Unraveling the inhibitory mechanism of adenylyl cyclase 8E: New insights into regulatory pathways of cAMP signal integration.

Adenylyl Cyclase 8E (AC8E), which lacks part of M1 transmembrane domain, has been previously shown to dimerize with AC3 and down-regulate its activity, but the molecular mechanism of this inhibitory effect has remained elusive. Here, we first show that AC8E also inhibits AC2 and AC6, highlighting the functional importance of this novel regulatory mechanism in the cAMP signaling pathway across AC families. We then completed the partial structure of Bos taurus AC9 using combinations of comparative modeling and fold recognition methods, and used this as a template to build the first full 3D-models of AC8 and AC8E. These models evidenced that the lack of M1 transmembrane domain of AC8E shifts the N-terminal domain, which impacts the orientation of the helical domains, thus affecting the catalytic site. This was confirmed in living cells with cAMP imaging, where we showed that the N-terminal domain is required for reducing cAMP production. Our data also show that AC8E prevents the translocation of other ACs towards the plasma membrane, further reducing the cAMP responsiveness to extracellular signals. This newly discovered dual inhibitory mechanism provides an additional level of regulation of cAMP-dependent signals integration.

Identification of a novel BCL2-specific inhibitor that binds predominantly to the BH1 domain.

The antiapoptotic protein BCL2 is overexpressed in several cancers and contributes to prolonged cell survival and chemoresistance, lending itself as an excellent target for cancer therapy. Here, we report the design, synthesis, and characterization of Disarib, a novel BCL2 inhibitor. Disarib showed selective cytotoxicity in BCL2 high cancer cell lines, and CLL patient primary cells, as compared to BCL2 low cell lines. BCL2 knockdown in cells rendered remarkable resistance to Disarib, while sensitivity was regained upon its ectopic expression, establishing target specificity. In silico, biochemical and biophysical studies demonstrated strong affinity of Disarib to BCL2, but not to other antiapoptotic BCL2 family members viz., BCL-xL, BCL2A1 etc. Interestingly, biophysical studies showed that BH1 domain deletion mutant demonstrated ~ 67-fold reduction in BCL2-Disarib interaction, while it was only ~ 20-fold in the case of BH3 deletion mutant, suggesting predominant involvement of the BH1 domain for Disarib binding. Thus, we report identification of a novel BCL2 inhibitor with a unique mechanism of BCL2 inhibition, as opposed to the well-studied BH3 domain targeting.