Hijacking Cancer Signaling: First-in-class JPRK-targeting Ligands for Fibrolamellar Hepatocellular Carcinoma

Described herein is a drug screening project focused on an oncoprotein beginning with in silico fragment screening against an un-drugged target and concluding with a collaboration to evaluate single digit nanomolar potent and >500-fold selective drug candidates in patient derived xenografts. Fibrolamellar hepatocellular carcinoma is a rare un-treated liver cancer driven by a fusion kinase known as JPRK, a mutant of protein kinase A (PKA). Targeting this fusion kinase selectively is challenging due to the structural similarity of the catalytic domain to other kinases, most significantly, PKA, whose function regulates cardiac activity. In silico screening of the predicted binding of 10,000 fragments to an allosteric site on JPRK was used to advance 51 fragments for in vitro for potency and selectivity and led to a hit compound. A subsequent Structure Activity Relationship (SAR) guided by biophysical assays led to an 8 nM KD inhibitor with > 500-fold selectivity against PKA. Compounds were also developed which selectively kill oncogenic liver cells which bear the JPRK mutation over the same cell line without this mutation. Crystallography and NMR provided atomic-level resolution of how the ligand was binding in a unique allosteric site, leading to allosteric signal transmission through the protein, and the subsequent change in protein-conformation. Furthermore, alongside this change in conformation, key protein-protein interactions with onco-proteins, such asSTAT5B and HDAC6, were perturbed. Target engagement studies confirmed that the compounds were not only altering the protein-protein interaction with HDAC6, but also hampering its phosphorylation. To further probe how the compounds selectively trigger cell death, global proteomics were performed studies to examine the effects of the ligands, and phosphorylation events on key protein-protein interactions. The JPRK-specific drugs downregulated proteins involved in metabolism and the Warburg effect while upregulating critical epigenetic apoptosis drivers leading to cancer cell death. These compounds will be advanced for mouse safety, pharmacokinetics, and patient derived xenograft studies.

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