Molecular Modeling Evaluation of the Enantiomers ... - ACS Publications

Article pubs.acs.org/jcim

Molecular Modeling Evaluation of the Enantiomers of a Novel Adenylyl Cyclase 2 Inhibitor Neha Rana,† Jason M. Conley,† Monica Soto-Velasquez,† Francisco León,‡ Stephen J. Cutler,‡ Val J. Watts,† and Markus A. Lill*,† †

Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy, Purdue University, West Lafayette, Indiana 47907, United States ‡ Department of BioMolecular Sciences, School of Pharmacy, The University of Mississippi, Oxford, Mississippi 38677, United States S Supporting Information *

ABSTRACT: Adenylyl cyclase 2 (AC2) is one of nine membrane-bound isoforms of adenylyl cyclase that converts ATP into cyclic AMP (cAMP), an important second messenger molecule. Upregulation of AC2 is linked to cancers like pancreatic and small intestinal neuroendocrine tumors (NETs). The structures of the various isoforms of adenylyl cyclases are highly homologous, posing a significant challenge to drug discovery efforts for an effective, isoform-selective modulator of AC2. In a previous study, a screen identified a potential isoform-selective and noncompetitive inhibitor of AC2, SKF83566. In the present study, molecular modeling is used to explore the mode of inhibition of AC2 by SKF83566 and to investigate the active enantiomer of SKF83566. Homology models of hAC2 were built based on canine AC5-C1a and rat AC2-C2a templates. With these models, a combination of flexible docking, molecular dynamics simulations, and free energy calculations using the MM/GBSA methodology suggested an allosteric mechanism in which (S)-SKF83566 binds to an allosteric site near ATP and alters the protein conformation of the ATP binding site, potentially preventing the adenosine moiety of ATP from forming an archlike shape to form cAMP. The predicted binding preference for the (S)-SKF83566 enantiomer and the predicted free energy are consistent with the experimental data.

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INTRODUCTION

The transmembrane adenylyl cyclases (ACs) are enzymes that upon activation convert intracellular ATP into cyclic AMP (cAMP), an important second messenger involved in numerous physiological responses.1 Adenylyl cyclases are important downstream effectors of G protein-coupled receptor signaling pathways and are consequently modulated by the actions of G proteins, protein kinases, and Ca2+. These regulatory properties, as well as their sequence homology, form the basis for the classification of the nine mammalian membrane-bound isoforms of AC into four families.1 Group I adenylyl cyclases (AC1, AC3, and AC8) are often called the Ca2+-stimulated family. The Group II adenylyl cyclase isoforms (AC2, AC4, and AC7) are characterized by their ability to be conditionally activated by Gβγ subunits. Group III adenylyl cyclases (AC5 and AC6) share the feature of being inhibited by free Ca2+. AC9 makes up Group IV, the only adenylyl cyclase isoform that is not robustly activated by forskolin. Structurally, mammalian ACs are a single polypeptide chain containing an intracellular N-terminus followed by two membrane-spanning domains alternating with two cytoplasmic domains that can be further divided into “a” and “b” regions (Figure 1). The two cytoplasmic “a” domains (C1a and C2a) form a heterodimer that is sufficient for catalytic activity. The C1a and C2a monomers contain a region of ∼200 amino acids and are highly homologous. These domains were used to solve the AC crystal structure in the presence of its © 2017 American Chemical Society

Figure 1. Scheme of structural domains of membrane-bound adenylyl cyclase.

activator, GαS, forming the ternary complex of GαS−AC5.C1− AC2.C2 (Figure 2). The structure contained the C1a subunit of type-5 AC from canine (VC1) and the C2a subunit of type-2 AC from rat (IIC2). The structure was also bound with a forskolin analogue, revealing a C1a/C2a dimer face that forms a Received: August 5, 2016 Published: January 9, 2017 322

DOI: 10.1021/acs.jcim.6b00454 J. Chem. Inf. Model. 2017, 57, 322−334

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Journal of Chemical Information and Modeling

Figure 2. (left) Proposed structure of hAC2 (C1a subdomain in yellow, C2a subdomain in orange). The green structure is the GαS subunit crystallized with the VC1−IIC2 complex (PDB entry 3C16), obtained by superimposing the crystal structure onto the hAC2 model. (right) The ventral face is rotated about the horizontal axis to show the forskolin (cyan), ATP (pink), and proposed SKF83556 (green) binding sites.

Figure 3. Flowchart of the molecular modeling study.

domains enclose only one catalytic site at their interface. The other site, homologous and symmetric to the catalytic site, is nonactive because of the lack of catalytic residues such as Asp295 and Asp-339 (hAC2 numbering) and is therefore called the pseudosymmetric site. The pseudosymmetric site is exploited by the nonphysiological diterpene activator forskolin, which results in an increase in the level of cAMP production.2 The conversion of ATP to cAMP is facilitated by two cations (Mg2+/Mg2+ or Mg2+/Mn2+) in the catalytic site, which in turn

long, shallow, diagonally running trough containing both the forskolin and substrate binding sites (Figure 2). The extensive contacts between C1a and C2a restrict the entry of the substrates from the dorsal surface, which is also believed to be interacting with the membrane because the Ntermini of both subdomains (C1a and C2a) project away from the dorsal surface. The ventral surface facing the cytoplasm allows access of forskolin and substrates from the cell interior to the binding site. The inverted C1a and C2a cytoplasmic 323


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Figure 4. Chemical structures of ligands used in the flexible docking study.

racemic mixture of SKF83566, which in addition to inhibiting AC2 is also a potent D1 dopamine receptor antagonist.5 We hypothesized that molecular modeling and docking studies would allow us to identify the active enantiomer and to understand further the mechanism of inhibition by SKF83566.

are stabilized by two Asp residues, Asp-295 (β-strand 1) and Asp-339 (β-strand 3) (hAC2 numbering). Concurrently, the adenosine moiety of ATP is predicted to interact with Leu-337 (hAC2 numbering).2 The primary difference between the forskolin and ATP binding sites is that Thr-411 and Ser-941 (hAC2 numbering) in the forskolin binding site are substituted by Asn-1026 and Asp-339 (hAC2 numbering), respectively, in the ATP binding site. Forskolin thus cannot bind in the ATP binding site because the latter residues would result in steric conflict.2 Similarly, ATP cannot be catalytically converted into cAMP in the forskolin binding site because it lacks the catalytic residues Asp-295 and Asp-339.2 Notably, the C1b domain has a molecular weight of nearly ∼15 kDa. Its structure varies among different isozymes, and its primary function is predicted to be isoform-specific regulation of enzyme activity by G protein subunits and other proteins.1 The C2b domain is either very small or nonexistent in some isozymes, and its function still has to be characterized. All of the X-ray structures of hAC are devoid of atomic data on these regions. Thus, we have excluded these sections from our modeling study. There is a growing interest in pursuing AC isoforms as drug targets in a variety of disease states.3 This interest is based on the relationship of AC isoform expression patterns and the contribution of specific AC isoforms to distinct physiological processes.1 More direct evidence supporting adenylyl cyclases as therapeutic targets is derived from genetic mouse models. Over the past 15 years, a variety of knockout and transgenic mouse model studies have shown that AC isoforms uniquely contribute to diverse physiological processes, including aging, learning and memory, pain and analgesia, cardiac function, ethanol responses, and obesity.1 The AC isoform expression patterns, together with the genetic mouse model studies, suggest that small-molecule modulation of AC isoforms may yield therapeutics with novel efficacy and/or specificity.1 In an effort to identify inhibitors of AC2, we previously completed a successful small-molecule screening effort (ca. 720 compounds) in which we identified the phenylbenzazepine derivative SKF83566 as the most robust and potent inhibitor of AC2 known to date.4 However, the detailed atomistic mechanism of inhibition of AC2 by SKF83566 is unknown. Furthermore, our studies used the commercially available

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MATERIALS AND METHODS Molecular Modeling. The overall design of the molecular modeling process is shown as a flowchart in Figure 3. Homology Modeling. The target hAC2 sequence was downloaded from the UNIPROT KB database (ID Q08462) and curated by removing the transmembrane regions from each monomer (1−207 and 602−821), yielding the C1 (monomer 1, chain A, 208−601) and C2 (monomer 2, chain B, 822− 1091) cytoplasmic domains.6 Both chains C1 and C2 have two further domains, a and b; the b domains were not considered in this study because they are neither conserved nor close to the active site. The template structures were taken from Protein Data Bank (PDB) entries 1AZS and 3C16,2,7 both of which used the C1a domain from canine AC5 and the C2a domain from rat AC2. In 1AZS, only the activator forskolin is bound to the pseudosymmetric site, and no ATP or ions are present in the active site. In 3C16, both forskolin in the pseudosymmetric site and ATP/Ca2+ in the active site are cocrystallized with AC. The hAC2 sequence was aligned to the 1AZS and 3C16 sequences independently using BLASTp,8 and these alignments were used to generate two sets of 10 homology models of the hAC2 heterodimer. The homology models were generated using Modeller 9.10.9 DOPE scores were used to estimate the quality of the models. The models derived from 1AZS did not contain cations, whereas the models derived from 3C16 automatically have two cations in the binding site of the protein. All of the homology models were superimposed using PyMOL, 10 and the coordinates of the cations in 3C16 were added to the models derived from 1AZS. ATP coordinates were obtained from the aligned template AC2 structure 3C16. Ligand Library. SKF83566 was identified as the most active inhibitor of AC2 in the previous screening assay.4 Its threedimensional structure was built using PyMOL. The structure was corrected using the sculpting feature of PyMOL. Plausible protonation states of SKF83566 were generated using the H++ 324


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Journal of Chemical Information and Modeling 3.0 server,11 rendering positively charged SKF83566 as alternative protonation states at physiological pH. Furthermore, the stereoisomers of neutral SKF83566 (RC and SC isoforms) were generated along with the stereoisomers of the protonated N atom on the phenylbenzazepine moiety (RN and SN isoforms). The complete library of different enantiomers and protonation states is shown in Figure 4. Preliminary Binding Site Identification. All of the SKF83566 stereoisomers were docked into the extended heterodimer interface region of the homology models using AutoDock4.2.12 The protein was kept rigid through the initial docking study. The dimensions of the search volume (i.e., docking box) were chosen such that the ligand-accessible pockets along the dimer interface, including the pseudosymmetric and active sites, were enclosed within the box. On the basis of the size of the ligand and number of degrees of torsional freedom, each docking run was performed using the Lamarckian genetic algorithm (LGA) search method for 250 000 steps of energy evaluations or 27 000 generations, whichever was reached first. This yielded 100 final poses, which were clustered on the basis of their root-mean-square deviations (RMSD = 2 Å). The clusters were ranked on the basis of the score of the top-scored pose within each cluster. Protein Flexibility and Limoc Ensemble Generation. To include protein flexibility into docking, MD simulations using Limoc were performed on all of the selected homology models as starting structures. Limoc probes were distributed to the binding site, which was defined by the energetically lowest docking poses of SKF83566 obtained in the previous docking runs. The Limoc ensembles were clustered separately for the 3C16-based and 1AZS-based homology models using QT clustering with a cluster radius of 1 Å, thus generating two ensembles for subsequent docking. A detailed description of the Limoc molecular dynamics (MD) approach can be found in our previous publications.13,14 Flexible Docking in the Limoc Ensemble. First, the ensemble of Limoc structures based on the 3C16 template was used for docking all of the stereoisomers of SKF83566. The reason for selecting this ensemble was the larger confidence in providing favorable ligand-engaging interactions due to 3C16’s prior ATP-bound conformation. As Limoc might undersample the rotameric states of flexible side chains, the following nine residues of the ATP binding site, which were found within a distance of 5 Å of ATP, were additionally treated as flexible entities during docking: Leu-337, Asp-339, Lys-939, Ile-941, Met-946, Val-1025, Asn-1026, Ser-1029 and Arg-1030. Because of the significant increase in the number of torsions, the number of energy evaluations in the LGA was increased to 25 000 000 steps. In an analysis of the side-chain flexibility throughout this docking run, the observed side-chain variability for five out of the nine residues was well-represented by the Limoc ensembles. Thus, the final docking procedure on the complete Limoc ensemble treated the side-chain flexibility explicitly only for the remaining four residues: Lys-939, Met946, Asn-1026, and Arg-1030. All of the poses of the final docking run were clustered on the basis of the 2 Å RMSD criterion for each ligand using AutoDockTools.12 Charge Fitting for Modeling of Potential Halogen Bonds. SKF83566 contains a bromine atom, a halogen, which displays a unique electronic distribution and is poorly modeled by current atom-centered partial charge force fields. Halogen atoms such as bromine and iodine display partial negative electrostatic potential perpendicular to the axis of the carbon−

halogen bond and partial positive charge in the center, the socalled σ hole, which can potentially act as a donor to form a halogen bond with a negatively charged acceptor as described in Figure 5.15 To model these electrostatic properties more

Figure 5. Sigma hole of a halogen atom: X is the halogen, A is an acceptor, and H−D is a donor moiety.16

accurately compared with standard force fields, we computed the electrostatic potentials of the initial ligand conformations obtained from the two top-ranked docking poses of SKF83566 isomers using quantum mechanics and redistributed the partial charges on the ligand atoms and a newly introduced virtual point on the halogen surface along the carbon−halogen axis using a previously established procedure.16 In detail, density functional theory calculations using Gaussian 0917 were performed with a mixed basis set of 6-311+G** for C, H, N, and O atoms and an augmented correlation-consistent valence double-ζ basis set with a polarization function, aug-cc-pVDZPP, for the bromine atom.16 A geometry optimization and minimization was followed by electrostatic field calculation. An additional dummy atom was created at the center of the periphery of the bromine atom along the C−Br axis at a distance of 2.22 Å from the bromine atom center, and charges were fitted to the new distribution of atoms (including the dummy atom) using the RESP charge fitting method in Antechamber.18 Molecular Dynamics Simulations. MD simulations were carried out for selected binding poses of SKF83566. The ligand, enzyme, and complex topology files were prepared using tleap and the Antechamber program of AmberTools 1319 with the Amber99SB force field. The parameter file of the halogenated ligand was modified by adding the parameters for the dummy atom (cf. Table 1). The protein−ligand complex was placed in a rectangular box with a minimum distance of 8 Å between the face and nearest atom. The box was solvated using the TIP3P water model, and the system was neutralized by adding two and three Na+ ions for protonated SKF83566 and neutral SKF83566, respectively. Table 1. Molecular Mechanics Parameters of the Extra Atom Representing the σ Hole

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parameter of the extra atom

value

mass vdW radius vdW well depth (ε) Br−EP bond length Br−EP bond force constant (kcal mol−1 Å−2) C−Br−EP angle phase C−Br−EP angle force constant (kcal mol−1 deg−2) C−C−X−EP dihedral phase C−C−X−EP dihedral periodicity

0.00 amu 1.00 Å 0.00 Å 2.22 Å 600.0 180.0° 150.0 180.0° 2


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(Phenomenex), eluting with CH3OH/isopropyl alcohol/formic acid (90:10:0.1 e.c.) at a flow rate of 0.8 mL/min. The eluate was properly partitioned according to the UV profile. Sample volumes of 10 μL were injected onto the column. The 10 mg SKF83566 sample was dissolved in 2 mL of methanol. The obtained fractions containing the enantiomers were evaporated at reduced pressure. (−)-(S)-8-Bromo-2,3,4,5-tetrahydro-3-methyl-5-phenyl1H-3-benzazepin-7-ol [(−)-(S)-SKF83566]. Colorless solid. 1 HPLC: tR = 13.72 min. [α]25 D : −4.0 (c 0.01, MeOH). H NMR (400 MHz, CD3OD): δ = 7.47 (t, J = 7.6 Hz, 2H), 7.40 (s, 1H), 7.39 (m, 2H), 7.27 (d, J = 7.6 Hz, 2H), 6.28 (s br, 1H), 4.61 (d, J = 10.4 Hz, 1H), 3.89−3.73 (m, 2H), 3.71−3.51 (m, 1H), 3.31−3.27 (m, 1H), 3.23−3.13 (m, 1H), 3.04 (dd, J = 7.2, 16.0 Hz, 1H), 2.97 (s, 3H) ppm. 13C NMR (100 MHz, CD3OD): δ = 155.3, 142.1, 139.4, 133.8, 129.9, 129.0, 128.0, 127.4, 116.4, 107.6, 60.1, 56.1, 45.4, 44.5, 30.3 ppm. CD (MeOH, c 0.14): [θ]279, 7535 (max); [θ]257, 157 (max). (+)-(R)-8-Bromo-2,3,4,5-tetrahydro-3-methyl-5-phenyl1H-3-benzazepin-7-ol [(+)-(R)-SKF83566]. Colorless solid. HPLC: tR = 14.70 min. [α]25 D : +8.0 (c 0.01, MeOH). CD (MeOH, c 0.14): [θ]272, 3800 (max); [θ]257, 5573 (max); [θ]233, 701 (max). Cell Culture and Pharmacology. HEK293 cells were cultured in DMEM supplemented with 5% bovine calf serum, 5% fetal clone I, and 1% antibiotic−antimycotic 100× solution and maintained in a humidified incubator at 37 °C and 5% CO 2 . HEK-AC2 cells were constructed as previously described,4 and HEK-hD1 cells were previously described.23 Cyclic AMP Assays. Cellular cAMP levels were measured using the Cisbio HTRF cAMP dynamic 2 assay kit. For transient transfections, HEK cells were plated in a 12-well plate at a cell density of 3 × 105 cells/well. After 24 h, cells were transiently transfected for 48 h with Gαs and rat AC2 (rAC2) or the forskolin-insensitive rAC2 mutant S942P in a 1:10 [DNA] ratio, respectively. Cells were harvested in Opti-MEM, plated in a low-volume 384-well plate, and incubated for 1 h in a humidified incubator at 37 °C and 5% CO2. The cells were then treated with 30 μM (±)-SKF83566 or 100 μM SQ22536 for 30 min at room temperature followed by the addition of 100 nM PMA and 500 μM IBMX to stimulate cAMP production. Assays were terminated after 1 h of stimulation as described below. For assays in stable cell lines, cryopreserved cells were rapidly thawed at 37 °C and resuspended in OptiMEM. Cells were centrifuged at 500g, and the supernatant was aspirated. Cells were washed by resuspension in Opti-MEM and centrifuged at 500g. The supernatant was aspirated, and cells were seeded into a 384-well plate and allowed to incubate at 37 °C and 5% CO2 for 1 h. Cells were then treated as indicated with the SKF83566 enantiomers for 30 min, followed by the addition of PMA (HEK-AC2) diluted in stimulation buffer (Opti-MEM, 500 μM IBMX) or dopamine (HEK-D1) diluted in stimulation buffer plus 0.02% ascorbic acid. The cells were incubated for 1 h at room temperature, and the stimulation was terminated by sequential addition of equal parts of cAMP-d2 and anti-cAMP cryptate conjugate (5−10 μL), each diluted (1:39) in lysis buffer. Following a 1 h incubation at room temperature, the time-resolved fluorescence resonance energy transfer (TR-FRET) was measured with a lag time of 100 μs and an integration time of 300 μs using a Synergy4 (BioTek) fluorescence plate reader (excitation filter, 330/80 nm; emission filters, 620/10 nm and 665/8 nm). The resulting cAMP concentrations were calculated in GraphPad

After a minimization of 1000 steps (500 steepest-descent and 500 conjugate-gradient), the system was slowly heated for 50 ps from 0 to 300 K and then equilibrated under NPT conditions for 550 ps using the isotropic position scaling to keep the pressure constant at 1 atm. Final production MD simulations were performed for 50 ns under NPT conditions using the GPU-supported pmemd MD module.20,21 All of the bonds involving hydrogen atoms were constrained using the SHAKE algorithm. Particle mesh Ewald methodology was used to calculate all of the long-range electrostatic interactions with a distance larger than 8.0 Å. For temperature scaling, Langevin dynamics was used with a collision frequency of 2 ps. Binding Free Energy Calculations. The binding free energies of the different ligand poses were estimated using the molecular mechanics/generalized Born surface area (MM/ GBSA) method.22 The trajectory of each protein−ligand complex simulation was used to extract 5000 snapshots for the complex, enzyme, and ligand. The free energy without ligand configurational entropy was estimated by the average difference between the complex and individual protein plus ligand interactions, where solvation effects were treated using an implicit GB solvation model. To estimate the vibrational entropy of the ligand, the normal-mode analysis method of the Amber suite was used; 48 snapshots were used to estimate the translational, rotational, and vibrational components of the entropy. The significance of the difference between binding free energies of poses was statistically tested using Student’s t test. Experimental Methods. Materials. Dopamine, 3-isobutyl1-methylxanthine (IBMX), G418, and L-ascorbic acid were purchased from Sigma-Aldrich (St. Louis, MO). Phorbol 12myristate 13-acetate (PMA), SQ22536, and (±)-SKF83566 were purchased from Tocris Bioscience (Ellisville, MO). OptiMEM, Dulbecco’s modified Eagle’s medium (DMEM), and antibiotic−antimycotic 100× solution were purchased from Life Technologies (Grand Island, NY). Lipofectamine 2000 was purchased from Thermo Fisher Scientific. FetalClone I serum and bovine calf serum were purchased from Hyclone (Logan, UT). The HTRF cAMP kits were purchased from Cisbio Bioassays (Bedford, MA). Chemistry. 1H NMR spectra were recorded on a Bruker model AMX 400 NMR spectrometer with standard pulse sequences operating at 400 MHz; chemical shifts are reported in parts per million with the solvent reference relative to tetramethylsilane employed as the internal standard (CD3OD, δ = 3.31 ppm). 13C NMR spectra were recorded on a 400 MHz spectrometer operating at 100 MHz with complete proton decoupling; chemical shifts are reported in parts per million. High-performance liquid chromatography (HPLC) runs were conducted on a Waters LC Module I autosampler equipped with a Waters 486 UV detector. Experimental data were acquired and processed using Millenium 32 Chromatography Manager software (Waters). Solvents used for chiral chromatography were HPLC-grade and supplied by Fisher Scientific (Hanover Park, IL). All of the HPLC analyses were performed at room temperature. Optical rotation values were measured on a Rudolph Research Analytical Autopol V polarimeter (Hackettstown, NJ) with a 1 cm cell at the sodium D line (λ = 589 nm); sample concentration values (c) are given in 1 mg/mL. Circular dichroism (CD) spectra were recorded on a 202SF spectrometer (Aviv Biomedical, Lakewood, NJ). Chiral Chromatography. The enantiomers of SKF83566 were completely resolved by a semipreparative process using a Quiral Lux Cellulose-1 column (Ø = 1 cm, l = 2 cm, 5 μm) 326


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Journal of Chemical Information and Modeling Prism by applying the 620/665 nm fluorescence ratio values to a standard curve of known cAMP concentrations.

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RESULTS AND DISCUSSION Homology Modeling. Because of the presence of ATP and Ca2+ in the template structure 3C16, there is a slight conformational difference between the 1AZS and 3C16 X-ray crystal structures, as shown in Figure S1 in the Supporting Information. Both structures were employed in our study, as they allowed consideration of both conformations of the template structure, i.e., in the absence (apo) or presence (holo) of the substrate. The alignment using BLASTp showed high sequence similarity between the target and template sequences, as illustrated in Figure S2 (overall sequence identity 79%; 63% identity between the respective chains A (C1a domain) and 93% between the respective chains B (C2a domain)), which in turn resulted in highly homologous structures of hAC2. The following functionally and structurally important residues, as annotated by PROSITE, are conserved between the template and target sequences: Asp-295, Asp-339, Leu-337, Asn-1026, and Arg-1030. As is noted, the rat AC2-C2a template is missing residues near the beginning of chain B in the template structure, from Pro-955 to Glu-964. Different homology models showed slight variations in the side chains of the residues, including the ATP binding site residues Asp-295, Asp-339, Leu-337, Asn-1026, and Arg-1030, as shown in Figure 6. An overlay of the per-residue DOPE scores of the template and homology models (Figure S3) demonstrates the structural integrity of the model. Preliminary Binding Site Identification. Docking of SKF83566 into all 20 homology models of hAC2 identified two preferred sites for binding, the active site where ATP binds and the pseudosymmetric site where forskolin binds (Figure 7). After clustering, we obtained 53 different ligand configurations

Figure 7. All of the docking poses in the ensemble of homology models are shown for different stereoisomers of SKF83566. They are located in two different binding sites, the ATP binding site and the forskolin (FSK) binding site, in all of the homology models. Similar poses in different homology models are shown in the same colors: pose A (pink), pose B (green), pose C (yellow), pose D (brown), pose E (cyan), pose F (purple), pose G (light pink), pose H (dark green), and pose I (dark blue). (top left) Poses of (SC,SN)-SKF83566 in the ensemble of hAC2 homology models shown in two different sites in five different conformations (A, B, C, D, and E). (top right) Poses of (SC,RN)-SKF83566 shown in two binding sites in six conformations (A, B, C, D, E, and F). (bottom left) Poses of (RC,SN)-SKF83566 shown in two binding sites in six conformations (A, B, D, G, H, and I). (bottom right) Poses of (RC,RN)-SKF83566 shown in two binding sites in four conformations (A, B, D, and G).

in the two preferred binding sites. One of the conformations, located in the active site (marked in brown in Figure 7), was the most frequently observed conformation across all stereoisomers of SKF83566 and all protein structures (30% of 53 conformations, lowest Autodock energy = −6.72 kcal/mol). It was followed by two conformations located in the pseudosymmetric site (both 18%; −6.46 kcal/mol, marked in pink in Figure 7, and −6.68 kcal/mol, marked in green in Figure 7). Protein Flexibility in Docking: Limoc Ensemble Generation Plus Flexible Side Chains. Limoc generated two ensembles of 10 protein structures (EPS = ensemble of protein structures) representing potential binding site conformations by using the top docked protein−ligand conformation (colored brown) in each set of homology models. The significant differences between the conformations of the side chains in the binding site of the EPS and the previous homology models are highlighted in Figure S4. The major differences occur in the binding site in the loop regions between β-strands 2 and 3 and β-strands 10 and 12. Flexible docking of SKF83566 to the EPS based on the 3C16 template homology model with nine flexible residues revealed that out of the nine residues, the conformational space of the

Figure 6. Ensembles of 10 homology models based on the 1AZS (apo) and 3C16 (holo) templates. Different homology models have different side-chain conformations, providing a pregenerated ensemble of structures, thus implicitly including a fraction of the flexibility of the protein. 327


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Journal of Chemical Information and Modeling five residues Leu-337, Asp-339, Ile-941, Val-1025, and Ser-1029 observed during flexible docking was covered in the complete Limoc ensemble derived from the 1AZS and 3C16 templates. The remaining four residues, Lys-939, Met-946, Asn-1026, and Arg-1030, showed significantly larger conformational variations in the flexible side-chain docking protocol compared with the Limoc ensemble (Figure 8). Consequently, all of the

Figure 9. Evaluation of SKF83566 and SQ22536 inhibitory effects on PMA-stimulated cAMP production in HEK cells transiently transfected with rat AC2 or the forskolin-insensitive AC2 S942P mutant. Data shown represent the mean and standard error of the mean (SEM) of three independent experiments.

an angle of approximately 45° between their planes and a distance of 3.7 Å between their nearest heavy atoms, representing potential π−π interactions. The same phenyl ring is also stacked under the aromatic side chain of Trp-1021, with an angle of 22° between their planes and a distance of 4.3 Å between their closest heavy atoms. Because of these two aromatic residues and other surrounding hydrophobic residues, the phenyl moiety rests in a hydrophobic pocket. In the other conformation, shown in orange color, there is a potential hydrogen bond between the −NH group of the ligand and the carboxylic acid group of Asp-1019. The planes of the phenyl ring and Phe-299 are nearly parallel (at an angle of approximately 30°) and have a distance of 4.4 Å between their closest heavy atoms. The Trp-1021 aromatic ring is perpendicular to the phenyl group. The hydroxyl group of the ligand is not involved in any particular protein−ligand interaction, while the bromine atom points in the direction of the −NH group of Arg-1030. Like the conformations shown in green color, the phenyl group was located in the same hydrophobic pocket. MM/GBSA Free Energy Calculations. To determine which stereoisomer and protonation state of SKF83566 has the highest binding affinity, and which pose from the flexible docking simulations is the most likely pose, we performed MD simulations and evaluated the free energy of binding for different ligand poses using the MM/GBSA method. We present the free energy calculations of all stereoisomers and conformations first, and then describe the results of the MD simulation of only the most energetically favorable pose in the subsequent section. The enthalpic and entropic contribution, and predicted free energies of binding are tabulated in Table 2. Among the set of stereoisomers, the neutral forms of (SC/RC)SKF83566 are more energetically favorable than the simulated poses of protonated (SC/RC,SN/RN)-SKF83566. The poses of protonated (SC,SN/RN)-SKF83566 generally are energetically more favorable than the poses of protonated (RC,SN/RN)-SKF83566. The energetically favorable form of neutral (SC)-SKF83566 has a predicted free energy of binding of −9.15 kcal/mol, while that of (RC)-SKF83566 has a predicted binding free energy of −12.2 kcal/mol. The two enantiomers reside in different pockets. Interestingly, each enantiomer’s less favorable pose coincides with the other’s favorite binding pose, e.g., the less favorable pose of (SC)SKF83566 occupies the same region as the energetically favorable form of (RC)-SKF83566 and vice versa. In the next section, we will discuss the binding conformations of the most

Figure 8. Comparison of the Limoc ensemble (light brown and light green; cf. Figure S4) and residue conformations sampled during flexible docking (pink). The flexible docking results demonstrate that the flexibility of the side chains of four residues (Lys-939, Ile-941, Met946, and Arg-1030) need to be considered explicitly during docking, as these residues occupy a larger conformational space than sampled by Limoc.

subsequent docking was performed with these four residues kept flexible, whereas for the other five residues only the flexibility inherent to the Limoc ensemble was utilized. In the final docking run in the complete Limoc ensemble, the representative conformations of the top five ranked clusters were analyzed. Combining the poses of all of the protonation states and stereoisomers of SKF83566, 10 distinct binding poses were identified, four in the ATP binding site and six in the pseudosymmetric site. In an effort to reduce the number of potential binding poses for future analyses, we experimentally examined the ability of SKF83566 to inhibit a forskolininsensitive mutant of AC2, S942P (rAC2 numbering). Substitution of the proline is predicted to distort the forskolin binding site and has previously been shown to reduce forskolinstimulated AC2 activity.24 Our hypothesis was that if SKF83566 acts via the pseudosymmetric forskolin binding site, disrupting that site would decrease its inhibitory activity. Our experiments showed that SKF83566 inhibited the S942P mutant to the same extent as wild-type rat AC2 (Figure 9). Similar results were also obtained with the P-site inhibitor SQ22536 (Figure 9). These results suggest that the binding site(s) for SKF83566 and SQ22536 are distinct from the forskolin binding site. Thus, only those poses of SKF83566 that were located in the ATP binding site were further investigated. The two highest-ranked poses for each SKF83566 stereoisomer were selected for stability analysis and free energy calculations using MD simulations, shown in green and orange color in the ATP binding site in Figure 10. Both poses were observed in all of the SKF83566 stereoisomers and partially overlap with the ATP binding site of the X-ray structure of AC. In the pose shown in green color, the hydroxyl group of the SKF83566 enantiomers is at a distance of 4 Å from the carboxyl group of Asp-1019 at an approximate angle of 150°. The phenyl group is partially stacked with the side chain of Phe-299, with 328


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Figure 10. Docking poses of SKF83566 stereoisomers as a result of flexible docking: (SC)-SKF83566 (top left), (SC,SN)-SKF83566 (top middle), (SC,RN)-SKF83566 (top right), (RC)-SKF83566 (bottom left), (RC,SN)-SKF83566 (bottom middle), and (RC,RN)-SKF83566 (bottom right). Similar conformations are shown in the same color.

Table 2. MM/GBSA Free Energy Analysis for Different Forms of SKF83566 and Their Selected Posesa ligand (SC)-SKF83566 (RC)-SKF83566 (SC,SN)-SKF83566 (SC,RN)-SKF83566 (RC,SN)-SKF83566 (RC,RN)-SKF83566 a

pose (cf. Figure 10) green orange green orange orange green orange orange green green

ΔH (kcal/mol)

TΔS(kcal/mol)

ΔG (kcal/mol)

−25.84 −4.27 −19.58 −31.08 −21.47 −17.87 −21.71 −13.53 −18.31 −11.23

−16.69 −16.39 −19.23 −18.89 −17.97 −17.13 −19.99 −17.55 −17.99 −14.90

−9.15 12.12 −0.35 −12.20 −3.50 −0.74 −1.72 4.03 −0.32 3.67

± ± ± ± ± ± ± ± ± ±

0.05 0.05 0.05 0.04 0.04 0.05 0.09 0.04 0.05 0.04

± ± ± ± ± ± ± ± ± ±

0.96 1.18 1.25 1.08 2.54 2.19 2.84 0.97 1.21 0.98

± ± ± ± ± ± ± ± ± ±

0.96 1.18 1.25 1.08 2.54 2.19 2.84 0.97 1.21 0.98

Values of ΔH, TΔS, and ΔG are shown as mean ± SEM.

(SC)-SKF83566. For MD, the starting conformation of the energetically most favorable pose corresponds to the flexible docking pose shown in green in the ATP binding site in Figure 10 (top left). During the MD simulation, no significant change in the conformation of the starting pose was observed for the first 20 ns. In the subsequent 10 ns, the configuration of the ligand in the binding site transitioned into another, stable

favorable poses of (SC)-SKF83566 and (RC)-SKF83566 and less favorable poses of (RC)-SKF83566. MD Simulations. In general, we observed that the overall structural stabilities of the SKF83566 stereoisomers are correlated to their MM/GBSA free energy values. In this section, the structural details of the MD simulation results for (SC)-SKF83566 and (RC)-SKF83566 are described. 329


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Journal of Chemical Information and Modeling conformation. This final conformation differs from the initial docking pose by an RMSD of about 4 Å (Figure 11).

ATP in the X-ray structure of the homologous AC7 and binds in a site adjacent to ATP. Most Stable Pose of (RC)-SKF83566. For MD, the starting conformation of the energetically most favorable pose corresponds to the flexible docking pose in the ATP binding site, shown in orange in Figure 10 (bottom left). During the MD simulation, (RC)-SKF83566 marginally deviates from the starting position. Finally, it forms two hydrogen bonds through the hydroxyl group: one with the carboxyl group of Asp-1019 and one with the backbone −NH of Ile-1020. The closest aromatic amino acid residue facing the phenyl moiety of SKF83566 is Trp-1021 in a displaced T-shaped orientation. The methylamino group is solvent-exposed, and the Br atom is buried in the interior of hAC2, as shown in Figure 13. This pose significantly overlaps with ATP in the X-ray structure, which may suggest binding in a competitive manner.

Figure 11. Conformational transition of (SC)-SKF83566 during the MD simulation (from blue to red). The starting pose of (SC)SKF83566 (green in Figure 10) undergoes conformational changes and finally stabilizes in the pose shown in thick red sticks.

In the final pose (Figure 12), the methylamino group extends into a small hydrophobic pocket formed by residues Phe-299,

Figure 13. Most stable pose of (RC)-SKF83566 (shown in green). ATP and forskolin (FSK), whose positions were derived from the Xray structure of the template, are shown as gray sticks. As seen, (RC)SKF83566 directly overlaps with the ATP. The final positions of cations are shown as spheres against the surface representation of AC2. Residues that directly interact with the ligand (Ile-938, Asp-1019, Ile1020, Trp-1021, Gly-1022, and Asn-1026) are shown as gray sticks.

Second Pose of (RC)-SKF83566. The second pose of (RC)SKF83566, which is barely energetically favorable (−0.35 kcal/ mol), is close to the (SC)-SKF83566 site at the end of the simulation and does not overlap with the ATP conformation. The starting conformation of this simulation is displayed in green in Figure 10 (bottom left). The final pose indicates that the bromine group points toward Asp-295 and Asp-339 and the hydroxyl group forms hydrogen bonds with the solvent. Hydrophobic and aromatic residues (Phe-299, Leu-315, and Trp-1021) surround the phenyl moiety. The amine group is exposed to the solvent to form a hydrogen bond between −NH and water. Pharmacological Assessment of the Resolved SKF83566 Enantiomers. We previously used a limited AC selectivity profile to identify racemic SKF83566 as the most selective compound for AC2 over AC1 and AC5 in cell-based assays.4 A cell-free reconstituted system was also used to assess the direct modulation of AC isoforms (i.e., AC1, AC2, and AC5), which revealed that SKF83566 inhibited forskolinstimulated AC2 activity by more than 40%, consistent with a direct mode of inhibition. The mechanism of SKF83566 inhibition of AC2 was also explored by determining inhibition

Figure 12. Most stable pose of (SC)-SKF83566 (shown in red). ATP and forskolin (FSK), whose positions were derived from the X-ray structure of the template, are shown as gray sticks. The final positions of cations are shown as spheres against the surface representation of AC2. Residues that directly interact with the ligand (Phe-299, Leu-315, Trp-1021, Gly-1022, and Asn-1026) are shown as gray sticks.

Leu-315, Ile-296, and Phe-319. The phenyl group of the ligand rests in an open hydrophobic pocket that is fairly exposed to the solvent. The hydroxyl group of the benzazepine ring forms a hydrogen bond with the backbone −NH of Gly-1022. The phenyl group of the ligand is positioned perpendicular to the benzazepine moiety of the ligand and the aromatic side chains of Phe-299 and Trp-1021 in T-shape-like aromatic interactions. The Br substituent has a dual electrostatic nature and is involved in strong electrostatic interactions with the different electrostatic components of the −CONH side chain of Asn1026. This pose no longer overlaps with the conformation of 330


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Figure 14. cAMP accumulation vs SKF83566 stereoisomer concentration. HEK293 cells expressing AC2 (A) or the D1 dopamine receptor (B) were incubated with increasing concentrations of the purified enantiomers of SKF-83566 followed by stimulation of cAMP accumulation using PMA (left) or dopamine (right).

Figure 15. (left) Seifert et al.3 suggested that 2′- and 3′-O ribosyl substituents of the ligand MANT-GTP (light pink, PDB entry 1TL7) bind in a hydrophobic patch, which can provide a novel site of inhibition. SKF83566 (shown in dark red) binds exclusively in that hydrophobic region, supporting our simulation results. SKF83566 is shown to bind exclusively to the 2′- and 3′-O ribosyl inhibitor’s hydrophobic patch. (right) In 50 ns simulations of the P-site inhibitors SQ22536 (cyan) and CB783340720 (pink) and of SKF83566 (yellow) in the presence of PPi molecules (same perspective view of the protein as in A), the P-site inhibitors bind in the adenosine binding site, but SKF83566 binds in the hydrophobic patch near the ATP binding site.

favorable pose was located in a hydrophobic region near the ATP binding site, but not overlapping with ATP, supporting the noncompetitive inhibition observed in the biochemical assays.4 The hypothesis of Seifert et al.3 further supports our claim that the hydrophobic site adjacent to the ribosyl moiety, 2′- and 3′-O substituents, is capable of binding potent and isoform-selective inhibitors (Figure 15 left). Ligand-Induced Conformational Changes Render the Binding Site Inaccessible to ATP Catalysis. After determining the binding modes of (SC)- and (RC)-SKF83566, we investigated the mechanism of inhibition of AC2 by SKF83566. We first examined the configurational change in binding conformations of ATP in the inhibitor-free AC2. It is to be noted that the starting template bound to ATP is only an initial binding conformation, found in the inactive open conformation of AC5 inhibited by Ca2+ (characteristic of the AC5 family). The above comparison to this binding site is simply to locate the initial relative SKF binding site. It, however, does not reveal the actual dynamics of the protein that occurs during substrate and/or inhibitor binding. The mechanism of inhibition of AC5 by Ca2+, as explained by Mou et al.,7 is caused by the expanded coordination sphere of two Ca2+ ions (radius 1.14 Å), which forces the β1−α1 loop (carrying Asp-396 and Ile-397 (canine AC5 numbering)) away from the catalytic site. The α1 helix is moved 10° further away

under varying concentrations of ATP and SKF83566. These studies revealed that SKF83566 behaves as a noncompetitive AC2 inhibitor.4 To explore our enantioselectivity predictions, we experimentally separated the SC and RC enantiomers of SKF83566 and measured their inhibitory activities against AC2. Similar to our previous studies, we used the phorbol ester PMA to selectively stimulate AC2 in HEK-hAC2 cells.4 PMA-stimulated cAMP accumulation was approximately 10-fold greater than basal cAMP levels. Studies with the resolved enantiomers of SKF83566 showed a dose-dependent inhibition of cAMP accumulation. The resulting IC50 values revealed that (SC)SKF83566 (∼5 μM) is about 4-fold more potent than (RC)SKF83566 (∼19 μM). Because SKF83566 was originally reported as an antagonist at D1 dopamine receptors, we also evaluated the enantiomers for D1 antagonist activity using HEK cells expressing the human D1 dopamine receptor (HEK-hD1 cells). These studies revealed that in contrast to AC2, (R)SKF83566 is more potent than (S)-SKF83566 (>15-fold). Thus, these studies demonstrate a reversal of the enantioselectivity for AC2 versus D1 receptors (Figure 14). The computational studies presented here reveal that (SC)SKF83566 binding in an allosteric site displays a lower free energy than (RC)-SKF83566 binding in the allosteric site, which is in agreement with the experimental data. The most 331


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Figure 16. (left) Conformational changes of ATP. After 50 ns of MD simulation, the ATP (gray) purine ring follows the trajectory (gray-to-silver-togreen) to adopt a bent conformation (green), which is assumed to be required for cAMP formation. The triphosphate tails of the ribose moiety are not displaced significantly. The light helices show the final conformation of the α1 helix, α4′ helix, and β2−β3 loop. (right) In the presence of SKF83566 (red), ATP (gray) cannot bend because SKF83566 occupies the hydrophobic site next to it and occludes the region sterically for the bent ATP (cAMP transition) conformation. The dark helices and loop indicate the displacements of the α1 helix, α4′ helix, and β2−β3 loop. The molecular interactions of SKF83566 (red) with neighboring residues are shown. Asp-295, Ile-296, Phe-299, Asp-339, ASP1019, Trp-1021, Asn-1026, Arg-1030, and Lys-1066 are shown in the absence of SKF83566 (gray lines) and in the presence of SKF83566 (dark red lines). Asp-295, Ile-296, Asp-339, and Asp-1019 interact with ATP (absence of SKF83566, gray stick model; presence of SKF83566, red stick model). Phe-299, Trp-1021, Asn-1026, Arg-1030, and Lys-1066 interact with SKF83566.

position of the purine nucleoside deviated approximately 40° from its original plane and, unlike the initial elongated conformation, acquired a bent shape. The β- and γ-phosphates are held in their original positions (Figure 16 left). Our simulation is in agreement with the catalytic mechanism hypothesized by Sprang et al. The change in conformation of the ATP is not an artifact of a bad starting conformation of ATP. It is validated in a dozen crystal structures of AC bound with nucleotide analogues that have conserved interactions between nucleotides as in our starting ATP position and activesite residues, such as hydrogen bonds between adenosine nitrogen and Asp-1019 and Ile-1020.7,25−28 Next, we simulated (SC)-SKF83566 and (RC)-SKF83566 in the forskolin- and ATP-containing AC2. In the (SC)SKF83566-ATP simulation, the presence of the inhibitor sterically hinders the bending of the purine nucleoside that was observed in the inhibitor-free ATP simulation (Figure 16 right). The proximity of (SC)-SKF83566 and a bent adenine ring (proposed by Sprang and confirmed by our simulations) makes it physically impossible for the 3′-OH and α-phosphate to come into an orientation for a chemical reaction to occur, preventing the catalysis. The bridging inhibitor between Phe299 and Trp-1021 leads to a very strong aromatic interaction that is otherwise absent without the inhibitor. We attempted to explore these potential interactions further by constructing mutants of AC2 in which Phe-299 or Trp-1021 was replaced with alanine. Unfortunately, expression of the mutants failed to reveal sufficient adenylyl cyclase activity for inhibition studies. The lack of activity may reflect the general importance of these amino acids for adenylyl cyclase expression and function because both are highly conserved across AC isoforms.2 Previously, simulations with (RC)-SKF83566 but without ATP (Figure 13) revealed the presence of the inhibitor in the crystallized ATP binding site. Thus, a simulation with both compounds in the same site is not technically feasible because of steric collisions. As also previously mentioned, the less favorable pose of (RC)-SKF83566 occupies the same site as the preferred pose of (SC)-SKF83566. When we performed the

from the active interface, with the collateral loss of interaction between the β- and γ-phosphates and the Gly-399 and Phe-400 (canine AC5 numbering) backbone amines. On the contrary, the AC2 class is insensitive to Ca2+ inhibition because the movement of the α1 helix is restricted by substitution of Ala409 of AC5-C1a with Pro-307 of AC2-C1a, found in the connection between the α1 and α2 helices.7 To understand the ATP and inhibitor binding, we first performed 50 ns MD simulations of AC2 with ATP and forskolin and compared the sampled protein conformations with those of the ATP-free counterpart simulation. The catalytic mechanism of action of adenylyl cyclase was originally proposed by Sprang and coworkers.2 They speculated that cyclization of the adenine molecule was preceded by inversion of the α-phosphate for a direct in-line attack by the 3′-oxyanion group of the αphosphate, facilitated by a basic residue like Asp-339. The electronegative environment of Asp-295, Asp-339, and the αphosphate, which might hinder the deprotonation of 3′-OH, is counteracted by one Mg2+ cation, which also stabilizes the pentavalent intermediate. In addition, Arg-1030 shares the role of stabilizing the pentacoordinate intermediate of the reaction. The second Mg2+ stabilizes the removed polyphosphate tail. In the proposed catalytic mechanism of AC2, the critical residues that coordinate with the cations, which in turn interact with the phosphates of ATP, are Asp-295, Asp-339, and Ile-296. Asn1026 also plays an important role in stabilization of the reactants upon the conformational change induced by ATP binding. As ATP−metal coordination brings the α1 helix closer to ATP, similar to closing of a lid, and the β7′−β8′ loop carrying Lys-1066 moves forward to interact with pyrophosphate group, Asn-1026 stabilizes the purine base via a watermediated network.2 In our forskolin- and ATP-containing AC2 simulation, the conformations of the catalytically important residues except Asn-1026 remained unperturbed. Asn-1026, located on the α4′ helix, shifted slightly, which caused a small movement of Asn-1026. In a concerted reaction, the purine nucleoside shifted significantly and induced a slight change in the ribose sugar and α-phosphate conformation. The final 332


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Journal of Chemical Information and Modeling simulation of this pose of (RC)-SKF83566 in the presence of ATP, the pose slipped further away from the ATP and slightly deeper into the protein compared with (SC)-SKF83566. However, it also prevented the cyclization of ATP by presenting steric clashes. The main interaction of the inhibitor with Phe-299 and Trp-1021 was lost, and the phenyl moiety was now surrounded by Phe-299 and Phe-319. The amine, hydroxyl, and bromine groups did not form any close connections to the surrounding residues. In Silico Comparison with the P-Site Inhibitor Mechanism. Our previous in vitro studies suggested that the inhibitory effects of SKF83566 are noncompetitive, like those of P-site inhibitors. However, our modeling studies suggested that (S)-SKF83566 binds to a unique site. To further explore the predicted mechanism of inhibition, we simulated two P-site inhibitors, SQ22536 and CB7833407,3,24 and the novel inhibitor (SC)-SKF83566 in the cAMP location in the presence of a PPi analogue and two cations. While the two P-site inhibitors showed overall structural stability in the cAMP binding site, (SC)-SKF83566 drifted to the predicted location in the hydrophobic region under the ATP binding site. Also, the PPi analogue started to leave the binding site in the (SC)SKF83566 simulation and moved closer to the solvent-exposed surface of the protein (Figure 15 right). This suggests that the binding mode of (SC)-SKF83566 and its mechanism of inhibition may differ from those of P-site inhibitors. On the basis of the results of the computational and experimental studies, it can be deduced that (S)-SKF83566 inhibits the activity of hAC2 in an allosteric manner by preventing cyclization of ATP.

prevents ATP from adopting the coiled conformation necessary for undergoing the catalyzed cyclization reaction. The key feature of (SC)-SKF83566 is its aromatic interaction with Phe299 and Trp-1021. In summary, our study was able to discover a novel mode of inhibition in AC2 that is different from that of conventional MANT- or TNP-nucleotide-based inhibitors as well as that of P-site inhibitors. It should be pointed out that although (SC)SKF83566 is not a P-site inhibitor, it prevents cAMP formation sterically. Moreover, the requirement of the presence of PPi for P-site inhibition is not valid for (SC)-SKF83566. This identified site is unique and may function as an AC-isoform-specific inhibitor binding site. Thus, this class of inhibitors may provide a new direction for identifying selective inhibitors for other ACs.

CONCLUSION Despite the plethora of studies suggesting the utility of targeting AC isoforms, advancements are limited by the overall lack of potent and isoform-selective small-molecule modulators.3 The development of new small-molecule modulators of AC isoforms is expected to offer investigators a complementary approach to transgenic and knockout mouse studies for the exploration of individual AC isoforms in physiological, pathological, and behavioral studies. Furthermore, the importance of small-molecule modulators is even greater for AC2 because there are no published studies of AC2 knockout or transgenic mice but AC2 is linked to several diseases. For example, AC2 appears to be involved in cancer, where ADCY2 expression is upregulated in pancreatic and small intestinal NETs.29,30 Studies have also suggested a role for AC2 in airway function, as AC2 mediates IL-6 expression in hBSMCs.31 In this study, we investigated the stereoselectivity of the novel AC2-selective inhibitor SKF83566 and the molecular mechanism of inhibition of the enzymatic conversion of ATP to cAMP by AC2. We found that while (RC)-SKF83566 exhibits a stronger overall binding affinity to AC2 compared with (SC)SKF83566, (RC)-SKF83566 displays a competitive inhibition mechanism by binding to the ATP binding site. Because of the relatively low binding affinity of (RC)-SKF83566 compared with ATP (biological affinity = 1−2 nM), the inhibitory potential of (SC)-SKF83566 dominates over that of (RC)SKF83566, as (SC)-SKF83566 displays an allosteric inhibition mechanism and does not directly compete with ATP for binding to the same site. The novel allosteric inhibition mechanism of (SC)-SKF83566, elucidated using molecular modeling and biochemical techniques, may be due to induction of a conformational change in the ATP binding site that

ORCID

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jcim.6b00454. Differences between template structures used for homology modeling, BLASTp alignment of human AC2 with the template sequence, Modeller DOPE profile plot for prediction of homology models, and comparison of homology models and Limoc conformations of the binding site (PDF)

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AUTHOR INFORMATION

Corresponding Author

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*E-mail: [email protected].

Francisco León: 0000-0002-5064-2381 Markus A. Lill: 0000-0003-3023-5188 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Wild-type and S942 rat AC2 plasmids were obtained from Dr. Carmen Dessauer (University of Texas, Houston). The authors gratefully acknowledge grants from Purdue University and the NIH (MH101673 and MH060397) for partial support of this research.

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ABBREVIATIONS: AC, adenylyl cyclase; FSK, forskolin; hAC2, human adenylyl cyclase type 2; rAC2, rat adenylyl cyclase type 2; cAMP, cyclic adenosine monophosphate; ATP, adenosine triphosphate; DMEM, Dulbecco’s modified Eagle’s medium; HBSS, Hank’s balanced salt solution; IBMX, 3-isobutyl-1-methylxanthine; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; SKF83566, 8-bromo-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H3-benzazepin-7-ol; TR-FRET, time-resolved fluorescence resonance energy transfer; SQ22536, 9-(tetrahydro-2-furanyl)-9Hpurin-6-amine); CB7833407, 2-[(4-amino-6-oxo-1,6-dihydro-2pyrimidinyl)thio]-N-(3- chlorophenyl)propanamide; amu, atomic mass unit; Å, angstrom

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REFERENCES

(1) Sadana, R.; Dessauer, C. W. Physiological Roles for G ProteinRegulated Adenylyl Cyclase Isoforms: Insights from Knockout and Overexpression Studies. Neurosignals 2009, 17, 5−22.

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