Discovery of Natural Products and Natural Product Derivatives as Inhibitors of KLK6 to Prevent the Progression of Preneoplastic Ductal Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDA) is one of the deadliest diseases in the United States with a five-year survival rate of only 13%. High mortality is due largely to late-stage diagnosis and the refractoriness of disease to current systemic therapies. Hence, early detection, prevention, and intervention strategies are crucial to improve patient outcomes in PDA. Current research efforts have primarily focused on biomarker screening and therapies for advanced PDA, leaving prevention strategies understudied. Furthermore, a lack of models that accurately represent early disease progression in human samples has hindered investigations in this area. We previously have established intraductally grafted human organoid (IGO) models of PDA in mice, which is the first xenograft animal model that recapitulates the early disease progression of human PDA. In the IGO models we found that the serine protease Kallikrein related peptidase 6 (KLK6) is upregulated during the PDA progression, and is a negative prognostic factor for PDA survival following resection. We hypothesize that KLK6, an extracellular matrix (ECM) remodeling enzyme, regulates ECM and basement membranes to liberate paracrine factors and biophysically enable the migration and invasion of preneoplastic ductal pancreatic cells. Therefore, we nominate KLK6 as a pancreatic cancer interception target that can prevent cancer progression from high- grade preneoplasms to invasive ductal carcinoma. Natural Products (NPs) have demonstrated significant potential in cancer chemotherapy. Here, we aim to fully exploit their benefits towards cancer prevention by identifying NPs that can inhibit the ECM protease activity of KLK6. However, unfavorable toxicity and pharmacokinetics of some NPs can prove limiting. Thus, we plan to perform a pseudo-evolution of the NP library using SuFEx click chemistry (CC) to modify a portion of NPs through semi-synthetic derivatization to enhance their efficacy and drug-like properties (Aim 1). We aim to establish an efficient and cost-effective protocol for functionalizing NP libraries, which serves two key purposes: uncovering new NP scaffold functions and produce hits with improved pharmacological properties and lower toxicity. To support the novel development of NP-CCs we will establish an innovative HRMS/MassQL analytical strategy to identify functionalized molecules. For our screening and validation approach (Aim2), we plan to develop and validate two independent cell-free pilot assay systems (Aim 2) prior to transferring to NCATS to utilize their resources and expertise in high-throughput screening of the NCI’s prefractionated NP library. The first assay is a cell-free protease activity assay that will identify KLK6 inhibitors associated with pre-invasive to invasive PDA interception. To exclude false-positives and cytotoxic NPs, we will employ a cytotoxicity and a counter assay. Following the high throughput screening at NCATs, we will validate the efficacy of non-cytotoxic NPs and NP-CCs that inhibit KLK6 as preventive agents using multiple cell-based assays and in vivo IGO models of human PanIN in mice. Identifying NPs and NP-CCs for the interception of early-stage pancreatic cancer may help prevent disease progression in at-risk patients.