Selective inhibition of p97 by chlorinated analogues of dehydrocurvularin.

The ATPase p97 is a ubiquitin targeted segregase that uses the energy of ATP binding and hydrolysis to extract ubiquitylated substrates from biological membranes, from other proteins, or from protein complexes to carry out myriad tasks in eukaryotes. Increased p97 activity has been linked to a poor prognosis in cancer patients, making p97 an anti-neoplastic target. In the present study, we show that dehydrocurvularin (DHC) and its chlorinated variants are covalent inhibitors of p97, interfering with its ATPase activity. Interestingly, cellular studies revealed both DHC and its monochloro analogue interfere with both the proteasome and p97, whereas its dichloro analogue showed p97 specificity.

Functional chromatography reveals three natural products that target the same protein with distinct mechanisms of action.

Access to lead compounds with defined molecular targets continues to be a barrier to the translation of natural product resources. As a solution, we developed a system that uses discrete, recombinant proteins as the vehicles for natural product isolation. Here, we describe the use of this functional chromatographic method to identify natural products that bind to the AAA+ chaperone, p97, a promising cancer target. Application of this method to a panel of fungal and plant extracts identified rheoemodin, 1-hydroxydehydroherbarin, and phomapyrrolidone A as distinct p97 modulators. Excitingly, each of these molecules displayed a unique mechanism of p97 modulation. This discovery provides strong support for the application of functional chromatography to the discovery of protein modulators that would likely escape traditional high-throughput or phenotypic screening platforms.