This proposal aims to develop the first selective substrates and activity-based probes (ABPs) of the STriatal- Enriched Tyrosine Phosphatase (STEP), a novel therapeutic target for Alzheimer's disease (AD) and other neurological disorders. STEP is a neuron-specific protein tyrosine phosphatase (PTP) targeted in part to postsynaptic terminals of excitatory glutamatergic synapses. Recent studies indicate that STEP is overactive in AD and other neurodegenerative and neuropsychiatric disorders. The emergent model from these studies suggests that the increased STEP activity interferes with synaptic function and contributes to the characteristic cognitive and behavioral deficits in these diseases. A major challenge in STEP research is the lack of specific tools to further investigate the function of STEP in cells or in vivo, which would provide proof of principle studies for a STEP-based therapeutic approach in AD. Generating specific chemical probes or ABPs for PTPs has been difficult, because the active site is highly conserved among the >100 PTP family members in humans. As a result, compounds that target the PTP catalytic site typically exhibit poor selectivity for their target. Indeed, reported chemical probes for STEP suffer from lack of selectivity as well as cellular efficacy. Prior studies using combinatorial peptide libraries have not resulted in any specific substrates or probes. We are proposing a novel approach based on our previously established and highly successful hybrid combinatorial substrate library (HyCoSuL) method. Our platform is unique, innovative, and addresses the major weaknesses of prior combinatorial peptide library strategies, primarily by covering an exponentially larger chemically space by using unnatural amino acids (UAAs). This is the first time that such libraries will be generated for a tyrosine phosphatase. Assays to fully characterize the generated STEP substrates and probes are in place, as well as crucial collaborations, ensuring the greatest likelihood of success. In Aim 1, we will generate and test fluorogenic HyCoSuL libraries, select optimal UAAs, and synthesize individual UAA peptide substrates. Substrates with the greatest STEP activity and selectivity will be converted into STEP-specific ABPs in Aim 2 and characterized in a battery of in vitro and cellular assays. We will also attempt to obtain and solve crystal structures of STEP:ABP complexes, which will aid in further optimization of the probes. Our overall goal is to develop at least one STEP probe with a minimum of 100-fold selectivity for STEP and proven activity on cells. Future plans include the development of STEP probes with activity in vivo.