Using Small Molecule Probes To Study The Biological Functions Of Cd38

Abstract

CD38 gene knockout studies in mice have identified physiological functions including insulin secretion, susceptibility to bacterial infection caused by loss of neutrophil chemotaxis, and social behavior through modulating neuronal oxytocin secretion. These physiological functions are explained by its NAD-degrading enzymatic activity and transmembrane signaling activity. Despite the large amount of literature on CD38, there still exists fundamental questions. Developing chemical tools to address these questions is the goal of my thesis research. Most notable is the previously reported link between CD38 robust hydrolysis of NAD and the intracellular localization that would cause regulation of intracellular NAD and affect other NAD-dependent enzymes. Consequently, whether CD38 is intracellular and regulating NAD deserves careful investigation. Development and usage of a cell permeable, fluorescent small molecule probe (SR101-F-araNMN) that covalently labels CD38 in cells and reveals CD38 intracellular localization indicated that CD38 is predominantly on the plasma membrane with very little intracellular present in Raji and retinoic acid treated HL-60 cell lines. The discovery in these two human cancer cell lines suggests the major enzymatic function of CD38 is to hydrolyze extracellular NAD rather than intracellular NAD. Further, CD38 has a single transmembrane domain with a short cytoplasmic tail and has a role in activating mitogen activated protein kinase (MAPK) signaling that is important for cellular differentiation induced by retinoic acid. However, the question remains as to how CD38 induces MAPK signaling. Development of dimeric small molecule probes that can dimerize two cell surface CD38 molecules allowed for investigation of whether dimerization of CD38 is sufficient to induce MAPK signaling. Finally, since CD38 is highly expressed in hematologic cancers, a novel approach using an antibody-recruiting small molecule (ARM) that can covalently and specifically label CD38 was used to target CD38-overexpressing cancer cells. As part of an integral three-body complex - CD38, ARM and antibody - capable of enacting immune-effector cells for target cell cytotoxicity, the ARM molecule showed a 2.5 fold increase in target cell cytotoxicity. Through employment of a chemical biology approach, my work contributed to elucidation of the mechanisms of CD38 function and increased knowledge of CD38 function in various normal and pathological conditions.