Cyclic AMP (cAMP) phosphodiesterase-4 (PDE4) enzymes degrade cAMP and underpin the compartmentalization of cAMP signaling through their targeting to particular protein complexes and intracellular locales. We describe the discovery and characterization of a small-molecule compound that allosterically activates PDE4 long isoforms. This PDE4-specific activator displays reversible, noncompetitive kinetics of activation (increased Vmax with unchanged Km), phenocopies the ability of protein kinase A (PKA) to activate PDE4 long isoforms endogenously, and requires a dimeric enzyme assembly, as adopted by long, but not by short (monomeric), PDE4 isoforms. Abnormally elevated levels of cAMP provide a critical driver of the underpinning molecular pathology of autosomal dominant polycystic kidney disease (ADPKD) by promoting cyst formation that, ultimately, culminates in renal failure. Using both animal and human cell models of ADPKD, including ADPKD patient-derived primary cell cultures, we demonstrate that treatment with the prototypical PDE4 activator compound lowers intracellular cAMP levels, restrains cAMP-mediated signaling events, and profoundly inhibits cyst formation. PDE4 activator compounds thus have potential as therapeutics for treating disease driven by elevated cAMP signaling as well as providing a tool for evaluating the action of long PDE4 isoforms in regulating cAMP-mediated cellular processes.

A survey of PDE4 inhibitors reveals that some compounds trigger intracellular aggregation of PDE4A4 into accretion foci through association with the ubiquitin-binding scaffold protein p62 (SQSTM1). We show that this effect is driven by inhibitor occupancy of the catalytic pocket and stabilization of a "capped state" in which a sequence within the enzyme's upstream conserved region 2 (UCR2) module folds across the catalytic pocket. Only certain inhibitors cause PDE4A4 foci formation, and the structural features responsible for driving the process are defined. Switching to the UCR2-capped state induces conformational transition in the enzyme's regulatory N-terminal portion, facilitating protein association events responsible for reversible aggregate assembly. PDE4-selective inhibitors able to trigger relocalization of PDE4A4 into foci can therefore be expected to exert actions on cells that extend beyond simple inhibition of PDE4 catalytic activity and that may arise from reconfiguring the enzyme's protein association partnerships.

The propensity of human embryonic stem cells to die upon enzymatic disaggregation or low-density plating is an obstacle to their isolation and routine use in drug discovery and basic research. Equally, the very low rate of establishment of implanted cells hinders cell therapy. In the present study we have developed a high-content assay for human embryonic stem cell survival and used this to screen a range of libraries of 'lead-like' small molecules and known bioactives. From this we identified 18 confirmed hits with four structural classes being represented by multiple compounds: a series of 5-(acyl/alkyl-amino)indazoles, compounds with a 4-(acylamino)pyridine core, simple N⁶,N⁶-dialkyladenines and compounds with a 5-(acylamino)indolinone core. In vitro kinase profiling indicated that the ROCK (Rho-associated kinase)/PRK2 (protein kinase C-related kinase 2) protein kinases are of pivotal importance for cell survival and identified previously unreported compound classes that inhibited this important biological activity. An evaluation using an extensive panel of protein kinases showed that six of our hit compounds exhibited better selectivity for ROCK inhibition than the routinely used commercially available ROCK inhibitor Y-27632. In this screen we also identified the K(+)-ATP channel opener pinacidil and show that it probably promotes cell survival, by 'off-target' inhibition of ROCK/PRK2. We have therefore identified novel pro-survival compounds of greater specificity, equivalent potency and reduced toxicity relative to the routinely employed ROCK inhibitor Y-27632.

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