Small-molecule modulation of 14-3-3 Protein-Protein Interactions
In comparison to the size of the human proteome (~25,000), the estimated numbers of PPIs in the human interactome (650,000) is much larger and therefore offers many opportunities for chemical perturbations of biological systems. Examples for successful small-molecule modulators of PPIs currently under investigation are the Nutlins or ABT-737. Approved drugs that have been found to modulate PPIs are the natural products taxanes and epothilones, which stabilize micro-tubules or the immunosuppressants rapamycin and FK506.
The 14-3-3 adapter proteins comprise a ubiquitous eukaryotic protein family (7 isoforms in humans) that is involved in a wide array of regulatory functions in diverse signalling pathways, cell cycle control and apoptosis. 14-3-3 proteins have no intrinsic enzymatic activity but rather exert their physiological function by directly binding to other proteins. As a consequence, their target proteins are modulated in their enzymatic activity, their subcellular localization or their ability to further bind other protein partners. In this sense 14-3-3 proteins are switchable docking modules controlling the biological activity of their target proteins. To date there are more than 200 protein partners of 14-3-3 proteins known, many of which play prominent roles in the development of diseases like the RAF kinases, p53, Cdc25 phosphatases, FOXO, BAD, YAP/TAZ and TSC1/2. Small-molecule modulation of these interactions by either inhibiting or stabilizing PPIs will produce new tools for the study of cells, tissues and whole organisms.
By protein crystallography we have deciphered how the natural product fusicoccin A (FC-A) stabilizes the activated protein complex between the plant plasma membrane H+-ATPase (PMA2) and 14-3-3 adapter proteins. The FC-A molecule binds to the interface of the complex by simultaneously contacting both proteins. We have also discovered chemically less complex molecules in a highthroughput-screening (HTS) that were able to substitute the stabilizing activity of FC-A (epibestatin and pyrrolidone1). Cotylenin A (CN-A), a molecule related to FC-A and showing anti-cancer activity was also crystallized in our group in complex with its protein target. As inhibitors of 14-3-3 PPIs we found a molecule that binds near the phosphate-accepting pocket, and a supramolecular tweezer that binds to lysine near the central binding channel of the 14-3-3 protein. We have furthermore developed a Chemical Induced Dimerisation (CID) system with FC-A as the dimerizing molecule.