Small-molecule modulation of protein-protein interactions (PPIs) is one of the most promising new areas in drug discovery. In the vast majority of cases only inhibition or disruption of PPIs is realized, whereas the complementary strategy of targeted stabilization of PPIs is clearly under-represented. Here, we report the example of a semi-synthetic natural product derivative--ISIR-005--that stabilizes the cancer-relevant interaction of the adaptor protein 14-3-3 and Gab2. The crystal structure of ISIR-005 in complex with 14-3-3 and the binding motif of Gab2 comprising two phosphorylation sites (Gab2pS210pT391) showed how the stabilizing molecule binds to the rim-of-the-interface of the protein complex. Only in the direct vicinity of 14-3-3/Gab2pT391 site is a pre-formed pocket occupied by ISIR-005; binding of the Gab2pS210 motif to 14-3-3 does not create an interface pocket suitable for the molecule. Accordingly, ISIR-005 only stabilizes the binding of the Gab2pT391 but not the Gab2pS210 site. This study represents structural and biochemical proof of the druggability of the 14-3-3/Gab2 PPI interface with important implications for the development of PPI stabilizers.

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Abstract. Cotylenin A, a plant growth regulator, and rapamycin, an inhibitor of the mammalian target of rapamycin, are potent inducers of differentiation in myeloid leukemia cells and also synergistically inhibit the proliferation of several human breast cancer cell lines including MCF-7 in vitro and in vivo. However, the mechanisms of the combined effects of cotylenin A and rapamycin are still unknown. Activated Akt induced by rapamycin has been suggested to attenuate the growth-inhibitory effects of rapamycin, serving as a negative feedback mechanism. In this study, we found that cotylenin A could suppress rapamycin-induced phosphorylation of Akt (Ser473) in MCF-7 cells and lung carcinoma A549 cells and that cotylenin A also enhanced the rapamycin-induced growth inhibition of MCF-7 and A549 cells. ISIR-005 (a synthetic cotylenin A-derivative) was able to enhance rapamycin-induced growth inhibition and could also markedly inhibit rapamycininduced phosphorylation of Akt. We also found that the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) or arsenic trioxide (ATO) in combination with rapamycin markedly inhibited the growth of MCF-7 cells and 17-AAG or ATO suppressed rapamycin-induced phosphorylation of Akt. The PI3K inhibitor LY294002 also suppressed rapamycin-induced phosphorylation of Akt and combined treatment showed synergistic growth inhibition of MCF-7 cells. Rapamycin inhibited growth more significantly in Akt siRNA-transfected MCF-7 cells than in control siRNA-transfected MCF-7 cells. These results suggest that the inhibition of rapamycin-induced Akt phosphorylation by cotylenin A correlates with their effective growth inhibition of cancer cells.