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

The DNAJB1-PRKACA oncogenic gene fusion results in an active kinase enzyme, J-PKAc alpha, 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-PKAc alpha 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 Aplidium sp. marine tunicate led to the discovery of two unprecedented alkaloids, aplithianines A (1) and B (2). Aplithianine A (1) showed potent inhibition against J-PKAc alpha with an IC50 of similar to 1 mu M in the primary screening assay. In kinome screening, 1 inhibited wild-type PKA with an IC50 of 84 nM. Further mechanistic studies including cocrystallization and X-ray diffraction experiments revealed that 1 inhibited PKAc alpha catalytic activity by competitively binding to the ATP pocket. Human kinome profiling of 1 against a panel of 370 kinases revealed potent inhibition of select serine/threonine kinases in the CLK and PKG families with IC50 values in the range similar to 11-90 nM. An efficient, four-step total synthesis of 1 has been accomplished, enabling further evaluation of aplithianines as biologically relevant kinase inhibitors.