Discovery and Synthesis of a Naturally Derived Protein Kinase Inhibitor that Selectively Inhibits Distinct Classes of Serine/Threonine Kinases.

The oncogenic gene fusion results in an active kinase enzyme, J-PKAcα, that has been identified as an attractive antitumor target for fibrolamellar hepatocellular carcinoma (FLHCC). A high-throughput assay was used to identify inhibitors of J-PKAcα catalytic activity by screening the NCI Program for Natural Product Discovery (NPNPD) prefractionated natural product library. Purification of the active agent from a single fraction of an sp. marine tunicate led to the discovery of two unprecedented alkaloids, aplithianines A () and B (). Aplithianine A () showed potent inhibition against J-PKAcα with an IC of ∼1 μM in the primary screening assay. In kinome screening, inhibited wild-type PKA with an IC of 84 nM. Further mechanistic studies including cocrystallization and X-ray diffraction experiments revealed that inhibited PKAcα catalytic activity by competitively binding to the ATP pocket. Human kinome profiling of against a panel of 370 kinases revealed potent inhibition of select serine/threonine kinases in the CLK and PKG families with IC values in the range ∼11-90 nM. An efficient, four-step total synthesis of has been accomplished, enabling further evaluation of aplithianines as biologically relevant kinase inhibitors.