Project Summary/ Abstract Osteoporosis is a systemic skeletal disease characterized by low bone mass and microarchitectural deterioration of bone tissue with a consequent increase in bone fragility and susceptibility to fracture. One in three women and one in five men over the age of 50 years are predicted to suffer a fracture leading to limitations in the quality of life, pain, morbidity, and increased mortality. However, it is difficult to treat most diseases of the skeletal system with non-bone selective drug delivery. Visceral organs may be exposed to the bulk of the pharmaceutical drug, while very low drug concentrations reach the bone compartment. Mechanical loading is well known as a key factor that can increase bone formation and bone mass. Astronauts lose bone mass during space flight due to reduced gravity and skeletal unloading. Recently, we demonstrated that inhibiting FAK (Focal adhesion kinase) mimics the effects of mechanical loading in bone cells (Sato et al. Nat Commun, 2020). However, FAK and its homolog, PYK2 are widely expressed in almost all tissues and organs. Therefore, FAK inhibitor treatment may cause severe side-effects outside of bone. To address this problem, we will develop bone-targeted FAK inhibitors via bisphosphonate (BP) conjugation. This high-risk, high-reward proposal brings considerable potential benefit to the musculoskeletal research field. (1) Bone targeting via BP-conjugation will eliminate extra-skeletal side effects of FAK inhibitors. (2) Bisphosphonates can cause side effects such as osteonecrosis of the jaw and cytotoxicities. However, our novel pharmacologically-inactive BP addresses this potential concern. (3) The BP-conjugates may increase FAK inhibitors' efficacy to around 10 to 1000 times higher bone formation than the original because this conjugation method delivers to the target sites and releases the active drug (FAK inhibitor) selectively at high bone metabolic sites. (4) There can be no serum calcium elevation by BP-FAK inhibitors, although current bone formation inducible drugs like teriparatide (PTH1-34) and abaloparatide (PTH analog). (5) FAK inhibitors are under several clinical trials for certain cancer therapies. So, bisphosphonate-conjugated FAK inhibitors are clinically relevant drugs as bone-specific bone formation induced agents. (6) This BP-conjugation can reduce administration frequencies due to bisphosphonate biomaterial property that binds to hydroxyapatite and stays on the sites. (7) Our new formulation strategies may be pursued to address oral availability limitations. Aim 1 of this proposal will develop and evaluate BP-conjugated bone-targeting FAK inhibitor (VS-6063) in vitro (osteoblasts, osteoclasts, and osteocytes). In aim 2, we will perform hindlimb unloading animlal model as disuse osteoporosis to define the therapeutic action of FAK inhibitors in bone. This will offer thousands of new candidate FAK substrates for the final development of a druggable BP-FAK inhibit...