PROJECT SUMMARY/ABSTRACT Targeting the hallmark biomarker prostate specific membrane antigen (PSMA) has been a successful small molecule drug-delivery strategy for various payloads to prostate tumor cells, which is evident in the FDA's recent approval of 177Lu-PSMA-617 and the clinical advancement of our 18F-CTT1057 (an irreversible-binding phosphoramidate-based) PSMA-targeted PET-imaging agent (NCT04838626, NCT04838613). However, efforts to develop PSMA-targeted chemotherapeutic nanomedicine have had limited clinical therapeutic efficacy. Our long-term goal is to develop a versatile and intelligently designed platform for the selective delivery of synergistic therapeutic payloads to offer novel treatment options for lengthening and improving the quality of life for metastatic castration-resistant prostate cancer (mCRPC) patients. The overall objective of this project is to develop a PSMA-targeted small-molecule dual drug conjugate (SMDDC) to serve as a companion therapeutic to our PSMA-targeted PET-imaging agent (NCT02916537, NCT03427476). Our central hypothesis is that tumor-site activation and release of two distinct chemotherapeutic payloads for prostate cancer can be achieved and will be more effective than small molecule drug conjugates (SMDCs) releasing only a single chemotherapeutic payload. The rationale for developing SMDDCs is to set the groundwork for a new, dual- drug therapeutic strategy for patients with mCRPC and advanced malignancies with PSMA(+) neovasculature. Two specific aims will be pursued to test the central hypothesis: 1) Assess the spatio-temporal cargo-release in prostate tumor cells with a PSMA-targeted small-molecule dual probe conjugate (SMDPC); and 2) Determine the potency enhancement of a SMDDC bearing dual payloads in prostate cancer cell lines. For Aim 1, PSMA(+) and PSMA(-) prostate cancer cell lines will be used to assess the spatio-temporal cargo-release of a PSMA-targeted SMDPC bearing two distinct turn-on probes, 7-Amino-4-methylcoumarin and hydroxymethyl rhodamine green. Established PSMA activity assays will be used to determine the IC50 and mode of binding for the PSMA-targeted SMDPC. In Aim 2, in vitro effectiveness of an SMDDC bearing both MMAE and Exatecan vs. an SMDC bearing either MMAE or Exatecan alone will be evaluated in PSMA(+) and PSMA(-) prostate cancer cell lines. The proposed work is innovative because it aims to utilize a unique combination of drug- delivery strategies; PSMA-ligand promoted internalization, a novel acid-labile linker for pH-triggered drug release, and two distinct chemotherapeutic drugs with differing mechanisms of action. This will be a significant achievement because it will provide proof-of-concept for developing clinically relevant chemotherapy for mCRPC and other malignancies with PSMA(+) neovasculature. Future plans include expanding the selection of drug payloads for PSMA-targeted SMDDCs and initiate preclinical IND-enabling studies. The expected positive impact of the...