Stephen Boulton

Biochemical and Pharmacological Characterizations of ESI-09 Based EPAC Inhibitors: Defining the ESI-09 “Therapeutic Window”

The cAMP signaling cascade is one of the most frequently targeted pathways for the development of pharmaceutics. A plethora of recent genetic and pharmacological studies suggest that exchange proteins directly activated by cAMP (EPACs) are implicated in multiple pathologies. Selective EPAC inhibitors have been recently developed. One specific inhibitor, ESI-09, has been shown to block EPAC activity and functions, as well as to recapitulate genetic phenotypes of EPAC knockout mice when applied in vivo. However, a recent study raised concern that ESI-09 might act as a non-specific protein denaturant. Herein, we present a detailed biochemical and pharmacological characterization, as well as a structure-activity relationship (SAR) analysis of ESI-09. Our studies show that ESI-09 dose-dependently inhibits activity of both EPAC1 and EPAC2 with apparent IC50 values well below the concentrations shown to induce “protein denaturation”. Moreover, the ESI-09s action towards EPAC proteins is highly sensitive to minor modifications of the 3-chlorophenyl moiety. Taken together, these results demonstrate that ESI-09 indeed acts as an EPAC specific antagonist and does not significantly destabilize/denature proteins at pharmacological effective concentrations. This conclusion is further supported by NMR data showing that ESI-09 induces residue-dependent chemical shift changes at low concentrations, while preserving well dispersed peaks.

A mechanism for the auto-inhibition of hyperpolarization-activated cyclic nucleotide-gated (HCN) channel opening and its relief by cAMP.

Background: Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels control electrical activity through tetramerization of an intracellular linker. Results: NMR shows that the apo-cAMP-binding domain (CBD) of HCN4 destabilizes the tetramer through steric clashes. Conclusion: The apo-HCN4 CBD structure is compatible with monomeric and dimeric but not with tetrameric HCN4. Significance: The proposed mechanism explains HCN auto-inhibition and its relaxation by cAMP. Hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels control neuronal and cardiac electrical rhythmicity. There are four homologous isoforms (HCN1–4) sharing a common multidomain architecture that includes an N-terminal transmembrane tetrameric ion channel followed by a cytoplasmic “C-linker,” which connects a more distal cAMP-binding domain (CBD) to the inner pore. Channel opening is primarily stimulated by transmembrane elements that sense membrane hyperpolarization, although cAMP reduces the voltage required for HCN activation by promoting tetramerization of the intracellular C-linker, which in turn relieves auto-inhibition of the inner pore gate. Although binding of cAMP has been proposed to relieve auto-inhibition by affecting the structure of the C-linker and CBD, the nature and extent of these cAMP-dependent changes remain limitedly explored. Here, we used NMR to probe the changes caused by the binding of cAMP and of cCMP, a partial agonist, to the apo-CBD of HCN4. Our data indicate that the CBD exists in a dynamic two-state equilibrium, whose position as gauged by NMR chemical shifts correlates with the V½ voltage measured through electrophysiology. In the absence of cAMP, the most populated CBD state leads to steric clashes with the activated or “tetrameric” C-linker, which becomes energetically unfavored. The steric clashes of the apo tetramer are eliminated either by cAMP binding, which selects for a CBD state devoid of steric clashes with the tetrameric C-linker and facilitates channel opening, or by a transition of apo-HCN to monomers or dimer of dimers, in which the C-linker becomes less structured, and channel opening is not facilitated.

A Tool Set to Map Allosteric Networks through the NMR Chemical Shift Covariance Analysis

Allostery is an essential regulatory mechanism of biological function. Allosteric sites are also pharmacologically relevant as they are often targeted with higher selectivity than orthosteric sites. However, a comprehensive map of allosteric sites poses experimental challenges because allostery is driven not only by structural changes, but also by modulations in dynamics that typically remain elusive to classical structure determination methods. An avenue to overcome these challenges is provided by the NMR chemical shift covariance analysis (CHESCA), as chemical shifts are exquisitely sensitive to redistributions in dynamic conformational ensembles. Here, we propose a set of complementary CHESCA algorithms designed to reliably detect allosteric networks with minimal occurrences of false positives or negatives. The proposed CHESCA toolset was tested for two allosteric proteins (PKA and EPAC) and is expected to complement traditional comparative structural analyses in the comprehensive identification of functionally relevant allosteric sites, including those in otherwise elusive partially unstructured regions.

Allosteric linkers in cAMP signalling.

Weak interactions mediated by dynamic linkers are key determinants of allosteric regulation in multidomain signalling proteins. However, the mechanisms of linker-dependent control have remained largely elusive. In the present article, we review an allosteric model introduced recently to explain how signalling proteins effectively sense and respond to weak interactions, such as those elicited by flexible linkers flanking globular domains. Central to this model is the idea that near degeneracy within the free energy landscape of conformational selection maximally amplifies the response to weak (~2RT), but conformation-selective interactions. The model was tested as proof of principle using the prototypical regulatory subunit (R) of protein kinase A and led to the unanticipated finding that dynamic linkers control kinase activation and inhibition by tuning the inhibitory pre-equilibrium of a minimally populated intermediate (apo R). A practical implication of the proposed model is a new strategy to design kinase inhibitors with enhanced potency through frustration-relieving mutations.

Tapping the translation potential of cAMP signalling: molecular basis for selectivity in cAMP agonism and antagonism as revealed by NMR.

Eukaryotic CBDs (cAMP-binding domains) control multiple cellular functions (e.g. phosphorylation, guanine exchange and ion channel gating). Hence the manipulation of cAMP-dependent signalling pathways has a high translational potential. However, the ubiquity of eukaryotic CBDs also poses a challenge in terms of selectivity. Before the full translational potential of cAMP signalling can be tapped, it is critical to understand the structural basis for selective cAMP agonism and antagonism. Recent NMR investigations have shown that structurally homologous CBDs respond differently to several CBD ligands and that these unexpected differences arise at the level of either binding (i.e. affinity) or allostery (i.e. modulation of the autoinhibitory equilibria). In the present article, we specifically address how the highly conserved CBD fold binds cAMP with markedly different affinities in PKA (protein kinase A) relative to other eukaryotic cAMP receptors, such as Epac (exchange protein directly activated by cAMP) and HCN (hyperpolarization-activated cyclic-nucleotide-modulated channel). A major emerging determinant of cAMP affinity is hypothesized to be the position of the autoinhibitory equilibrium of the apo-CBD, which appears to vary significantly across different CBDs. These analyses may assist the development of selective CBD effectors that serve as potential drug leads for the treatment of cardiovascular diseases.

‘AND’ logic gates at work: Crystal structure of Rad53 bound to Dbf4 and Cdc7

Forkhead-associated (FHA) domains are phosphopeptide recognition modules found in many signaling proteins. The Saccharomyces cerevisiae protein kinase Rad53 is a key regulator of the DNA damage checkpoint and uses its two FHA domains to interact with multiple binding partners during the checkpoint response. One of these binding partners is the Dbf4-dependent kinase (DDK), a heterodimer composed of the Cdc7 kinase and its regulatory subunit Dbf4. Binding of Rad53 to DDK, through its N-terminal FHA (FHA1) domain, ultimately inhibits DDK kinase activity, thereby preventing firing of late origins. We have previously found that the FHA1 domain of Rad53 binds simultaneously to Dbf4 and a phosphoepitope, suggesting that this domain functions as an ‘AND’ logic gate. Here, we present the crystal structures of the FHA1 domain of Rad53 bound to Dbf4, in the presence and absence of a Cdc7 phosphorylated peptide. Our results reveal how the FHA1 uses a canonical binding interface to recognize the Cdc7 phosphopeptide and a non-canonical interface to bind Dbf4. Based on these data we propose a mechanism to explain how Rad53 enhances the specificity of FHA1-mediated transient interactions.

Free energy landscape remodeling of the cardiac pacemaker channel explains the molecular basis of familial sinus bradycardia

The hyperpolarization-activated and cyclic nucleotide-modulated ion channel (HCN) drives the pacemaker activity in the heart, and its malfunction can result in heart disorders. One such disorder, familial sinus bradycardia, is caused by the S672R mutation in HCN, whose electrophysiological phenotypes include a negative shift in the channel activation voltage and an accelerated HCN deactivation. The outcomes of these changes are abnormally low resting heart rates. However, the molecular mechanism underlying these electrophysiological changes is currently not fully understood. Crystallographic investigations indicate that the S672R mutation causes limited changes in the structure of the HCN intracellular gating tetramer, but its effects on protein dynamics are unknown. Here, we utilize comparative S672R versus WT NMR analyses to show that the S672R mutation results in extensive perturbations of the dynamics in both apo- and holo-forms of the HCN4 isoform, reflecting how S672R remodels the free energy landscape for the modulation of HCN4 by cAMP, i.e. the primary cyclic nucleotide modulator of HCN channels. We show that the S672R mutation results in a constitutive shift of the dynamic auto-inhibitory equilibrium toward inactive states of HCN4 and broadens the free-energy well of the apo-form, enhancing the millisecond to microsecond dynamics of the holo-form at sites critical for gating cAMP binding. These S672R-induced variations in dynamics provide a molecular basis for the electrophysiological phenotypes of this mutation and demonstrate that the pathogenic effects of the S672R mutation can be rationalized primarily in terms of modulations of protein dynamics.

Lipid binding protein response to a bile acid library: a combined NMR and statistical approach

Primary bile acids, differing in hydroxylation pattern, are synthesized from cholesterol in the liver and, once formed, can undergo extensive enzyme‐catalysed glycine/taurine conjugation, giving rise to a complex mixture, the bile acid pool. Composition and concentration of the bile acid pool may be altered in diseases, posing a general question on the response of the carrier (bile acid binding protein) to the binding of ligands with different hydrophobic and steric profiles. A collection of NMR experiments (H/D exchange, HET‐SOFAST, ePHOGSY NOESY/ROESY and 15N relaxation measurements) was thus performed on apo and five different holo proteins, to monitor the binding pocket accessibility and dynamics. The ensemble of obtained data could be rationalized by a statistical approach, based on chemical shift covariance analysis, in terms of residue‐specific correlations and collective protein response to ligand binding. The results indicate that the same residues are influenced by diverse chemical stresses: ligand binding always induces silencing of motions at the protein portal with a concomitant conformational rearrangement of a network of residues, located at the protein anti‐portal region. This network of amino acids, which do not belong to the binding site, forms a contiguous surface, sensing the presence of the bound lipids, with a signalling role in switching protein–membrane interactions on and off.

Implementation of the NMR CHEmical Shift Covariance Analysis (CHESCA): A Chemical Biologist’s Approach to Allostery

Mapping allosteric sites is emerging as one of the central challenges in physiology, pathology, and pharmacology. Nuclear Magnetic Resonance (NMR) spectroscopy is ideally suited to map allosteric sites, given its ability to sense at atomic resolution the dynamics underlying allostery. Here, we focus specifically on the NMR CHEmical Shift Covariance Analysis (CHESCA), in which allosteric systems are interrogated through a targeted library of perturbations (e.g., mutations and/or analogs of the allosteric effector ligand). The atomic resolution readout for the response to such perturbation library is provided by NMR chemical shifts. These are then subject to statistical correlation and covariance analyses resulting in clusters of allosterically coupled residues that exhibit concerted responses to the common set of perturbations. This chapter provides a description of how each step in the CHESCA is implemented, starting from the selection of the perturbation library and ending with an overview of different clustering options.

Mechanism of Selective Enzyme Inhibition through Uncompetitive Regulation of an Allosteric Agonist

Classical uncompetitive inhibitors are potent pharmacological modulators of enzyme function. Since they selectively target enzyme-substrate complexes (E:S), their inhibitory potency is amplified by increasing substrate concentrations. Recently, an unconventional uncompetitive inhibitor, called CE3F4R, was discovered for the exchange protein activated by cAMP isoform 1 (EPAC1). Unlike conventional uncompetitive inhibitors, CE3F4R is uncompetitive with respect to an allosteric effector, cAMP, as opposed to the substrate (i.e., CE3F4R targets the E:cAMP rather than the E:S complex). However, the mechanism of CE3F4R as an uncompetitive inhibitor is currently unknown. Here, we elucidate the mechanism of CE3F4Rs action using NMR spectroscopy. Due to limited solubility and line broadening, which pose major challenges for traditional structural determination approaches, we resorted to a combination of protein- and ligand-based NMR experiments to comparatively analyze EPAC mutations, inhibitor analogs, and cyclic nucleotide derivatives that trap EPAC at different stages of activation. We discovered that CE3F4R binds within the EPAC cAMP-binding domain (CBD) at a subdomain interface distinct from the cAMP binding site, acting as a wedge that stabilizes a cAMP-bound mixed-intermediate. The mixed-intermediate includes attributes of both the apo/inactive and cAMP-bound/active states. In particular, the intermediate targeted by CE3F4R traps a CBDs hinge helix in its inactive conformation, locking EPAC into a closed domain topology that restricts substrate access to the catalytic domain. The proposed mechanism of action also explains the isoform selectivity of CE3F4R in terms of a single EPAC1 versus EPAC2 amino acid difference that destabilizes the active conformation of the hinge helix.