Hydroxamate based inhibitors of adenylyl cyclase. Part 2: the effect of cyclic linkers on P-site binding

The adenylyl cyclases (ACs) are a family of enzymes that are key elements of signal transduction by virtue of their ability to convert ATP to cAMP. The catalytic mechanism of this transformation proceeds through initial binding of ATP to the purine binding site (P-site) followed by metal mediated cyclization with loss of pyrophosphate. Previous work in our group identified novel inhibitors which possess an adenine ring joined to a metal-coordinating hydroxamic acid through flexible linkers. Considering the spatial positioning of the metals with respect to the adenine binding site coupled with potentially favorable entropic factors, conformational restriction of the tether through a stereochemistry based SAR employing a rigid cyclic scaffold was explored.     

Comparative electrostatic analysis of adenylyl cyclase for isoform dependent regulation properties

The enzyme adenylyl cyclase (AC) plays a pivotal role in a variety of signal transduction pathways inside the cell, where it catalyzes the cyclization of adenosine triphosphate (ATP) into the second‐messenger cyclic adenosine monophosphate (cAMP). Among other roles, AC regulates processes involved in neural plasticity, innervation of smooth muscles of the heart and the endocrine system of the pancreas. The functional diversity of AC is manifested in its different isoforms, each having a specific regulation pattern. There is an increasing amount of data available concerning the regulatory properties of AC isoforms, however little is known about the interactions on a structural level. Here, we conducted a comparative electrostatic analysis of the catalytic domains of all nine transmembrane AC isoforms with the aim of detecting, verifying and predicting the binding sites of molecular regulators on AC. The results provide support for the positioning of the binding site of the inhibitory protein G_iα at a pseudo‐symmetric position to the stimulatory G_sα binding site. They also provide a structural interpretation of the Gβγ interaction with ACs 2, 4, and 7 and suggest a new binding site for RGS2. Comparison of the small molecule binding sites on AC shows that overall they have high electrostatic similarity, but regions of electrostatic differences are identified. These could provide a basis for the development of novel compounds with isoform‐specific modulatory effects on AC. Proteins 2016; 84:1844–1858. © 2016 Wiley Periodicals, Inc.     

A novel mechanism for adenylyl cyclase inhibition from the crystal structure of its complex with catechol estrogen

Catechol estrogens are steroid metabolites that elicit physiological responses through binding to a variety of cellular targets. We show here that catechol estrogens directly inhibit soluble adenylyl cyclases and the abundant trans-membrane adenylyl cyclases. Catechol estrogen inhibition is non-competitive with respect to the substrate ATP, and we solved the crystal structure of a catechol estrogen bound to a soluble adenylyl cyclase from Spirulina platensis in complex with a substrate analog. The catechol estrogen is bound to a newly identified, conserved hydrophobic patch near the active center but distinct from the ATP-binding cleft. Inhibitor binding leads to a chelating interaction between the catechol estrogen hydroxyl groups and the catalytic magnesium ion, distorting the active site and trapping the enzyme substrate complex in a non-productive conformation. This novel inhibition mechanism likely applies to other adenylyl cyclase inhibitors, and the identified ligand-binding site has important implications for the development of specific adenylyl cyclase inhibitors.     

Differential inhibition of adenylyl cyclase isoforms and soluble guanylyl cyclase by purine and pyrimidine nucleotides

Mammals express nine membranous adenylyl cyclase isoforms (ACs 1-9), a structurally related soluble guanylyl cyclase (sGC) and a soluble AC (sAC). Moreover, Bacillus anthracis and Bacillus pertussis produce the AC toxins, edema factor (EF), and adenylyl cyclase toxin (ACT), respectively. 2'(3')-O-(N-methylanthraniloyl)-guanosine 5'-[gamma-thio]triphosphate is a potent competitive inhibitor of AC in S49 lymphoma cell membranes. These data prompted us to study systematically the effects of 24 nucleotides on AC in S49 and Sf9 insect cell membranes, ACs 1, 2, 5, and 6, expressed in Sf9 membranes and purified catalytic subunits of membranous ACs (C1 of AC5 and C2 of AC2), sAC, sGC, EF, and ACT in the presence of MnCl(2). N-Methylanthraniloyl (MANT)-GTP inhibited C1.C2 with a K(i) of 4.2 nm. Phe-889 and Ile-940 of C2 mediate hydrophobic interactions with the MANT group. MANT-inosine 5'-[gamma-thio]triphosphate potently inhibited C1.C2 and ACs 1, 5, and 6 but exhibited only low affinity for sGC, EF, ACT, and G-proteins. Inosine 5'-[gamma-thio]triphosphate and uridine 5'-[gamma-thio]triphosphate were mixed G-protein activators and AC inhibitors. AC5 was up to 15-fold more sensitive to inhibitors than AC2. EF and ACT exhibited unique inhibitor profiles. At sAC, 2',5'-dideoxyadenosine 3'-triphosphate was the most potent compound (IC(50), 690 nm). Several MANT-adenine and MANT-guanine nucleotides inhibited sGC with K(i) values in the 200-400 nm range. UTP and ATP exhibited similar affinities for sGC as GTP and were mixed sGC substrates and inhibitors. The exchange of MnCl(2) against MgCl(2) reduced inhibitor potencies at ACs and sGC 1.5-250-fold, depending on the nucleotide and cyclase studied. The omission of the NTP-regenerating system from cyclase reactions strongly reduced the potencies of MANT-ADP, indicative for phosphorylation to MANT-ATP by pyruvate kinase. Collectively, AC isoforms and sGC are differentially inhibited by purine and pyrimidine nucleotides.     

Towards selective inhibitors of adenylyl cyclase toxin from Bordetella pertussis

Whooping cough is a very important medical problem that requires novel approaches for treatment. The disease is caused by Bordetella pertussis, with the calmodulin (CaM)-activated adenylyl cyclase (AC) toxin (also known as CyaA) being a major virulence factor. Hence, CyaA inhibitors could constitute novel therapeutics, but it has been difficult to develop potent drugs with high selectivity over mammalian membranous ACs (mACs). Recent studies have shown that bis-anthraniloyl-substituted nucleoside 5'-triphosphates are potent and selective CyaA inhibitors. In addition, the interaction of CyaA with CaM is very different from the interaction of membranous mAC1 with CaM. Accordingly, compounds that interfere with the CyaA-CaM interaction may constitute a novel class of drugs against whooping cough.     

A point-mutated guanylyl cyclase with features of the YC-1-stimulated enzyme: implications for the YC-1 binding site?

Guanylyl cyclases (GCs) and adenylyl cyclases (ACs) play key roles in various signaling cascades and are structurally closely related. The crystal structure of a soluble AC revealed one binding site each for the substrate ATP and the activator forskolin. Recently, YC-1, a novel activator of the heterodimeric soluble GC (sGC), has been identified which acts like forskolin on AC. Here, we investigated the respective substrate and potential activator domains of sGC using point-mutated subunits. Whereas substitution of the conserved Cys-541 of the beta(1) subunit with serine led to an almost complete loss of activity, mutation of the respective homologue (Cys-596) in the alpha(1) subunit yielded an enzyme with an increased catalytic rate and higher sensitivity toward NO. This phenotype exhibits characteristics similar to those of the YC-1-treated wild-type enzyme. Conceivably, this domain which corresponds to the forskolin site of the ACs may comprise the binding site for YC-1.     

Molecular basis for P-site inhibition of adenylyl cyclase

P-site inhibitors are adenosine and adenine nucleotide analogues that inhibit adenylyl cyclase, the effector enzyme that catalyzes the synthesis of cyclic AMP from ATP. Some of these inhibitors may represent physiological regulators of adenylyl cyclase, and the most potent may ultimately serve as useful therapeutic agents. Described here are crystal structures of the catalytic core of adenylyl cyclase complexed with two such P-site inhibitors, 2'-deoxyadenosine 3'-monophosphate (2'-d-3'-AMP) and 2',5'-dideoxyadenosine 3'-triphosphate (2',5'-dd-3'-ATP). Both inhibitors bind in the active site yet exhibit non- or uncompetitive patterns of inhibition. While most P-site inhibitors require pyrophosphate (PP(i)) as a coinhibitor, 2',5'-dd-3'-ATP is a potent inhibitor by itself. The crystal structure reveals that this inhibitor exhibits two binding modes: one with the nucleoside moiety bound to the nucleoside binding pocket of the enzyme and the other with the beta and gamma phosphates bound to the pyrophosphate site of the 2'-d-3'-AMP.PP(i) complex. A single metal binding site is observed in the complex with 2'-d-3'-AMP, whereas two are observed in the complex with 2', 5'-dd-3'-ATP. Even though P-site inhibitors are typically 10 times more potent in the presence of Mn(2+), the electron density maps reveal no inherent preference of either metal site for Mn(2+) over Mg(2+). 2',5'-dd-3'-ATP binds to the catalytic core of adenylyl cyclase with a K(d) of 2.4 microM in the presence of Mg(2+) and 0.2 microM in the presence of Mn(2+). Pyrophosphate does not compete with 2',5'-dd-3'-ATP and enhances inhibition.     

Adenylyl cyclase, a coincidence detector for nitric oxide.

Nitric oxide (NO) donors inhibit hormone- and forskolin-stimulated adenylyl cyclase activity in purified plasma membrane preparations from N18TG2 neuroblastoma cells. Northern blot analyses indicate that the predominant isoform of adenylyl cyclase in N18TG2 cells is the type VI. Our experiments eliminate all the known regulatory proteins for this isoform as possible targets of NO. NO decreases the Vmax of the enzyme without altering the Km for ATP. Occupancy of the substrate-binding site protects the enzyme from the inhibitory effects of NO, suggesting that the conformation of the enzyme determines its sensitivity. The inhibition is reversed by reducing agents, implicating a Cys residue(s) as the target for nitric oxide and an S-nitrosylation as the underlying modification. These findings implicate NO as a novel cellular regulator of the type VI isoform of adenylyl cyclase.     

Discovery of a potent and selective adenylyl cyclase type 8 agonist by docking-based virtual screening

Adenylyl cyclases (ACs), which are responsible for catalyzing the conversion of adenosine triphosphate (ATP) into the second messenger cyclic adenosine monophosphate (cAMP), play a critical role in cell signal transduction. In this study, a combined approach involving docking-based virtual screening, with the combination of homology modeling followed by an in-vitro, and cell-based biological assay have been performed for discovering a class of novel potent and selective isoform adenylyl cyclase type 8 (AC8) agonist. The computer-aided virtual screening was used to identify fourteen virtual cluster compounds as potential hits which were further subjected to rigorous bioassays. A novel hit compound VHC-7 (ethyl 3-(2,4-dichlorobenzyl)-2-oxoindoline-3-carboxylate) was identified as a highly potent selective AC8 agonist with EC₅₀ value of 0.1052 ± 0.038 µM. Remarkably, the molecule herein reported can be explored further to discover greater number of hit compounds with better pharmacokinetic properties as well as to serve as a promising novel hit agonist of AC8 for the treatment of various central nervous system disorders and its associated diseases.     

Physiological roles for G protein-regulated adenylyl cyclase isoforms: insights from knockout and overexpression studies

Cyclic AMP is a universal second messenger, produced by a family of adenylyl cyclase (AC) enzymes. The last three decades have brought a wealth of new information about the regulation of cyclic AMP production by ACs. Nine hormone-sensitive, membrane-bound AC isoforms have been identified in addition to a tenth isoform that lacks membrane spans and more closely resembles the cyanobacterial AC enzymes. New model systems for purifying and characterizing the catalytic domains of AC have led to the crystal structure of these domains and the mapping of numerous interaction sites. However, big hurdles remain in unraveling the roles of individual AC isoforms and their regulation in physiological systems. In this review we explore the latest on AC knockout and overexpression studies to better understand the roles of G protein regulation of ACs in the brain, olfactory bulb, and heart.     

Tyrosine kinase-mediated serine phosphorylation of adenylyl cyclase

Receptor tyrosine kinase (RTK) activation is associated with modulation of heptahelical receptor-stimulated adenylyl cyclase responses. The mechanisms underlying the RTK-mediated enhancement of adenylyl cyclase function remain unclear. In the present studies, we show that the tyrosine kinase-dependent enhancement of adenylyl cyclase isoform VI function parallels an enhancement in serine phosphorylation of the enzyme. This effect was mediated by both RTK activation, with IGF-1, and by tyrosine phosphatase inhibition, with sodium orthovanadate. This enhancement of adenylyl cyclase function was not attenuated by inhibitors of ERK, PKC, PKA, or PI3 kinase activity but was blunted by inhibition of endogenous p74(raf-1)() activity. To characterize the molecular site of this effect we identified multiple candidate serine residues in and adjacent to the adenylyl cyclase VI C1b catalytic region and performed serine-to-alanine site-directed mutagenesis using adenylyl cyclase VI as a template. Mutation of serine residues 603 and 608 or serine residues 744, 746, 750, and 754 attenuated both the tyrosine kinase-mediated enhancement of enzyme phosphorylation as well as the sensitization of function. Together, these data define a novel tyrosine kinase-mediated mechanism leading to serine phosphorylation of adenylyl cyclase isoform VI and the sensitization of adenylyl cyclase responsiveness.     

Nitric oxide-dependent allosteric inhibitory role of a second nucleotide binding site in soluble guanylyl cyclase

The mechanism of desensitization of the nitric oxide (NO) receptor (alpha1.beta1 isoform of soluble guanylyl cyclase, sGC) is not known. Models of the structure of alpha1.beta1, based on the x-ray crystal structure of adenylyl cyclase (AC) suggest the existence of a nucleotide-like binding site, in addition to the putative catalytic site. We have previously reported that mutating residues that coordinate Mg(2+)GTP (substrate) binding in alpha1.beta1 into those present in AC fully reverts GC activity to AC activity. The wild-type form of alpha1.beta1 (GC-wt) and the mutant form (AC-mut, alpha1R592Q.beta1E473K,C541D) were purified, and their sensitivities to various nucleotides were assessed. In using the AC-mut as well as other mutants that coordinate purine binding, we were able to distinguish allosteric inhibitory effects of guanine nucleotides from competitively inhibitory effects on catalytic activity. Here we report that several nucleotide analogs drastically alter sGC and AC-mut activity by acting at a second nucleotide site, likely pseudosymmetric to the catalytic site. In particular, Mg(2+)GTP gamma S and Mg(2+)ATP gamma S inhibited cyclase activity through a mixed, non-competitive mechanism that was only observable under NO stimulation and not under basal conditions. The non-competitive pattern of inhibition was not present in mutants carrying the substitution beta1D477A, the pseudosymmetric equivalent to alpha1D529 (located in the substrate-binding site and involved in substrate binding and catalysis), or with the double mutations alpha1E525K,C594D, the pseudosymmetric equivalent to beta1E473K,C541D. Taken together these data suggest that occupation of the second site by nucleotides may underlie part of the mechanism of desensitization of sGC.     

Particulate and soluble adenylyl cyclases participate in the sperm acrosome reaction

cAMP is important in sea urchin sperm signaling, yet the molecular nature of the adenylyl cyclases (ACs) involved remained unknown. These cells were recently shown to contain an ortholog of the mammalian soluble adenylyl cyclase (sAC). Here, we show that sAC is present in the sperm head and as in mammals is stimulated by bicarbonate. The acrosome reaction (AR), a process essential for fertilization, is influenced by the bicarbonate concentration in seawater. By using functional assays and immunofluorescence techniques we document that sea urchin sperm also express orthologs of multiple isoforms of transmembrane ACs (tmACs). Our findings employing selective inhibitors for each class of AC indicate that both sAC and tmACs participate in the sperm acrosome reaction.     

Bicarbonate activation of adenylyl cyclase via promotion of catalytic active site closure and metal recruitment

In an evolutionarily conserved signaling pathway, 'soluble' adenylyl cyclases (sACs) synthesize the ubiquitous second messenger cyclic adenosine 3',5'-monophosphate (cAMP) in response to bicarbonate and calcium signals. Here, we present crystal structures of a cyanobacterial sAC enzyme in complex with ATP analogs, calcium and bicarbonate, which represent distinct catalytic states of the enzyme. The structures reveal that calcium occupies the first ion-binding site and directly mediates nucleotide binding. The single ion-occupied, nucleotide-bound state defines a novel, open adenylyl cyclase state. In contrast, bicarbonate increases the catalytic rate by inducing marked active site closure and recruiting a second, catalytic ion. The phosphates of the bound substrate analogs are rearranged, which would facilitate product formation and release. The mechanisms of calcium and bicarbonate sensing define a reaction pathway involving active site closure and metal recruitment that may be universal for class III cyclases.     

Direct inhibition of type 5 adenylyl cyclase prevents myocardial apoptosis without functional deterioration

Adenylyl cyclase, a major target enzyme of beta-adrenergic receptor signals, is potently and directly inhibited by P-site inhibitors, classic inhibitors of this enzyme, when the enzyme catalytic activity is high. Unlike beta-adrenergic receptor antagonists, this is a non- or uncompetitive inhibition with respect to ATP. We have examined whether we can utilize this enzymatic property to regulate the effects of beta-adrenergic receptor stimulation differentially. After screening multiple new and classic compounds, we found that some compounds, including 1R,4R-3-(6-aminopurin-9-yl)-cyclopentanecarboxylic acid hydroxyamide, potently inhibited type 5 adenylyl cyclase, the major cardiac isoform, but not other isoforms. In normal mouse cardiac myocytes, contraction induced by low beta-adrenergic receptor stimulation was poorly inhibited with this compound, but the induction of cardiac myocyte apoptosis by high beta-adrenergic receptor stimulation was effectively prevented by type 5 adenylyl cyclase inhibitors. In contrast, when cardiac myocytes from type 5 adenylyl cyclase knock-out mice were examined, beta-adrenergic stimulation poorly induced apoptosis. Our data suggest that the inhibition of beta-adrenergic signaling at the level of the type 5 adenylyl cyclase isoform by P-site inhibitors may serve as an effective method to prevent cardiac myocyte apoptosis induced by excessive beta-adrenergic stimulation without deleterious effect on cardiac myocyte contraction.     

Novel short isoforms of adenylyl cyclase as negative regulators of cAMP production

Here, we cloned a new family of four adenylyl cyclase (AC) splice variants from interleukin-1β (IL-1β)-transdifferentiated vascular smooth muscle cells (VSMCs) encoding short forms of AC8 that we have named “AC8E-H”. Using biosensor imaging and biochemical approaches, we showed that AC8E-H isoforms have no cyclase activity and act as dominant-negative regulators by forming heterodimers with other full-length ACs, impeding the traffic of functional units towards the plasma membrane. The existence of these dominant-negative isoforms may account for an unsuspected additional degree of cAMP signaling regulation. It also reconciles the induction of an AC in transdifferentiated VSMCs with the vasoprotective influence of cAMP. The generation of alternative splice variants of ACs may constitute a generalized strategy of adaptation to the cell's environment whose scope had so far been ignored in physiological and/or pathological contexts.     

Mutational Analysis Gives Insight into Substrate Preferences of a Nucleotidyl Cyclase from Mycobacterium avium

Mutational, crystallographic and phylogenetic analysis of nucleotidyl cyclases have been used to understand how these enzymes discriminate between substrates. Ma1120, a class III adenylyl cyclase (AC) from Mycobacterium avium, was used as a model to study the amino acid residues that determine substrate preference, by systematically replacing ATP specifying residues with those known to specify GTP. This enzyme was found to possess residual guanylyl cyclase (GC) activity at alkaline pH. Replacement of key residues lysine (101) and aspartate (157) with residues conserved across GCs by site directed mutagenesis, led to a marked improvement in GC activity and a decrease in AC activity. This could be correlated to the presence and strength of the hydrogen bond between the second substrate binding residue (157) and the base of the nucleotide triphosphate. This is substantiated by the fact that the pH optimum is highly dependent on the amino acid residues present at positions 101 and 157.     

Molecular modeling of manganese regulation of calmodulin-sensitive adenylyl cyclase from mammalian sperm

The soluble calmodulin-sensitive isoform of adenylyl cyclase isolated from equine sperm is unique because it requires Mn(2+) rather than Mg(2+) for activity. To gain insight into the molecular action of metals on sperm adenylyl cyclase, the kinetics of Mn(2+) and ATP effect was examined. A biphasic response to increases in ATP concentration was observed when metal was held constant. When [Mn(2+)] exceeded [ATP], however, greatly enhanced enzyme activity was observed. The kinetic profiles were consistent with allosteric activation of adenylyl cyclase by Mn(2+). Linear transformation of the data yielded an apparent K(m) for Mn-ATP of 5.8 mM and calculated V(max) of 12 nM cyclic AMP formed/min/mg. Data analysis using calculated equilibrium concentrations of free and complexed reactants provided similar estimates of these kinetic parameters.     

Genetic selection in<Emphasis Type="Italic"> Saccharomyces</Emphasis> of mutant mammalian adenylyl cyclases with elevated basal activities

We show that co-expression of rat Galphas together with type I, II, IV, or VI mammalian adenylyl cyclase (AC) can suppress the growth defect of cyr1 strains of Saccharomyces cerevisiae, which lack a functional endogenous AC. Complemention of cvr1 is not observed in the absence of Galphas, indicating that the mammalian ACs retain their normal regulatory behavior in yeast. Selection for Galphas-independent growth of (cyr1 strains expressing type IV AC yielded several ACIV mutants with enhanced basal activity, each of which had a single amino acid substitution in the conserved C1a or C2a region of the protein. Expression of two of the mutant ACs in HEK293 cells resulted in increased levels of cAMP and elevated adenylyl cyclase activity. Further selection for reverting mutations in one of these constitutively active AC mutants yielded three independent intragenic suppressor mutations. The distribution of the activating and suppressor mutations throughout both C1a and C2a is consistent with a model in which the enhanced basal activity results from an increase in the affinity between C1a and C2a. These results demonstrate the utility of Saccharomyces as a tool for the identification of informative mutant forms of mammalian ACs.