In Silico Prediction, Molecular Docking and Dynamics Studies of Steroidal Alkaloids of Holarrhena pubescens Wall. ex G. Don to Guanylyl Cyclase C: Implications in Designing of Novel Antidiarrheal Therapeutic Strategies

The binding of heat stable enterotoxin (STa) secreted by enterotoxigenic Escherichia coli (ETEC) to the extracellular domain of guanylyl cyclase c (ECDGC-C) causes activation of a signaling cascade, which ultimately results in watery diarrhea. We carried out this study with the objective of finding ligands that would interfere with the binding of STa on ECDGC-C. With this view in mind, we tested the biological activity of a alkaloid rich fraction of Holarrhena pubescens against ETEC under in vitro conditions. Since this fraction showed significant antibacterial activity against ETEC, we decided to test the screen binding affinity of nine compounds of steroidal alkaloid type from Holarrhena pubescens against extracellular domain (ECD) by molecular docking and identified three compounds with significant binding energy. Molecular dynamics simulations were performed for all the three lead compounds to establish the stability of their interaction with the target protein. Pharmacokinetics and toxicity profiling of these leads demonstrated that they possessed good drug-like properties. Furthermore, the ability of these leads to inhibit the binding of STa to ECD was evaluated. This was first done by identifying amino acid residues of ECDGC-C binding to STa by protein–protein docking. The results were matched with our molecular docking results. We report here that holadysenterine, one of the lead compounds that showed a strong affinity for the amino acid residues on ECDGC-C, also binds to STa. This suggests that holadysenterine has the potential to inhibit binding of STa on ECD and can be considered for future study, involving its validation through in vitro assays and animal model studies.

Guanylyl cyclase C as a biomarker for immunotherapies for the treatment of gastrointestinal malignancies

Gastrointestinal cancers encompass a diverse class of tumors arising in the GI tract, including esophagus, stomach, pancreas and colorectum. Collectively, gastrointestinal cancers compose a high fraction of all cancer deaths, highlighting an unmet need for novel and effective therapies. In this context, the transmembrane receptor guanylyl cyclase C (GUCY2C) has emerged as an attractive target for the prevention, detection and treatment of many gastrointestinal tumors. GUCY2C is an intestinally-restricted protein implicated in tumorigenesis that is universally expressed by primary and metastatic colorectal tumors as well as ectopically expressed by esophageal, gastric and pancreatic cancers. This review summarizes the current state of GUCY2C-targeted modalities in the management of gastrointestinal malignancies, with special focus on colorectal cancer, the most incident gastrointestinal malignancy.

122 Guanylyl cyclase C as a target for CAR-T cell therapy in a metastatic gastric cancer model

BackgroundGastric cancer is the sixth most common cancer and second-leading cause of cancer-related mortality worldwide.1 The heterogenous and genetically complex nature of this disease underlies the challenges in developing effective therapies for metastatic gastric cancer. In the majority of cases, stomach tumors evolve from intestinal metaplasia resulting in ectopic expression of the enterocyte differentiation antigen guanylyl cyclase C (GUCY2C) by ~50% of primary and metastatic gastric cancers.2–4 In the context of the efficacy of GUCY2C-directed chimeric antigen receptor (CAR)-T cells against metastatic colorectal cancer in animal models,5,6 we hypothesized that this adoptive cell therapy may be effective against metastatic gastric cancer.MethodsHere, we explored the efficacy of GUCY2C-directed CAR-T cells for gastric cancer in a patient derived xenograft (PDX) tumor model. Also, we interrogated translational GUCY2C biomarker assays using RT-qPCR, immunoblot analysis, and immunohistochemistry (IHC) for the intended purpose of identifying patients whose tumors express GUCY2C and could benefit from GUCY2C-directed CAR-T cell therapy.ResultsGUCY2C-directed CAR-T cells significantly reduced subcutaneous T84 colorectal tumor growth, producing a 5-fold reduction in tumor volume, compared to control treated tumors. GUCY2C-directed CAR-T cells produced no response in tumors produced from the GUCY2C-deficient colorectal cancer cell line, SW480. Importantly, GUCY2C-directed CAR-T cells controlled gastric cancer PDX growth, maintaining a >12-fold reduction in tumor volume compared to control and in some cases produced complete tumor elimination. Furthermore, IHC based assays, indicate that antibodies developed in our laboratory may be suitable for development of a companion diagnostic for GUCY2C-directed CAR-T cells. Indeed, the commercial polyclonal antibody demonstrated robust, non-specific staining regardless of tissue type or GUCY2C mRNA profile, while novel monoclonal antibodies produced in our laboratory primarily detected protein localized to the membrane of glandular epithelial cells, demonstrating antigen specificity, and indicating their potential for further development in diagnostic companion assays to identify gastric cancer patients who may benefit from GUCY2C-directed CAR-T cell therapy.ConclusionsGUCY2C-directed CAR-T cells prevented the growth of, and at times eliminated, a subcutaneous gastric cancer PDX model. In the context of previously established safety in mouse models, additional studies defining the efficacy of GUCY2C-directed CAR-T cells in gastric cancer models may allow future translation of this therapy to patients with advanced gastric cancers. Concurrent development of a novel companion diagnostic IHC assay would permit identification of the ~50% of gastric cancer patients whose tumors express GUCY2C and could benefit from this therapy.AcknowledgementsThis work was supported by a DeGregorio Family Foundation Award and by the Department of Defense Congressionally Directed Medical Research Programs (W81XWH-17-1-0299, W81XWH-191-0263, and W81XWH-19-1-0067) to AES. SAW is supported by the National Institutes of Health (NIH) (R01 CA204881, R01 CA206026, and P30 CA56036), the Defense Congressionally Directed Medical Research Program W81XWH-17-PRCRP-TTSA, and Targeted Diagnostic & Therapeutics. SAW and AES were also supported by a grant from The Courtney Ann Diacont Memorial Foundation. SAW is the Samuel M.V. Hamilton Professor of Thomas Jefferson University. AZ was supported by NIH institutional award T32 GM008562 for Postdoctoral Training in Clinical Pharmacology.The authors thank the NCI Patient-Derived Models Repository for their support and resources to make this research possible. The authors also thank the Sidney Kimmel Cancer Center Translational Research & Pathology Core Facility, and the Office of Animal Resources at Thomas Jefferson University for their continued technical assistance and support in this research.Ethics ApprovalThis study was approved by the Thomas Jefferson University Institutional Review Board (#14.0204) and the Instituational Animal Care and Use Commitee (Protocol #01529).ReferencesBray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin2018;68:394–424. doi:10.3322/caac.21492.Park J, Schulz S, Haaf J, Kairys JC, Waldman SA. 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