become available, the receptor affinities for CB1 and CB2 may compete with the naturally occurring endocannabinoid ligands and influence the GABA-ECS communication. In some instances this addition of phytocannabinoids may provide some therapeutic benefit while in other circumstances the presence of these plant derived ligands for the CB1 and CB2 receptors binding site may lead to disruption of important functions within the CNS. The regulatory approval of several THC products for nausea and vomiting and anorexia and CBD for rare pediatric seizure disorders are examples of some of the benefits of phytocannabinoids. Concerns regarding cannabis exposure in utero and in the child and adolescence are shrill warnings of the hazards associated with disrupting the normal maturation of the developing CNS.