Research Article pubs.acs.org/chemneuro
Aryl Sulfonamide Inhibitors of Insulin-Regulated Aminopeptidase Enhance Spine Density in Primary Hippocampal Neuron Cultures Shanti Diwakarla,† Erik Nylander,† Alfhild Grönbladh,† Sudarsana Reddy Vanga,‡ Yasmin Shamsudin Khan,‡ Hugo Gutiérrez-de-Terán,‡ Jonas Sav̈ marker,§ Leelee Ng,¶ Vi Pham,¶ Thomas Lundbac̈ k,# Annika Jenmalm-Jensen,# Richard Svensson,∇,○ Per Artursson,∇,○ Sofia Zelleroth,† Karin Engen,⊥ Ulrika Rosenström,⊥ Mats Larhed,∥ Johan Åqvist,‡ Siew Yeen Chai,¶ and Mathias Hallberg*,† †
The Beijer Laboratory, Department of Pharmaceutical Biosciences, Division of Biological Research on Drug Dependence, Department of Cell and Molecular Biology, §The Beijer Laboratory, Department of Medicinal Chemistry, ⊥Department of Medicinal Chemistry, and ∥Science for Life Laboratory, Department of Medicinal Chemistry, BMC, ∇Chemical Biology Consortium Sweden, Science for Life Laboratory, UDOPP at Institution of Pharmacy, and ○Drug Discovery and Development Facility, Science for Life Laboratory, Department of Pharmacy, Uppsala University, 751 24 Uppsala, Sweden # Chemical Biology Consortium Sweden, Science for Life Laboratory, Division of Translational Medicine and Chemical Biology, Department of Medicinal Biochemistry and Biophysics, Karolinska Institute, 171 77 Solna, Sweden ¶ Biomedicine Discovery Institute, Department of Physiology, Monash University, Clayton, Victoria 3800, Australia ‡
ABSTRACT: The zinc metallopeptidase insulin regulated aminopeptidase (IRAP), which is highly expressed in the hippocampus and other brain regions associated with cognitive function, has been identified as a high-affinity binding site of the hexapeptide angiotensin IV (Ang IV). This hexapeptide is thought to facilitate learning and memory by binding to the catalytic site of IRAP to inhibit its enzymatic activity. In support of this hypothesis, low molecular weight, nonpeptide specific inhibitors of IRAP have been shown to enhance memory in rodent models. Recently, it was demonstrated that linear and macrocyclic Ang IV-derived peptides can alter the shape and increase the number of dendritic spines in hippocampal cultures, properties associated with enhanced cognitive performance. After screening a library of 10 500 druglike substances for their ability to inhibit IRAP, we identified a series of low molecular weight aryl sulfonamides, which exhibit no structural similarity to Ang IV, as moderately potent IRAP inhibitors. A structural and biological characterization of three of these aryl sulfonamides was performed. Their binding modes to human IRAP were explored by docking calculations combined with molecular dynamics simulations and binding affinity estimations using the linear interaction energy method. Two alternative binding modes emerged from this analysis, both of which correctly rank the ligands according to their experimental binding affinities for this series of compounds. Finally, we show that two of these drug-like IRAP inhibitors can alter dendritic spine morphology and increase spine density in primary cultures of hippocampal neurons. KEYWORDS: Insulin-regulated aminopeptidase, aryl sulfonamides, molecular dynamics, ligand interaction energy simulations, dendritic spines, hippocampal neurons
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have few structural similarities to Ang IV, also enhance cognition in animal models.10−12 These studies support the hypothesis that IRAP plays an important role in memory facilitation; however, the precise mechanism of how these compounds exert their effects in vivo remains unclear. Ang IV-derived peptides and peptidomimetics can alter the shape and increase the number of dendritic spines in
nsulin-regulated aminopeptidase (IRAP, EC 3.4.11.3), a high-affinity binding site of angiotensin IV (1, Ang IV),1 is a single-transmembrane spanning zinc metalloenzyme that belongs to the M1 family of aminopeptidases.2 High levels of IRAP expression are found in areas of the brain associated with cognitive function including the hippocampus. 3−5 The hexapeptide Ang IV and other structurally related peptidomimetics that act as IRAP inhibitors have been shown to improve memory and learning performance and attenuate drug and lesion-induced memory deficits in rodents.6−9 Furthermore, low molecular weight, nonpeptide inhibitors of IRAP, which © XXXX American Chemical Society
Received: May 23, 2016 Accepted: July 22, 2016
A
DOI: 10.1021/acschemneuro.6b00146 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
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ACS Chemical Neuroscience hippocampal cultures.13,14 Moreover, a correlation between the procognitive activities of Ang IV-derived peptides and their capacity to increase spine number and head size has been observed.13,14 Spine morphology is thought to correlate with the strength and activity of the synapse,15 with mushroomshaped spines typically having a large spine head that contains more receptors and signaling proteins.16 These data suggest that the positive impact of these peptides on dendritic spines may account for the improved cognition observed in vivo. We recently disclosed that macrocyclic and linear peptides and pseudopeptides that are derived structurally from Ang IV (1, Ki = 62 nM) can act as very potent inhibitors of IRAP.17−22 Among the cyclic inhibitors, the very potent 13-membered macrocycle 2 (HA08; Ki = 3.3 nM),21 which comprises three amino acid residues and mimics the N-terminus of the IRAP substrate oxytocin,3,23,24 was found to alter dendritic spine density and morphology.25 However, because of its structural similarity to the N-terminus of Ang IV, it may have alternative macromolecular targets other than IRAP, as is the case with Ang IV, which may partly account for its demonstrated impact on dendritic spines. Indeed, it has been suggested that the procognitive and synaptogenic effects of peptides derived from the structure of Ang IV may be associated with inhibition of the hepatocyte growth factor/c-met system.26 A series of drug-like, low molecular weight, competitive IRAP inhibitors were reported in 2008 by Albiston et al.10−12 The benzopyran 4 (HFI-419; Ki = 420 nM), with a phenolic hydroxyl, exhibited pronounced enhancement of memory in two memory paradigms in the rat after intracerebroventricular administration10 (Figure 1).
tetrazole in the meta position of the aromatic ring as moderately potent IRAP inhibitors.28 Here, three of the most potent in vitro inhibitors from this series, the trichloro compound 5, the dimethyl compound 6, and the benzoxadiazole compound 7 (Figure 2), were selected for further structural and biological characterization.
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RESULTS AND DISCUSSION Inhibition of IRAP Activity by Compounds 5, 6, and 7. The sulfonamide compounds 5, 6, and 7 were evaluated as inhibitors of human IRAP, exhibiting IC50 values of 0.54 ± 0.05, 0.74 ± 0.02, and 0.42 ± 0.03 μM, respectively. Kinetic analysis of the inhibition of IRAP by compounds 5, 6, and 7 revealed that compound 5 is a competitive inhibitor of IRAP as shown by the relatively constant Vmax value with increasing compound concentration, while the binding of compounds 6 and 7 are more reflective of either mixed or noncompetitive inhibition as indicated by the relatively constant Km values and reduced Vmax (Figure 3). Inhibition of Aminopeptidase N and Leukotriene A4 Hydrolase by Compounds 5, 6, and 7. The specificity of the sulfonamide compounds 5, 6, and 7 was evaluated by inhibition of aminopeptidase N (APN) and leukotriene A4 hydrolase (LTA4H). All three compounds exhibited selectivity for IRAP over APN and LTA4H, with much lower efficacy in inhibiting the activity of the structurally related enzymes (Table 1). The percent inhibition was benchmarked against the aminopeptidase inhibitor bestatin (100 μM). Molecular Dynamics Simulations and Linear Interaction Energy Calculations. The binding modes of compounds 5, 6, and 7 were explored by a combination of extensive docking combined with molecular dynamics (MD) simulations and free energy calculations. The exhaustive docking exploration resulted in a wide range of potential binding modes, each of which was subject to a short MD sampling run. For poses that displayed structural stability at this stage, the MD sampling was replicated 10 times, and the binding free energy was calculated with the linear interaction energy (LIE) method.29 Initial docking with GLIDE30 provided up to 10 poses per compound that, despite being in the vicinity of the Zn2+ ion binding site, did not show any direct contact with the ion. Subsequent MD simulations discarded all these potential binding modes, since none of them showed stability of the corresponding docking pose, with protein−ligand interactions lost during the MD sampling. The second docking approach with GOLD31 identified binding modes governed by interactions between the negatively charged tetrazole and the Zn2+ ion, including the binding mode proposed earlier for this class of compounds.28 Even if this pose was compatible with some of the structure−activity relationships of the sulfonamide ligand series, the subsequent MD simulations clearly showed instability of the binding mode, and coordination with the Zn2+ ion was invariably lost in all cases. The above kinetic results only confirmed compound 5 as a competitive inhibitor, while compounds 6 and 7 behave as
Figure 1. Angiotensin IV (1), the macrocyclic IRAP inhibitor HA08 (2), the macrocyclic peptide and IRAP substrate oxytocin (3), and the benzopyran IRAP inhibitor HFI-419 (4).
Based on these findings, we searched for chemical entities that were structurally diverse from Ang IV analogues, for example, compounds lacking phenolic elements in proximity to peptide bonds. After screening a library of 10 500 drug-like compounds for their ability to inhibit the proteolytic activity of IRAP,27 we identified a series of aryl sulfonamides comprising a
Figure 2. Aryl sulfonamide IRAP inhibitors: trichloro compound 5, dimethyl compound 6, and benzoxadiazole compound 7. B
DOI: 10.1021/acschemneuro.6b00146 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
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ACS Chemical Neuroscience
Figure 3. Kinetic analysis of IRAP inhibition. Nonlinear regression and Lineweaver−Burk double-reciprocal plots (inset) show IRAP activity in the absence and presence of three concentrations of (A) compound 5, (B) compound 6, and (C) compound 7. Each point represents the mean ± SEM of three replicates from three independent experiments.
backbone of Arg439 and the side chain of Thr442. The substituted benzyl ring is adjacent to the GAMEN loop, allowing the para-position chlorine atom in compound 5 to interact with Gln293 (Cl−N distance ∼3.3 Å). The second stable binding mode (pose B, Figure 4B) is inverted compared with pose A; that is, it places the substituted aromatic ring in the 438−443 loop cavity and the tetrazole remains solvent exposed. Additionally, the −NH group of the sulfonamide interacts with the zinc-coordinating carboxylate of Glu487. Because these two poses were stable during the MD trajectories, the corresponding binding free energies could be estimated not only for compound 5 but also for compounds 6 and 7 in the analogous binding poses (Table 2). The mean unsigned error between calculated and observed binding free energies, utilizing the same values of the LIE α and β parameters as earlier,25 was 0.33 and 1.16 kcal/mol for pose A and B, respectively. The two binding modes considered here are further supported by other sulfonamide-containing ligands interacting with zinc binding sites.33,34 However, it differs from a previously hypothesized binding arrangement with a salt bridge between the negatively charged tetrazole ring and the positively charged Zn2+ ion,28 which was found to be unstable in MD simulations. Furthermore, the constrained docking of ligand 5 resulted in three poses, all of which showed sulfoxy− Zn2+ ion interactions, similar to the manually docked poses. After subsequent MD simulations, however, all three constrained docking poses fall into either pose A or pose B. Thus, our results suggest that the sulfoxy−Zn2+ ion interaction is essential for binding of compound 5 to IRAP. Although, the simulations and docking experiments suggest that compounds 6 and 7 indeed could bind in a similar fashion as the competitive inhibitor compound 5 to IRAP, the kinetic
Table 1. Percentage Inhibition of the Related Enzymes APN and LTA4Ha compound 5 enzyme APN (EC 3.4.11.2) LTA4H (EC 3.3.2.6)
compound 6
compound 7
100 μM 10 μM 100 μM 10 μM 100 μM 10 μM 28
6
8
2
28
0
27
18
0
0
23
0
a
Results are expressed as percentage inhibition of the catalytic activity of the enzyme at the given concentration (n = 3).
noncompetitive or have a mixed role, that is, they could bind in two alternative binding sites (competitive or allosteric). This led us to consider alternative poses, which were modeled by manual docking. The strategy here was guided by the recent crystal structure of the pseudopeptide inhibitor DG025 (PDB code 4Z7I), which contains a phosphinic group coordinating the Zn2+ ion.32 Initially, compound 5 was modeled in up to 70 alternative binding modes, satisfying the condition that one of the sulfone oxygens coordinates with the Zn2+ ion. This idea was further supported by crystal structures containing sulfonamide−Zn2+ ion interaction motifs (PDB codes 4R59, 4KAP, 4KUY),33,34 as well as by the O−Zn2+ ion interaction experimentally observed for Ang IV in the closely related human aminopeptidase-B.35 From the pool of manually docked poses, only two binding modes showed stability during the subsequent MD simulations and maintained the initial interactions with the protein, including the coordination with the Zn2+ ion. In pose A (Figure 4A), the sulfonamide is further stabilized by interactions between its −NH group and Glu465. The tetrazole is accommodated in the cavity defined by the loop region involving residues 438−443 and interacts with the
Figure 4. Average ligand structures from MD simulations of compound 5, superimposed in the enzymatic cleft of IRAP: (A) pose A; (B) pose B. Only one average protein structure is shown for clarity. C
DOI: 10.1021/acschemneuro.6b00146 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
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ACS Chemical Neuroscience Table 2. Experimental and Calculated Binding Free Energies (in kcal/mol) for the IRAP Inhibitorsa free
bound
ligand
⟨Uell−s⟩
⟨UvdW l−s ⟩
compound 5 compound 6 compound 7
−159.32 ± 1.2 −156.56 ± 0.4 −160.30 ± 1.1
−25.26 ± 0.0 −23.44 ± 0.1 −25.25 ± 0.1
compound 5 compound 6 compound 7
−159.32 ± 1.2 −156.56 ± 0.4 −160.30 ± 1.1
−25.26 ± 0.0 −23.44 ± 0.1 −25.25 ± 0.1
⟨Uell−s⟩ Pose −135.19 −132.97 −140.78 Pose −165.15 −158.82 −179.15
A ± ± ± B ± ± ±
⟨UvdW l−s ⟩
ΔGobs bind
ΔGcalc bind
0.8 1.0 1.1
−44.59 ± 0.2 −42.01 ± 0.5 −45.15 ± 0.3
−8.54 ± 0.1 −8.36 ± 0.0 −8.70 ± 0.1
−8.22 ± 0.1 −8.18 ± 0.1 −9.20 ± 0.2
1.4 1.2 2.4
−38.34 ± 0.5 −35.19 ± 0.5 −37.90 ± 0.2
−8.54 ± 0.1 −8.36 ± 0.0 −8.70 ± 0.1
−8.04 ± 0.2 −7.12 ± 0.2 −10.44 ± 0.3
a Calculated binding free energies obtained with an optimized LIE model, with α = 0.18, β = 0.19, and γ = −9.96 for pose A and −5.22 kcal/mol for pose B. Error bars denote the SEM for replicate simulations.
analyses reveal that it may not be the (only) case. A more extensive analysis would be required in order to allow for a proposal of alternative tentative binding modes of compounds 6 and 7, which both demonstrate more complex binding characteristics (i.e., a mixed or noncompetitive inhibition). Effect of Compounds 5, 6, and 7 on Spine Density and Morphology. Previous studies have shown that Ang IV and related analogs can augment synaptic plasticity,14,26 which may be one mechanism involved in the procognitive effects seen in vivo. However, these peptides are reported to have other targets. We have previously shown that a macrocyclic IRAP inhibitor (2, HA08), which mimics the N-terminal of the endogenous IRAP substrate 3 (oxytocin), can alter dendritic spine density and morphology in vitro.25 However, similar to Ang IV, this inhibitor is more peptidic in nature and may augment spine properties by binding to other macromolecular targets. Therefore, we used the newly developed, more druglike aryl sulfonamide IRAP inhibitors 5, 6, and 7 to assess whether IRAP inhibition affects dendritic spines. Changes in the number and morphology of dendritic spines have been linked to enhanced cognitive performance.13,36−38 Therefore, the effect of the aryl sulfonamide IRAP inhibitors on augmenting dendritic spine morphology in primary cultures of hippocampal neurons was assessed using immunocytochemical staining against drebrin, a protein highly expressed in dendritic spines. Immunostaining of cells following exposure to varying concentrations of compounds 5, 6, and 7 at 14, 17, and 20 days in vitro (DIV) revealed an obvious increase in spine density following exposure to 10 μM of compounds 5 and 6, compared with vehicle treated cultures. No marked differences were observed following exposure to compound 7 (Figure 5). Quantification confirmed these observations, with a significant increase in total spine density observed following treatment with 10 μM compound 5 (mean spine number = 21.4, P < 0.05; Figure 6A), and 0.1 and 10 μM compound 6 (mean spine number = 19.5 and 22.2, respectively, P < 0.05; Figure 6B) compared with the vehicle control (mean spine number = 15.5). No change was observed for compound 7 (mean spine number = 16.0;Figure 6C). A parallel effect was observed for the number of stubby/mushroom-like spines, a morphology typically associated with mature spines, which are believed to have strengthened synaptic connectivity.39 Exposure to 10 μM compound 5 increased stubby/mushroom-like spine density by 26.1% (P < 0.05; Figure 6D), and exposure to 0.1 and 10 μM compound 6 increased spine density by 27.3% (P < 0.05) and 33.4% (P < 0.01), respectively, compared with vehicle (Figure 6E). No change was observed for compound 7 (Figure 6F). The effects of compounds 5 and 6 on total and
Figure 5. Effect of compounds 5, 6, and 7 on dendritic spine number and morphology. Cultures were exposed to varying concentrations of compounds 5, 6, and 7 at 14, 17, and 20 DIV. At 21 DIV, hippocampal cells were fixed and immunostained against β-III tubulin (1:500, green) and drebrin (1:500, red) to visualize neuronal processes and dendritic spines, respectively. Nuclei were counterstained with DAPI (blue). Images indicate segments depicting changes in spine density and morphology at 10−7−10−5 M. Arrows indicate different spine morphologies (yellow = stubby/mushroom-like spines; white = filopodia/thin-like spines). Brain-derived neurotrophic factor (100 ng/mL) was included as a positive control. Scale bar = 10 μm.
stubby/mushroom-like spine density were similar to that seen with brain-derived neurotrophic factor (BDNF; 100 ng/mL) following 24 h exposure, a known inducer of spine development in hippocampal neurons.40,41 Unlike compound 7, the number of immature filopodia/thinlike spines increased in the presence of compounds 5 and 6. Exposure to 10 μM compound 5 increased spine number by 40.6% (P < 0.01) while exposure to 1 μM compound 6 increased filopodia/thin-like spine density by 33.8% (P < 0.05; Figure 6G−I). Filopodia/thin-like spines are typically expressed early in development42−44 and are thought to be an early stage of synaptic formation.43,45 A possible cause for this increase in immature spine formation could be because of the direct/ indirect effects of these compounds on glial-derived secreted factors that are essential for spine maturity.46−48 However, further studies are needed to demonstrate this hypothesis. Overall, given that previous studies have shown that the procognitive activity of a molecule in vivo correlates well with its ability in vitro to alter dendritic spine structure,13,14 these results suggest that compounds 5 and 6 may have cognitive enhancing effects. Effect of Compounds 5 and 6 on Dendritic Spine Functionality. One of the most important functions of dendritic spines is to sense glutamate released from presynaptic nerve terminals.49 To further investigate the effect of D
DOI: 10.1021/acschemneuro.6b00146 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
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Figure 6. Percentage change in spine number following exposure to compounds 5, 6, and 7. Spine number and morphology were assessed along 50 μm of dendrite in 10 neurons (3 dendrites per neuron; n = 3 independent cultures). Exposure to (A) compound 5 increased total spine density at 10−5 M, while exposure to (B) compound 6 increased total spine density at 10−7 and 10−5 M compared with the vehicle control (0.01% (v/v) DMSO). No change in total spine number was observed for (C) compound 7. Similarly, stubby/mushroom-like spine number increased at 10−5 M following exposure to (D) compound 5 and at 10−7 and 10−5 M when exposed to (E) compound 6. No change was observed for (F) compound 7. An increase in the number of filopodia/thin-like spines was observed at 10−5 and 10−6 M for (G) compound 5 and (H) compound 6, respectively. No change was observed for (I) compound 7 when compared with the vehicle control. Brain-derived neurotrophic factor (100 ng/mL) was included as a positive control. Data were analyzed by one-way ANOVA followed by Dunnett’s test. All data are expressed as the mean ± SEM, *P < 0.05, **P < 0.01, and ***P < 0.001 compared with vehicle.
Figure 7. Effect of compounds 5 and 6 on dendritic spine functionality. Cultures were exposed to 10 μM of compounds 5 and 6 at 14, 17, and 20 DIV. Spines were labeled for synapsin1 and vGLUT1, and the numbers of spines were counted on a 50 μm length of dendrite. The functionality of spines was assessed along 50 μm of dendrite in 10 neurons (3 dendrites per neuron; n = 3 independent cultures). Representative images of a dendritic segment treated with compound 5 indicating (A) drebrin-positive (red) and synapsin1-positive (green) spines and (B) drebrin-positive (red) and vGLUT1-positive (green) spines. Representative images of a dendritic segment treated with compound 6 indicating (C) drebrin-positive (red) and synapsin1-positive (green) spines and (D) drebrin-positive (red) and vGLUT1-positive (green) spines. With respect to stubby/ mushroom-like spines, there were no differences in the proportion of (E, G) synapsin1-positive spines or (F, H) vGLUT1-positive spines compared with vehicle. Overall, there was a significant increase in the overall number of (I, K) synapsin1-positive and (J, L) vGLUT1-positive spines compared with vehicle. Data were analyzed by one-way ANOVA followed by Dunnett’s test. All data are expressed as the mean ± SEM, **P < 0.01 compared with vehicle. Scale bar =10 μm.
E
DOI: 10.1021/acschemneuro.6b00146 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
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and 6. However, for compound 7 in the presence of HLM, which contains membrane bound glutathione transferase (GST)53 and glutathione (GSH), only 1% remained after 90 min, compared with approximately 70% with GSH alone. Because GST and other antioxidant enzymes exist in all cell types, a nucleophilic attack by GSH catalyzed by GST is likely to be the predominant clearance mechanism of compound 7. Notably, benzoxadiazole derivatives that are structurally very similar to compound 7 are known to act as powerful electrophiles and are prone to react with endogenous nucleophiles as thiols and amines in plasma proteins54 and have been developed as potential reactive anticancer agents.55 The electrophilicity testing clearly demonstrated the sensitivity of compound 7 toward activated nucleophiles in the form of the GST enzyme system, a system that is present in all cell types. Thus, the lack of efficacy of compound 7 on dendritic spine density and morphology is likely attributed to its high reactivity.
compounds 5 and 6 on dendritic spines, we performed functionality studies to identify their neurotransmitter signature. Dendritic spines that receive glutamatergic presynaptic input play an important role in memory formation.38 Given that only compounds 5 and 6 induced an increase in total and stubby/mushroom-like spines, we next labeled cells for the presynaptic markers synapsin1 and vesicular glutamate transporter 1 (vGLUT1) to determine their functionality. Cells were exposed to 10 μM of compounds 5 and 6, a concentration shown to significantly increase stubby/mushroom-like spines for both IRAP inhibitors. The numbers of stubby/mushroomlike spines staining positive for synapsin1, a universal marker of synaptic terminals, or vGLUT1, a measure of glutamatergic synapses, were counted to determine their functionality. Immunocytochemical staining revealed that the majority (>80%) of dendritic spines formed active synapses in the presence of 10 μM of compounds 5 and 6. Dendritic spines were also vGLUT1-positive, indicating that spines were receptive to glutamatergic signaling. Interestingly, there were fewer vGLUT1-positive spines compared with synapsin1positive spines, suggesting that some synapses act independently of glutamate signaling and may perhaps interact with cholinergic or GABAergic neurons, which are also known to play an important role in learning and memory processing.50,51 Compared with the vehicle control, there was no significant change in the percentage of drebrin-positive/synapsin1-positive or drebrin-positive/vGLUT1-positive spines (Figure 7A), indicating that neither compounds 5 nor 6 altered the ratio of functional synapses. Given that both compounds 5 and 6 induced an overall increase in the number of stubby/ mushroom-like spines, we also observed an overall increase in the number of synapsin1- and vGLUT1-positive spines compared with vehicle treated cells (P < 0.01; Figure 7B). These results suggest that cells exposed to 10 μM of compounds 5 or 6 have the capacity to receive increased excitatory synaptic input. ADME Profiling. In order to obtain better insight into why the IRAP inhibitors differed in their ability to enhance spine density, solubility and cell permeability tests were performed. The solubility of both compounds 5 and 6 was very high in buffer at pH 7.4, 0.83 and 1.3 mM. The solubility of compound 7 was likewise high (>0.12 mM), and 7 was completely dissolved at 0.1 mM, a concentration higher than that used in the in vitro experiments performed. Compounds 5 and 7 were moderately to highly stable against oxidative metabolism in human liver microsomes (HLM) and mouse liver microsomes (MLM), with a t1/2 of 88 and 44 min and t1/2 of 200 and 81 min for compounds 5 and 7 (human and mouse) respectively, while compound 6 showed a very high level of degradation in HLM, which was likely attributed to species specific benzylic oxidation. The intrinsic clearance (μL/(min·mg)) for compounds 5, 6, and 7 was 16, 900, and 7 for human and 29, 28, and 17 for mouse, respectively. Cell permeability tests were performed using Madin−Darby canine kidney (MDCK) cells, which express significant endogenous P-glycoprotein activity.52 MDCK cell monolayer permeability was fairly low for all compounds. The apical to basolateral apparent permeabilities were 3.3, 1.3, and 1.2 × 10−6 cm/s for compounds 5, 6 and 7, respectively. These differences were not significant. The permeability for all compounds was comparable in both directions across the monolayers, indicating that they were not significantly effluxed by P-glycoprotein. Electrophilicity testing indicated low reactivity of compounds 5
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CONCLUSION In this study, we report a thorough characterization of the enzymatic inhibition and phenotypic consequences of three low molecular weight IRAP inhibitors. The binding modes of the competitive inhibitor 5 to human IRAP were explored by docking calculations combined with MD simulations and binding affinity estimations with the LIE method. Two alternative binding modes emerged from the MD simulations and the binding free energy calculations. Notably, these binding modes differ from a previously suggested one. Although structurally very similar, we report that compounds 5 and 6, in contrast to the equally efficient IRAP inhibitor 7, increase the number of mushroom-shaped dendritic spines, a morphology associated with memory enhancement,36 which are capable of forming active synapses and are receptive to glutamatergic signaling. Overall, the data provides support for the hypothesis that there is a link between IRAP inhibition, and the morphology and density of dendritic spines, which may contribute to the improved cognition observed after administration of IRAP inhibitors in animal models.
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METHODS
Synthesis of Compounds. The aryl sulfonamide inhibitors 5, 6, and 7 were prepared as reported previously.28 The inhibitors were prepared from 3-amino phenyltetrazole and the corresponding aryl sulfonyl chlorides were produced in good yields (76−83%). Enzyme Assays. Insulin-Regulated Aminopeptidase. The efficacy of the candidate compounds to inhibit IRAP enzymatic activity was analyzed using solubilized, crude membranes isolated from HEK 293 cells (ATCC, Manassas, VA) transfected with pCI-IRAP. Membranes from vector-only transfected cells were used as a negative control. IRAP enzymatic activity was determined by the hydrolysis of the synthetic substrate L-leucine-4-methyl-7-coumarinylamide (SigmaAldrich, St Louis, MO) and monitored by the release of a fluorogenic product at excitation and emission wavelengths of 380 and 430 nm, respectively. Assays were performed in 96-well plates; each well containing 1 μg of solubilized membrane protein and 25 μM substrate in a final volume of 100 μL in 50 mM Tris-HCl buffer (pH 7.4). Reactions were performed at 37 °C for 30 min using a Wallac Victor3 V Multilabel counter (PerkinElmer, Waltham, MA). Inhibitory constants (Ki) for each of the inhibitors were determined over a range of concentrations (0.01−100 μM) and were calculated from the relationship IC50 = Ki(1 + [S]/Km) with GraphPad Prism 5 software (GraphPad Software Inc., San Diego, CA). Kinetic analysis was performed over a range of substrate concentrations (15.6 μM to 1 mM), in the absence and presence of F
DOI: 10.1021/acschemneuro.6b00146 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
Research Article
ACS Chemical Neuroscience compounds 5, 6, and 7 (0.25, 0.5, or 1 μM). Samples were assayed in at least three separate experiments in triplicate. The Michaelis− Menten equation and nonlinear regression were used to analyze the kinetic parameters Vmax and Km. Linear regression analysis of kinetic data was expressed in the form of a Lineweaver−Burk doublereciprocal plot (GraphPad Prism). Leukotriene A4 Hydrolase. Recombinant human LTA4H (Cayman Chemical, Ann Arbor, MI) (specific activity 166 U/mg) was incubated at room temperature with 100 μM alanine-β-naphthylamide as a substrate in 50 mM Tris-HCl buffer, pH 8.0, containing 100 mM KCl with or without increasing concentrations of the inhibitors. The fluorescence was measured at excitation and emission wavelengths of 320 and 405 nm, respectively. Aminopeptidase N. APN (specific activity 40 U/mg) (SigmaAldrich) was incubated with 250 μM of substrate alanine-βnaphthylamide (Sigma-Aldrich) in Tris buffered saline (50 mM TrisHCl, 150 mM NaCl pH 7.5) at 25 °C. IRAP inhibitors (1−10 μM) were added after 1 min, and fluorescence was monitored at excitation and emission wavelengths of 320 and 405 nm, respectively. Structural Modeling and Molecular Docking. The crystal structure of human IRAP was retrieved from the protein data bank (PDB code 4PJ6)35 and prepared as previously described for docking and molecular dynamics (MD) simulations.25 The ligands 5, 6, and 7 (Figure 2) were built in their most probable 3D conformation using Maestro version 9.2. (Schrödinger, LLC; NY) and a thorough docking exploration followed using three different approaches.56 The first approach made use of GLIDE,30 with a cubic search grid of 30 Å centered on the equivalent position of the Cα atom of His4 in Ang IV25 and 50 independent docking runs per compound using Glide-XP precision. The top 10 ranked poses were retained for further MD refinement. The second docking approach utilized the GOLD program31 with 20 independent runs using a search sphere of 15 Å radius around the same center. In this case, between 2 and 4 docking poses were retained per compound for further analysis. Finally, up to 70 manually docked poses were produced using the PyMol Molecular Graphics System, version 1.5.0.4 (Schrödinger, LLC) based on superposition of the sulfonamide group with the phosphinate group of the cocrystallized pseudopeptide DG025 (PDB code 4Z7I).32 To avoid bias in the manually docked poses, a constrained docking of ligand 5 was also performed in GLIDE using the same conditions as above, but with H-bond/Metal and Metal Coordination constraints in the receptor grid generation. With this setup, the ligand atoms were screened against the Zn2+ ion to ensure a metal−ligand interaction, preserving the initial metal coordination. Molecular Dynamics Simulations and Linear Interaction Energy Calculations. MD simulations were performed using the program Q57 with the OPLS-AA force field.58 Parameters for the Zn2+ ion and the ligands were retrieved from the automatic parametrization available in Macromodel, version 10.6 (Schrödinger LLC). Spherical MD boundary conditions were used, with a simulation sphere of 25 Å radius centered on the same point as defined for the docking calculations. This sphere was solvated with TIP3P water molecules59 and subjected to polarization and radial constraints according to the surface constrained all-atom solvent model57,60 to mimic the properties of bulk water at the sphere surface. Protein atoms outside the simulation sphere were restrained to their initial positions and only interacted with the system through bonds, angles, and torsions. Titratable residues within 20 Å of the Zn2+ ion were treated in their charged form. In addition, the residues Lys520, Lys726, Glu767, Asp773, Arg817, Glu818, Arg820, Glu825, Arg858, Glu887, Lys890, Lys892, Glu895, Arg933, and Glu1002 were also treated as ionized. With this setup, the simulation sphere was overall neutral, thus avoiding the consideration of additional Born terms in the calculation of free energies. Nonbonded interactions were calculated explicitly up to a 10 Å cutoff, except for the ligand atoms for which no cutoff was used. Beyond the direct cutoff, long-range electrostatics were treated with the local reaction field multipole expansion method.61 During a 175 ps equilibration stage, the system was slowly heated to the target temperature of 310 K while initial positional restrains on all solute heavy atoms were gradually released. In the subsequent data
collection phase an MD time step of 1 fs was used and no positional restraints were applied. Solvent bonds and angles were constrained using the SHAKE algorithm.62 Nonbonded pair lists were updated every 25 steps, and the ligand-surrounding interaction energies were sampled every 50 steps. In the initial screening phase, MD trajectories of 2 ns length were calculated for each selected docking pose. These poses were then filtered based on their stability during the simulations. Stable poses were subsequently subjected to 10 independent 2 ns MD simulations with different initial velocities, which were used for binding affinity calculations (see below). In parallel, reference calculations for each ligand in water were carried out using the same protocol as for the bound state (i.e., same sphere size, center, equilibration scheme, and 10 independent production runs lasting 2 ns each). Because the goal of this setup was to evaluate the binding affinities of the ligands but not to simulate the conformational changes distal to the binding site, spherical boundary conditions were employed. With this setup, we could efficiently sample the free energies of many ligands at a low computational cost, while attaining reliable statistics.63,64 Binding free energies were calculated using the LIE method:29,65 calcd el ΔG bind = α⟨ΔUlvdW − s ⟩ + β Δ⟨U l − s⟩ + γ
(1)
el where Δ⟨UvdW l−s ⟩ and Δ⟨Ul−s⟩ are the differences in the average nonpolar and polar ligand-surrounding interaction energies in the two states, that is, free and bound ligand. The coefficients α and β are scaling parameters65−67 for the nonpolar and polar terms, respectively. In the standard LIE model, α has a value of 0.18, while β depends on the chemical nature of the ligand. The IRAP active site has a divalent Zn2+ ion together with a cluster of carboxylates, causing very large electrostatic interaction energies with the ligands. Because these interaction energies, particularly those involving the Zn2+ ion, will be very sensitive to the force field parameters, we follow a protocol used earlier for binding sites containing ions.68 Thus, β was treated as a free parameter, allowing it to compensate for possible insufficient dielectric screening in the microscopic system, and we retained the value β = 0.19 calculated earlier for IRAP inhibitors.25 An electrostatic correction term57 was added to account for the long-range interactions of the charged sulfonamides with the neglected charges of the neutralized titratable residues. This term was calculated as ΔGelcorr = 0.64 kcal/mol as follows
el ΔGcorr = 332 ∑ ∑ p
l
qpql εrpl
(2)
where qp is the formal charge of the neglected ionic group, while ql is the partial charge of the ligand atom. The effective dielectric constant ε was set to 80. The last term γ is a protein-dependent constant that does not affect the relative binding free energies66 but is used to offset the resulting calculated energies to the experimental values. The reported nonbonded energies correspond to average values over all 10 replicates, and the corresponding errors are calculated as the standard error of the mean (SEM). Experimental binding free energies (ΔGexp bind) were extracted from IC50 values as exp ΔG bind = RT ln Kd = RT ln IC50 + c
(3)
where c is an assay-specific constant, which depends on the substrate concentration and the enzymatic Km value.69 This constant value does not affect the relative free energies and is implicitly included in the optimized value of γ in eq 1. Primary Hippocampal Cultures. Mixed primary hippocampal neuron−astrocyte cultures were prepared from embryonic day 17 Sprague−Dawley rats (Charles River, Sulzfeld, Germany) as previously described.25 Briefly, hippocampi were isolated, chemically and mechanically digested, and seeded on poly-D-lysine coated glass coverslips (50 μg/mL) in 24-well plates at a density of 1 × 105 cells per well. Cultures were grown in neurobasal medium (Invitrogen, Carlsbad, CA) supplemented with B27 (Invitrogen), 0.5 mM GlutaMAX supplement (Invitrogen), and 10% (v/v) fetal bovine serum (Invitrogen) for 24 h, after which the media was replaced with G
DOI: 10.1021/acschemneuro.6b00146 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
Research Article
ACS Chemical Neuroscience serum-free media containing an additional 2% (v/v) B27. Partial media changes were performed twice a week, and cultures were maintained at 37 °C/5% (v/v) CO2 in a humidified incubator. Hippocampal cells were grown for 13 DIV. The addition of varying concentrations of inhibitors was performed on day 14, 17, and 20, and treatments were ceased at 21 DIV. No noticeable effects on cell viability were observed, as assessed by the MTT assay. All animal experiments were approved by the Animal Ethics Committee at Uppsala University. Immunocytochemistry and Image Analysis. Immunocytochemistry and image analysis of spine density and functionality were performed as previously described.25 At 21 DIV, cells were fixed, permeabilized, and blocked with normal donkey serum (SigmaAldrich), followed by incubation with the neuronal marker rabbit antiβ III-tubulin (1:500; Sigma-Aldrich) and the dendritic spine marker mouse anti-drebrin (1:500; Enzo Life Sciences, Farmingdale, NY) for 90 min at room temperature. For spine functionality experiments, cells were either incubated with the presynaptic glutamatergic synapse marker rabbit anti-vGlut1 (1:500; Abcam, Cambridge, MA) or the universal presynaptic marker rabbit anti-synapsin1 (1:500; Abcam). Cells were then incubated for 1 h at room temperature with the appropriate fluorescent-conjugated secondary antibodies (1:500; Alexa 488 and Alexa 568; Invitrogen). Cultures were counterstained with 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI; Merck Millipore, Bedford, MA) and mounted with MOWIOL antifade mounting medium (Sigma-Aldrich). Images were captured using a Zeiss LSM700 inverted confocal microscope (Carl Zeiss Microscopy GmbH, Jena, Germany) with a 63× oil immersion lens. Stubby/mushroom-like spines and filopodia/thin-like spines were identified and counted as previously described.25 The number of spines was counted on three individual basal dendrites (primary and secondary) from 10 neurons per culture (3 independent cultures) using ImageJ 1.49e software (National Institutes of Health, Bethesda, MD). Quantification was performed on 50 μm dendritic segments that were at least 50 μm away from the cell body. All images were captured and analyzed by a researcher blinded to the treatments. Immunostaining where the primary antibodies were omitted was performed to ensure specificity of labeling. ADME Profiling. Metabolic stability in HLM and MLM was performed as previously described.70 The solubility of the compounds was determined using a miniaturized shake-flask method. In brief, 0.1 M potassium phosphate buffer was added to 0.2−0.5 mg of solid compound to yield a saturated solution. The vials were sealed and shaken for 24 h at ambient temperature and centrifuged at 10 000g, and the supernatant was quantified using an external standard curve. The MDCK (wt) cell permeability study was performed essentially as for Caco-2 in accordance with published protocols.71 MDCK cell monolayers (passage 11−22) were grown on permeable filter supports and used for transport studies on day 4 after seeding.52 The test compound concentration was 10 μM. To test for electrophilicity, compounds 5, 6, and 7 were incubated with 1 μM over 90 min at 37 °C in buffer (pH 7.4), buffer spiked with 2 mM glutathione (GSH), 1 mg/mL HLM, and 1 mg/mL HLM with 2 mM GSH. The percent compound remaining at 90 min was compared with a 0 min control incubated in the presence of 50% (v/v) acetonitrile. All assays were analyzed by LC-MS/MS using a Waters XEVO TQ coupled to an Acquity UPLC. Statistical Analyses. All data were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test using GraphPad Prism (GraphPad Prism Software Inc.). A P value
