Development of Heterogeneous Nuclear Ribonuclear Protein A18 (hnRNP A18) Small Molecule Inhibitors and Phosphorylation of hnRNP A18 by Casein Kinase-2
AdvisorWeber, David J., Ph.D.
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AbstractHeterogeneous nuclear ribonuclear protein A18 (hnRNP A18) is an RNA binding protein (RBP) is upregulated in response to cellular stressors such as cold, UV, and hypoxia. Upon cellular stress, hnRNP A18 is phosphorylated in the nucleus by casein kinase-2 (CK-2) and glycogen synthase kinase-3β (GSK-3β). Upon phosphorylation, hnRNP A18 translocates to the cytosol where it interacts with pro-survival mRNA transcripts and stabilizes them to increase their translation. hnRNP A18 is differentially upregulated in solid tumors in response to low oxygen tension where it stabilizes pro-survival mRNA transcripts, such as hypoxia inducible factor-1α (HIF-1α) and Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). Chemotherapeutics that target HIF-1α and CTLA-4 have demonstrated increased patient progression-free survival. hnRNP A18 presents a unique advantage in the ability to target both the hypoxic cellular responses and immune checkpoints in cancer. Toward the development of hnRNP A18 inhibitors, the X-ray crystal structure of the hnRNP A18 RNA recognition motif (RRM) was solved and used in the design of inhibitors for hnRNP A18 that disrupt RNA binding with the RRM. Initial inhibitors were identified through the Site Identified Ligand Competitive Saturation (SILCS) computational screening method. Nuclear magnetic resonance (NMR) screening of these compounds produced a lead compound that was further refined. Subsequently identified compounds were analyzed for hnRNP A18 binding, specificity, membrane permeability, and binding affinity. This work serves as a foundation for investigation of hnRNP A18 inhibitors in vivo and toward the development of an hnRNP A18 therapeutic approach. Phosphorylation by CK-2 and GSK-3β is required for nuclear to cytosolic translocation upon cellular stressors. CK-2 phosphorylation primes hnRNP A18 for GSK-3β phosphorylation, which subsequently increases hnRNP A18 affinity for target mRNA transcripts. To understand the structural and functional impact of such post translational modifications, hnRNP A18 was phosphorylated by CK-2 and the structural impacts of the phosphorylation were analyzed by NMR. RBPs, like hnRNP A18, are involved in a variety of cellular processes and disease pathologies, such as carcinogenesis and neurodegenerative disorders. Insight into the biological and structural mechanisms of RBP action can aid in the development of therapeutic strategies important for the treatment of such pathologies.
University of Maryland, Baltimore