Recent advances in the discovery of small molecules targeting exchange proteins directly activated by cAMP (EPAC)

Abstract

3',5'-Cyclic adenosine monophosphate (cAMP) is a pivotal second messenger that regulates numerous biological processes under physiological and pathological conditions, including cancer, diabetes, heart failure, inflammation, and neurological disorders. In the past, all effects of cAMP were initially believed to be mediated by protein kinase A (PKA) and cyclic nucleotide-regulated ion channels. Since the discovery of exchange proteins directly activated by cyclic adenosine 5'-monophosphate (EPACs) in 1998, accumulating evidence has demonstrated that the net cellular effects of cAMP are also regulated by EPAC. The pursuit of the biological functions of EPAC has benefited from the development and applications of a growing number of pharmacological probes targeting EPACs. In this review, we seek to provide a concise update on recent advances in the development of chemical entities including various membrane-permeable analogues of cAMP and newly discovered EPAC-specific ligands from high throughput assays and hit-to-lead optimizations.

Figure 1

Domain structures of EPAC proteins. Each EPAC family member composes an auto-inhibitory amino-terminal regulatory region and a carboxyl-terminal catalytic region for activation of Rap GTPase. The regulatory region contains a Dishevelled Egl-10 Pleckstrin (DEP) domain and at least one functional cyclic nucleotide–binding domain (CNBD). The carboxyl-terminal catalytic region consists of a Ras exchange motif (REM) domain and a Ras association (RA) domain as well as the CDC25-homology domain (CDC25-HD). The CDC25-homology domain is responsible for guanine nucleotide exchange activity and catalyzes the exchange of G-protein-bound GDP for GTP on the Ras-like small GTPases Rap1 and Rap2 isoforms.

Figure 2

The X-ray crystal structures of inactive EPAC2 (the full-length apo-EPAC2, PDB: 2BYV) and active EPAC2 (EPAC2Δ305:Sp-cAMPS:Rap1B complex, PDB: 3CF6). CNBD-A, DEP domain, CNBD-B, REM domain, RA domain, and CDC25-HD are colored in red, green, blue, yellow, cyan, and magentas, respectively. Upon binding of cAMP, a sequence of structural reorganizations within the cyclic nucleotide–binding domains allows the regulatory domain to open and move to the back side of the catalytic region. This conformational change leads to the exposure of the catalytic region for binding of Rap GTPase to catalyze the exchange of GDP for GTP.

Figure 3

Signaling pathway of EPAC proteins. EPAC1 only contains one functional cyclic nucleotide binding domain (CNBD-B), while EPAC2 contains two CNBDs (CNBD-A and CNBD-B).

Figure 4

Chemical structures of cAMP molecule (1) and cAMP analogues with modified exocyclic oxygen atom (2 and 3).

Figure 5

Chemical structures of 2′-deoxy-cAMP (4), 2′-O-Me-cAMP (5), 6-Bnz-cAMP (6) and N⁶-phenyl-cAMP (7).

Figure 6

Chemical structures of 8-Br-2′-O-Me-cAMP (8), 8-pCPT-2′-O-Me-cAMP (9), 8-pMeOPT-2′-O-Me-cAMP (10) and 8-pHPT-2′-O-Me-cAMP (11).

Figure 7

Chemical structures of 8-pCPT-2′-O-Me-cAMP-AM (12), Sp-8-pCPT-2′-O-Me-cAMPS (13) and 8-(2-[7-nitro-4-benzofurazanyl]aminoethylthio)adenosine-3′, 5′-cyclic monophosphate (14, 8-NBD-cAMP).

Figure 8

Chemical structures of identified EPAC inhibitors 15–17.

Figure 9

Chemical structures of HTS hits 18–24.

Figure 10

Chemical structures of 25 and 26.

Figure 11

Predicted binding mode and molecular docking of 25, and 23 into the cAMP binding domain B (CNBD-B) of EPAC2 protein. Important residues are drawn in sticks. Hydrogen bonds are shown as dashed green lines. (A) Binding mode of 25 (orange). The cyclopentyl group at the C-6 position interacts with the hydrophobic residues of Phe367, Ala415 and Ala416, while the hydrophobic S-benzyl moiety of 2-position forms interactions with Leu406 and Leu449. (B) Binding mode of 23 (pink). The tert-butylisoxazolyl moiety forms a hydrogen bond with the residue Gly404 and interacts with the hydrophobic residues of Phe367, Leu406, Ala407, and Ala415. Meanwhile, 3-chlorophenyl fragment forms hydrophobic interactions with residues Val386, Val394 and Leu397.

Figure 12

Chemical structures of representative diarylsulfones 27, 28 and N,N-diarylamines 29–32, and arylsulfonamides 33–35.

Figure 13

Chemical structures of tetrahydroquinoline analogues 36–39.