Therapeutic targeting of 3',5'-cyclic nucleotide phosphodiesterases: inhibition and beyond

Abstract

Phosphodiesterases (PDEs), enzymes that degrade 3',5'-cyclic nucleotides, are being pursued as therapeutic targets for several diseases, including those affecting the nervous system, the cardiovascular system, fertility, immunity, cancer and metabolism. Clinical development programmes have focused exclusively on catalytic inhibition, which continues to be a strong focus of ongoing drug discovery efforts. However, emerging evidence supports novel strategies to therapeutically target PDE function, including enhancing catalytic activity, normalizing altered compartmentalization and modulating post-translational modifications, as well as the potential use of PDEs as disease biomarkers. Importantly, a more refined appreciation of the intramolecular mechanisms regulating PDE function and trafficking is emerging, making these pioneering drug discovery efforts tractable.

Figure 1.. 11 families of PDEs degrade cyclic nucleotides.

Both of the canonical cyclic nucleotide signaling pathways (cAMP and cGMP) are composed of numerous molecules responsible for the synthesis, execution, and breakdown of their signals. cAMP is synthesized by transmembrane adenylyl cyclases (ACs) that are activated by Gαs and inhibited by Gαi as well as soluble ACs that are activated by bicarbonate and calcium. cGMP is synthesized by particulate guanylyl cyclases (pGCs) that are activated by natriuretic peptides and soluble guanylyl cyclases (sGCs) that are activated by nitric oxide (NO). Both cAMP and cGMP activate cyclic nucleotide gated channels and allosterically modulate activity of select PDEs. In contrast, only cGMP stimulates protein kinase G (PKG); whereas, cAMP activates protein kinase A (PKA), exchange protein activated by cAMP (Epac) and popeye domain-containing proteins (POPDC). Signaling through either the cAMP or cGMP pathways ultimately leads to phosphorylation of a myriad of downstream targets, including the transcription factor cAMP response element binding protein (CREB). In addition to cAMP and cGMP, several PDEs also hydrolyze the non-canonical cyclic nucleotides (not included) cUMP (PDE3A, PDE3B, PDE9A), cCMP (PDE7A), and c-di-GMPa (bacterial PDEs), albeit with much lower affinity^, .

Figure 2.. The 21 phosphodiesterase (PDE) genes are grouped into families (name and substrate specificity listed to right of each illustration) based on the homology of their C-terminal catalytic domain (represented as a semi-ellipse).

Due to alternate promoters and splicing events, each PDE family has multiple isoforms that differ in terms of the length and complexity of their N-terminal regulatory domains (depicted with different shapes), which are thought to regulate subcellular trafficking, substrate affinity, and catalytic activity. The relative size and domain distances were drawn based on estimations from the Pfam/uniprot database, with the exception of the REC domain of PDE8 (estimated from) and the second CaM domain of PDE1 (estimated from). Illustrations represent the longest isoform for gene A of each PDE family. CaM, calmodulin-binding domain; GAF, cGMP-binding PDEs Anabaena adenylyl cyclases and E. coli FhlA; TM, transmembrane domain of PDE3; UCR, upstream conserved region; REC, signal receiver domain; PAS, Per-Arnt-Sim domain.

Figure 3.. Mechanisms that activate phosphodiesterase (PDE) catalytic activity.

A) Calcium-calmodulin (CaM) binding to the CaM domains of PDE1 relieves N-terminal auto-inhibition of the catalytic site, thereby promoting enzymatic activity. B) Cyclic nucleotides binding to GAF domains of dimeric PDEs (shown here: cGMP binding the GAF-B domain of PDE2) are thought to promote catalytic activity by inducing an outward rotation of the catalytic domains and, thus, enabling access to substrates. C) Phosphorylation by PKA or PKG activates several PDEs. In the case of PDE4D, phosphorylation of the UCR1 domain by PKA causes UCR1 to bind its own UCR2 domain instead of the catalytic site of the other monomer, thereby locking the enzyme in an active state. D) PDE activity can also be modulated by protein-protein binding interactions. One such well-characterized example involves membrane-bound PDE6, where the rhodopsin-activated G-protein α-subunit transducin displaces the inhibitory PDE6γ C-termini from the catalytic sites on PDE6αβ, thus, promoting cGMP hydrolysis.

Figure 4.. Methods for targeting phosphodiesterase signaling with increasing specificity.

Given the vast diversity of PDE isoforms, each with unique tissue expression profiles, subcellular compartmentalization, and protein-protein interactions, it is becoming clear that selective targeting of PDE function will be required to achieve efficacy while diminishing undesirable side effects. Small molecule inhibitors (e.g., cilomilast) are readily developed with family-specific selectivity (e.g., targeting PDE4 over PDE3); however, isoform specificity remains a challenge (e.g., cilomilast inhibits PDE4D with only 7-fold selectivity versus PDE4B). Conversely, gene therapy (i.e., expressing a recombinant construct to knock down or restore expression of a given PDE isoform) and dominant negative approaches (i.e., expressing a catalytically inactive PDE4D5 that displaces the endogenous isoform from its interacting partners) can target isoform subtypes exclusively (e.g., targeting PDE4D5 but not PDE4D3 nor PDE4B). That said, dominant negative approaches would influence signalling within all microdomains regulated by that isoform. The greatest specificity can be achieved with peptide/small molecule binding disruptors or mutagenesis approaches (not shown) that are designed to prevent a specific PDE isoform from binding a specific partner, thus, altering signaling only within one specific complex. As shown here, a disruptor peptide that specifically prevents the interaction between PDE4D5 and β-arrestin would lead to the recruitment of EPAC1 to β2 adrenergic receptors (βAR), but would leave PDE4D5 regulation of heat shock protein 20 (HSP20) and RACK1 complexes intact^{, ,} .

Figure 5.. Phosphodiesterase (PDE) regulation by post-translation modification (PTM).

Cyclic nucleotide dynamics can be modulated by the addition of different functional groups to PDEs. Phosphorylation is a very common mechanism to control PDE activity as depicted by the action of PKA on PDE4D3. Both enzymatic activity and binding affinity of PDE4D3 for mAKAP are increased by PKA phosphorylation, allowing a faster signal termination in myocytes. Palmitoylation of PDE10A2 in its N-terminal region translocates the enzyme to the plasma membrane, although its phosphorylation by PKA can prevent the action of the palmitoyl acyltransferase (zDHHC). Ubiquitination can influence PDE function by controling stability. For example, the E3 ubiquitin ligase Smurf2 targets PDE4B for degradation which leads to the attenuation of liver fibrosis. S-nitrosylation can also tag PDEs for destruction. Thus, the covalent incorporation of nitric oxide (NO) to the GAF-A domain of a PKG-phosphorylated and active PDE5, directs the enzyme to the proteasome. Hydroxylation of proline residues has emerged as another PTM to stimulate turnover of PDEs. Prolyl hydroxylase domain protein 2 (PHD2) action on PDE4D increases its recognition by E3 ligase complexes in cardiomyocytes. Finally, SUMOylation can intensify the activity of PDE4A and PDE4D. The SUMO transfer from the E2 conjugase UBC9–E3 enzyme PIASy complex to the PDEs, enhances their activation by PKA phosphorylation and represses their inhibition induced by ERK activity.