Journal Pre-proof Neuroprotective effects of novel nitrones: In vitro and in silico studies Saira Cancela, Lucía Canclini, Gustavo Mourglia-Ettlin, Paola Hernández, Alicia Merlino PII:
To appear in:
European Journal of Pharmacology
Received Date: 13 November 2019 Revised Date:
11 January 2020
Accepted Date: 13 January 2020
Please cite this article as: Cancela, S., Canclini, Lucí., Mourglia-Ettlin, G., Hernández, P., Merlino, A., Neuroprotective effects of novel nitrones: In vitro and in silico studies, European Journal of Pharmacology (2020), doi: https://doi.org/10.1016/j.ejphar.2020.172926. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.
Neuroprotective effects of novel nitrones: in vitro and in silico studies Saira Cancelaa*, Lucía Canclinib, Gustavo Mourglia-Ettlinc, Paola Hernándeza*, Alicia Merlinodα. a
Laboratorio de Epigenética e Inestabilidad Genómica, Departamento de Genética,
Instituto de Investigaciones Biológicas Clemente Estable, Montevideo, Uruguay b
Departamento de Genética, Instituto de Investigaciones Biológicas Clemente Estable,
Montevideo, Uruguay c
Cátedra de Inmunología, Departamento de Biociencias, Facultad de Química,
Universidad de la República, Montevideo, Uruguay d
Instituto de Química Biológica, Facultad de Ciencias, Universidad de la República,
Professor Alicia Merlino passed away on July 8, 2018.
Corresponding authors: PhD. Paola Hernández. Phone: +589-24871616, email:
, [email protected]
Postal address: Laboratorio de Epigenética e Inestabilidad Genómica, Departamento de Genética, Instituto de Investigaciones Biológicas Clemente Estable, Avenida Italia 3318, Montevideo, 11600, Uruguay. Lic. Saira Cancela. Phone: +589-24871616, email: [email protected]
Postal address: Laboratorio de Epigenética e Inestabilidad Genómica, Departamento de Genética, Instituto de Investigaciones Biológicas Clemente Estable, Avenida Italia 3318, Montevideo, 11600, Uruguay.
Abstract Neurodegenerative diseases affect millions of people around the world. Several studies point out caspase-3 as a key player in the development and progression of neurological disorders including amyotrophic lateral sclerosis, Alzheimer’s, Parkinson’s and Huntington’s diseases. Furthermore, oxidative stress and mitochondrial dysfunction plays an important role in neurodegenerative pathologies leading to neuronal damage and cell death. Pharmacological properties of nitrones such as free radical trapping and neuroprotection has been previously described. In the present work, we have assessed ten non-cytotoxic nitrones for their ability to inhibit apoptosis plus their potential to reduce active caspase-3 and oxidative stress in the hippocampal neuronal cell line HT22. Our results highlight the faculty of nitrones to inhibit apoptosis by a mechanism that involves active caspase-3 reduction and decrease of reactive oxygen species. Moreover, docking and molecular dynamics approaches lead to a detailed analysis at the atomic level of the nitrones binding mode to caspase-3 suggesting that compounds bind in a region close to the catalytic site. All these data place these molecules as excellent hits for further efforts to redesign novel compounds in the search of a new therapy against neurodegenerative disorders.
Keywords: nitrones, apoptosis, oxidative-stress, caspase-3, molecular docking, molecular dynamics
1. Introduction Neurodegenerative diseases are common age-related pathologies that affects millions of people around the world (Poewe et al., 2017; World Alzheimer Report, 2019). These 2
pathologies including amyotrophic lateral sclerosis, Alzheimer’s, Parkinson’s and Huntington’s diseases are characterized by the progressive alteration of neuronal structure and function leading to cellular death. Currently used drugs, are unable to prevent or cure these neurological disorders and besides cause severe side effects. Thus, the need of alternative therapies, sets an urgent look into the identification and manipulation of new therapeutic targets that can help to overcome this scenario.
Previous studies have pointed out several roles of caspase-3 in the development and progression of these neurodegenerative disorders (D’Amelio et al., 2012). Overexpression and active participation of caspase-3 in the initial stages of these pathologies as well as the positive results following its inhibition highlight this enzyme as an interesting therapeutic target for prevention and/or treatment of these devastating diseases. In the last few years, huge efforts have been made to design caspase-3 reversible inhibitors molecules using experimental and computational methodologies (MacKenzie et al., 2010; Porȩba et al., 2013; Wu et al., 2014). While many of these inhibitors showed good activity in vitro against caspase-3, in most cases no comparative data to account for its selectivity or no information about compounds cytotoxicity or their activity in vivo is available. This points out the urgent need to find novel reversible and selective inhibitors able to modulate caspase-3 activity in cells.
The potent free radical trapping properties in biological systems exerted by nitrones like PBN (phenyl-tert-butylnitrone) was described by Robert A. Floyd (Carney and Floyd, 1991). In our search for potential inhibitors of caspase-3, nitrones emerged as suitable candidates due to their excellent pharmacological properties. These compounds show anti-oxidant and neuroprotective effects, can inhibit mitochondrial-dependent apoptotic 3
cascade and are able to cross the blood brain barrier (Barriga et al., 2010; Chavarría et al., 2012; Das et al., 2012; Porcal et al., 2008). Taking this into account, in the present work we study novel aspects concerning the neuroprotective potential of some (Z)-αaryl and heteroaryl N-alkyl or N-benzyl-nitrones previously synthesized in our research group (Fig. 1) (Barriga et al., 2010; Chavarría et al., 2012; Porcal et al., 2008).
FIG 1 SHOULD BE LISTED HERE
For this purpose, we determined the anti-apoptotic and anti-oxidant properties of these molecules in a mouse hippocampal cell line (HT22). Moreover, the capability of these compounds to directly bind and inhibit caspase-3 was studied by molecular docking and molecular dynamics simulations.
2. Materials and Methods 2.1 Materials Cell culture supplies were from Gibco (Invitrogen) and PAA Laboratories. MTT (3(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide), baicalein, camptothecin, and DCFH-DA (2′,7′-Dichlorodihydrofluorescein diacetate) were from Sigma. The annexin V-FITC and the FITC Active Caspase-3 apoptosis kit were purchased from BD Pharmingen. Nitrones, including PBN used as reference compound, were kindly supplied by Dr. Williams Porcal (Departamento de Química Orgánica, Facultad de Química, Universidad de la República, Uruguay).
2.2 Cell line 4
HT22 is a sub-line derived from HT4 neuronal cells that were originally immortalized from primary mouse hippocampus (Morimoto and Koshland 1990). Cells were kindly provided by Dr. David Schubert (The Salk Institute for Biological Studies, La Jolla, CA, USA). HT22 cells were maintained in DMEM high glucose medium supplemented with 10% FBS, 1% penicillin and streptomycin under humidified 5% CO2 atmosphere at 37 °C.
2.3 Cell viability Cell viability was evaluated using the MTT assay previously described (Mosmann, 1983). 5 × 103 cells/well were plated in 96-well plate in complete medium, and 24 h later, the increasing concentrations of compounds or vehicle and culture medium controls were added. Cells were incubated for additional 24 h and cell viability was assessed by measuring the reduction of MTT to formazan. All experiments were done in triplicate and repeated at least three times. The results are expressed as percentage of cell viability relative to the controls treated with DMSO 0.5%.
2.4 Apoptosis inhibition HT22 cells were plated into 12-well plates at 1.2 x 105 cells/well and 24 h later cells were pre-treated with the compounds for 2 h at non-cytotoxic concentration of 25 µM previously determined by the MTT assay. We used DMSO at 0.5% in culture medium as a negative control and baicalein at 50 µM as anti-apoptotic, anti-inflammatory and neuroprotective compound control (Sowndhararajan et al., 2017). After 2 h, apoptosis was induced using 5 µM camptothecin (Stefanis et al., 1999) and cells were incubated for additional 15 h and harvested by trypsinization. The assay was also performed in
absence of camptothecin cap to discard the pro-apoptotic potential of nitrones. This assay was performed by three independent experiments.
2.4.1 Annexin V-FITC/propidium iodine staining Cells were stained using FITC annexin V as recommended by the manufacturer. Briefly, cells were harvested, washed with PBS and after addition of annexin V-FITC (Vermes et al., 1995) in annexin buffer, cells were incubated for 15 min at room temperature in the dark. Previous to flow cytometry analysis propidium iodine was added.
2.4.2 Active caspase-3 inhibition To follow the intracellular active caspase-3 reduction, cells were fixed and permeabilized with Cytofix/Cytoperm solutions, washed with BD Perm/Wash buffer (1x), stained with FITC anti-active caspase-3 antibody and incubated for 30 min, according to the manufacturer’s protocol. Afterwards, cells were analyzed by flow cytometry. Experiments were performed by triplicates.
2.4.3 Acquisition and Statistical Analysis Samples were acquired using a FACS calibur cytometer collecting 10.000 events per sample. The results were normalized against camptothecin and are represented as the mean ± S.D. (Standard Deviation) from three independent experiments (n=3). Statistical significance was determined by one-way ANOVA followed by Dunnet test compared to camptothecin. P-values, P ≤ 0.01 ** P ≤ 0.001 *** P ≤ 0.0001 **** were regarded as statistically significant, and are indicated in the figures.
2.5. Anti-oxidant activity Cells were seeded into 35 mm plates at 150.000 cell/ml into 2 ml of complete culture medium and incubated for 24 h. Then, cells were treated with compounds at 25 µM, baicalein at 50 µM or DMSO. After 2 h, 250 µM of H2O2 was added to each plate and incubated for additional 1 h. Cultures were incubated with 10 µM of DCFH-DA in DMEM for 10 min at 37 ºC. Finally, cells were visualized using a Zeiss LSM800 confocal microscope, equipped with a 20X objective Plan Apo NA 0.5. Cellular fluorescence of 250 cells per condition was quantified using the ImageJ program (https://imagej.net/ImageJ). Statistical analysis was performed using one-way ANOVA.
2.6. Protein structures preparation Crystal structure of the human heterotetrameric active caspase-3 in complex with a nicotinic acid aldehyde inhibitor (PDB ID: 1RHM) was taken from the Protein Data Bank (http://www.rcsb.org/). Crystallographic waters and the inhibitor were eliminated. Due to some residues of substrate or ligand binding in the active site of caspase-3 present distinct rotameric states relative to the unliganded enzyme, molecular dynamics simulations were performed prior to docking calculations. Hydrogen atoms and six chloride (to neutralize charge) were added to caspase-3 using the leap utility from AmberTools14 package (Case et al., 2014). The enzyme was solvated in a truncated octahedral box of TIP3P water molecules (Jorgensen et al., 1983) extended 12 Å outside the protein on all sides. First, water and counterions were relaxed during 2000 steps (500 steepest descent steps, SD, and 1500 conjugate-gradient steps, CG) with the protein restrained with a force constant of 500 kcal/molÅ2. Subsequently, the system was minimized without restraints during 2000 steps (500 SD and 1500 CG). The cutoff distance for direct calculation of nonbonded interactions was set to 10 Å. Outside of this
distance, electrostatic interactions were calculated using the Particle-Mesh-Ewald method (Darden et al., 1993). Energy minimization was performed with the pmemd.MPI module of the AMBER 14 software suite (Case et al., 2014) using the ff03.r1 force field (Duan et al., 2003). Following minimization, the system was gently heated in a NVT ensemble from 0 to 300 K during 100 ps using the Langevin dynamic algorithm (Pastor et al., 1988). After this, 100 ns of NPT simulations at 300 K and 1 atm pressure using the Berendsen barostat were performed (Berendsen et al., 1984). The SHAKE algorithm was applied to fix all bond lengths involving hydrogen atoms (Ryckaert et al., 1977). The equations of motion were integrated with a time step of 2.0 fs and coordinates were saved every 10 ps. Molecular dynamics simulations were performed with the pmemd.cuda module in AMBER 14. Through cluster analysis using the hierarchical agglomerative algorithm with the average linkage method (Kelley et al., 1996), representative structures of caspase-3 from the stable part of the trajectory were obtained and used for subsequent docking calculations. Trajectories analyses were carried out using the cpptraj module in AmberTools14 and the VMD program was used for visualization (Humphrey et al., 1996).
2.7. Ligands preparation Previous to docking, compounds 1a, 2 and 4b (Fig. 1) were optimized at the ωB97XD/6-31+G(d,p) level (Chai and Head-Gordon, 2008; Krishnan et al., 1980) in water using the IEF-PCM continuum model (Tomasi et al., 1999) with Bondi atomic radii. These calculations were performed using the Gaussian09 software (Frisch et al., 2009).
2.8. Molecular docking 8
The representative structure of caspase-3 from molecular docking simulations (see Section 2.6) was employed as starting point for flexible-ligand docking of compounds 1a, 2 and 4b into the enzyme using the program AutoDock4.2 (Morris et al., 2009). Input files were prepared with AutoDockTools (Morris et al., 2009) using GasteigerMarsili (Gasteiger and Marsili, 1980) charges both for the ligands and the protein. A grid box of 126×126×126 points with a spacing of 0.5 Å between grid points was used in order to cover the entire protein surface. The box was centered on the macromolecule. For each ligand, a population size of 150 individuals and 2.5x106 energy evaluations were used for 50 independent search runs. Conformations varying by less than 2.0 Å in root-square deviation were clustered together. The conformation with the lowest binding energy from the most populated cluster was selected and the corresponding protein-ligand complex was used for future analyses.
2.9. Ligand-protein molecular dynamics Molecular docking simulations of compounds 1a, 2 and 4b complexed with caspase-3 were performed as described in Section 2.5. Ligands were treated with the General Amber Force Field (Wang et al., 2004) and charges were derived with the RESP fitting procedure (Wang et al., 2000) by a HF/6-31G* single point calculation on the previously optimized structures.
3. Results 3.1. Cell viability Initially, we determined the cytotoxicity of the nine nitrones shown in Fig. 1. PBN and baicalein were included as reference compounds. In order to define a non-cytotoxic concentration of nitrones to perform the next experiments, cells were incubated with
increasing concentrations of each compound. The results showed that most of these compounds did not affect cell viability in the range of 25-100 µM (Table 1). Based on these results, a 25 µM concentration was chosen to perform the anti-apoptotic studies.
% CELL VIABILITY COMPOUNDS
Table 1. Nitrones cytotoxicity determined by MTT assay
3.2. Apoptosis inhibition. The anti-apoptotic activity of nitrones at 25 µM was evaluated in HT22 cells. Results reveal that most compounds are able to inhibit apoptosis although the observed antiapoptotic behaviour varies among them (Fig. 2).
FIG. 2 SHOULD BE LISTED HERE
Nitrone derivatives 1a, 5 and 6 have a good anti-apoptotic activity, even better than PBN a well-known molecule with high anti-oxidant properties, causing a statistically significant 40% reduction in the number of apoptotic cells regarding camptothecin. Phenolic derivative 1b, quinoxalin derivative 3 and thiadiazole derivative 4c result inactives as apoptosis inhibitors. Undoubtedly, thiadiazole derivatives (4a and 4b) are the most potent compounds presenting percentages of apoptosis reduction about 60% which are similar to the positive control baicalein. In a likely way, furoxan derivative 2 is almost as good as thiadiazole derivatives.
3.3. Caspase-3 inhibition Compounds showing anti-apoptotic activity were evaluated to assess whether the antiapoptotic activity may be related to their capability of decreasing active caspase‐3 in cells. Results demonstrated that selected nitrones, as well as PBN and baicalein, reduce between 30 and 60% the active caspase‐3 levels in cells, except compound 2 which interestingly presents anti-apoptotic activity by a mechanism independent of active caspase-3 reduction (Fig. 3).
FIG. 3 SHOULD BE LISTED HERE
As in the previous experiment, thiadiazole derivatives 4a and 4b are the most effective compounds regarding their anti-apoptotic activity. It is worth mentioning that controls treated with nitrones without camptothecin stimulation were also included with the aim of evaluating the potential pro-apoptotic activity of the compounds, finding that nitrones were non pro-apoptotic per se (data non shown).
3.4. Anti-oxidant activity We further evaluated the capability of the nitrones to reduce reactive oxygen species in H2O2-treated HT22 cells. Fig. 4 indicates that pre-treatment with nitrones prior to the addition of H2O2 resulted in a statistically significant reduction of the intracellular reactive oxygen species. Except for compound 2, nitrones present an excellent antioxidant profile whose percentage of fluorescence intensity varies between 9-18%.
FIG. 4 SHOULD BE LISTED HERE
3.5. Molecular dynamics simulations In search of some clues about the mechanism in which nitrones reduce active caspase-3 we carry out molecular dynamics simulations of three different complexes combining caspase-3 with compounds 1a, 2 and 4b. Nitrones 1a and 4b were chosen for exhibiting good anti-apoptotic profile and causing one-half reduction in active caspase-3 whereas nitrone 2 was selected for being quite good as anti-apoptotic inhibitor but unable to decrease active caspase-3 in cells.
According to our computational studies caspase-3 binds two molecules of the corresponding nitrone in regions near the catalytic sites. When nitrones bind to the enzyme, loops involved in catalysis change their positions relative to the unliganded enzyme in order to accommodate the molecules. Through hydrogen bonds and hydrophobic interactions, nitrones were held in their binding sites throughout the entire simulation time. Fig. 5 shows that residues taking part of these interactions are from sites implicated in substrate binding and catalysis. 12
FIG. 5 SHOULD BE LISTED HERE
Binding of active nitrones 1a and 4b affects the overall dynamics of the macromolecule. In particular, loops L4, L2 and L2’ show a dramatic change in their relative positions regarding the unliganded enzyme. The correlated motion and appropriate positioning of those loops play an essential role during catalysis and help maintain the enzyme in an active conformation (Feeney et al., 2006). Compound 4b lies along the substrate binding cleft interacting with residues from all subsites including Phe256 from S5 and forms a hydrogen bond with Arg207 from S1 (Fig. 5A) whereas compound 1a appears to interact more weakly specially on one of the biding sites. This compound also forms hydrogen bonds with Ser205 and Gln161 from S1 (Fig. 5B). Finally for compound 2, one of the molecules leaves the binding site predicted by docking moving towards the surface of the enzyme after 50 ns where it remains feebly attached through a weak hydrogen bond with Lys224’ (Fig. 5C). In the other heterodimer, the nitrone remains bound to the site predicted by docking, and compared with active nitrones 4b and 1a, it establishes fewer interactions and could be easily removed from the enzyme.
Poisson Boltzmann electrostatic calculations carried out with the APBS package (Baker et al., 2001) and mapped onto the protein molecular surface evidences that loops rearrangement is accompanied by a clear change in the shape and charge distribution of the enzyme. In fact, mapped electrostatic potential of the caspase-3-4b complex after molecular dynamics simulations shows how catalytic sites are disturbed and charge distribution varies upon ligand binding. As depicted in Fig. 6 one of the catalytic clefts is completely distorted because of changes in loops conformation and becomes highly 13
enriched in positive charges. Regarding enzyme shape, ligand binding leads to an increase in caspase-3 globularity which also exhibits a smoother surface.
FIG. 6 SHOULD BE LISTED HERE
4. Discussion Uncontrolled apoptotic cell death is a hallmark of many neurodegenerative disorders. Thus, apoptosis modulation has been considered a beneficial therapeutic approach. Caspase-3 activation is an early event that triggers apoptotic damage, and overexpression of this enzyme is involved in the pathogenesis of amyotrophic lateral sclerosis, Alzheimer’s, Parkinson’s and Huntington’s diseases (D’Amelio et al., 2012). Therefore, a precise downregulation or modulation of caspase-3 activity during neuropathological processes could help to prevent and/or reduce the progression of these diseases. In this way, the use of reversible caspase-3 inhibitors emerge as an interesting pharmacological therapy.
Caspase-3, a cysteine-aspartic acid protease, exists as an inactive pro-enzyme that undergoes proteolytic processing at conserved aspartic residues to produce the active enzyme. All caspases in their active form are heterotetramers composed of dimers containing two small and two large subunits. They present two active sites at the interface between heterodimers, which consist of a catalytic Cys-His dyad (Fig. 7A) and an extensive pocket formed by four loops (L1-L4) where the amino acids belonging to the S1-S5 sites are able to accommodate P1-P5 residues of the substrate in an extended conformation (Fig. 7B) (Crawford and Wells 2011).
FIG. 7 SHOULD BE LISTED HERE
Loops L1-L4 have an active role in enzyme activation and catalysis. In fact, interactions between loops L2 and L4 with loop L2’ stabilize the active conformation (Feeney et al., 2006; Rotonda et al., 1996). A similar hydrophobic S5 site for caspase-3 and -2 has been described, while caspases 1, 7, 8 and 9 do not possess structurally equivalent hydrophobic residues at this subsite, causing a different selectivity for the recognition of the P5 position in substrates (Fang et al., 2006). This is a very important feature to be consider before starting novel initiatives about rational design of caspases selective inhibitors.
In the present work, we determined the anti-apoptotic and anti-oxidant effects of nine nitrone derivatives in HT22 mouse hippocampal cells. Initial screening to test the capability of the nitrones under study of inhibiting apoptosis showed that most of the evaluated compounds have good anti-apoptotic properties. Although it was previously reported that all the evaluated nitrones act as free radical scavengers, with good antioxidant activity in vitro and neuroprotective effects (Barriga et al., 2010; Chavarría et al., 2012; Porcal et al. 2008) these features are not enough to explain their anti-apoptotic activity. In the experiments using annexin-V compounds 1b, 3 and 4c were unable to reduce the percentage of apoptotic HT22 cells. In order to get a deeper insight upon the mechanisms mediating apoptosis inhibition, nitrones from each derivative group showing the best anti-apoptotic profile were selected to assay their potential to inhibit apoptosis by reducing the intracellular active caspase-3 and reactive oxygen species levels. Our results highlight that nitrone derivatives (PBN, 1a, 4a, 4b, 5 and 6) confer robust reactive oxygen species protection and reduce between 30 and 60% active
caspase-3 levels. Hence, these findings indicate that these nitrones confer neuroprotection in HT22 cells against oxidative stress and inhibited apoptosis via caspase-3. On the other hand, the furoxan derivative 2 did not reduce active caspase-3, although good anti-oxidant activity was shown, suggesting this as a possible antiapoptotic mechanism of action. From the novel synthetized nitrones the 1,2,4thiadiazolyl derivatives 4a and 4b were the most effective of them in reducing active caspase-3 levels. It is worth mentioning that heterocyclic compounds containing the 1,2,4-thiadiazole system have been considered of great interest in medicinal chemistry due to the high therapeutic potential and versatility of this system (Castro et al., 2006). Like the 1,2,4-thiadiazole system, benzofuroxan and furan chemotypes are widely used in organic chemistry and have been extensively studied as bioactive compounds (Cerecetto and Porcal 2005; Dall’lgna et al., 2003; Loğoğlu et al., 2010). Also, hydroxyphenyl nitrones as well as PBN have been reported to display good antioxidant, neuroprotective and anti-inflammatory profiles (Barriga et al., 2010; Porcal et al., 2008).
In order to confirm one of our hypothesis about the potential of active nitrones 1a and 4b to bind active caspase-3 and to understand the differential behavior of the inactive derivative 2, docking and molecular dynamics simulations were performed. According to our results, it is expected that nitrones binding affects the substrate processing since a proper arrangement of catalytic loops is key for substrate recognition and positioning. This would be indicating that these compounds could act as effective inhibitors of caspase-3. It is worth mentioning that the binding mode of each nitrone molecule and the conformation of residues lining the corresponding binding site differ from one active site to the other. This interesting finding leads to the conclusion that nitrones
interact differentially on both catalytic sites because they are not identical as commonly assumed. In fact, a thorough research carried out in our group has shown that notorious differences regarding the shape and charge distribution exist between both catalytic sites (Minini et al., 2017). The binding modes predicted by molecular docking for compounds 4b, 1a and 2 were unable to explain the inability of 2 to decrease active caspase-3 in cells. However, during molecular dynamics simulations it was observed that one of the molecules 2 leaves the initial binding site remaining barely attached to the protein surface. In the other heterodimer, although the compound remains bound to the site predicted by docking it establishes minor interactions and could be easily removed toward the solvent by the intrinsic dynamics of the enzyme. Interestingly, analyzing the Root Mean Square Fluctuations (R.M.S.F., a measure of the average atomic mobility of backbone atoms (Cα) during the molecular dynamics simulations) of the caspase-3-2 complex, a clear increase in the flexibility of the enzyme was evidenced (data not shown). This rise in enzyme mobility might not favor a strong and stable interaction of nitrone 2. As a consequence, this compound would not reside enough time bound to the enzyme to cause significant shape or charges alterations or even affect substrate binding.
Conclusions Our study highlights the anti-apoptotic and anti-oxidant capability of novel nitrones and their potential to act as caspase-3 inhibitors. Since these compounds are non-cytotoxic, have free radical scavenger capability and are able to cross the blood brain barrier they can be considered excellent molecular hits. In this sense, nitrones containing the thiadiazole moiety 4a and 4b appear to be the most promising compounds due to their excellent anti-apoptotic and anti-oxidant properties. Despite complementary studies 17
need to be done, results here presented here shed light into the molecular basis behind the neuroprotective effects of nitrones. Therefore, nitrones are promising candidates for future drug design against neurodegenerative diseases.
Credit Author Statement
Saira Cancela: Methodology, Validation, Formal analysis, Writing- Original draft preparation and Visualization. Lucía Canclini: Methodology, Writing - Review & Editing and Visualization. Gustavo Mourglia-Ettlin: Methodology and Writing Review & Editing. Paola Hernández: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Writing- Original draft preparation and Visualization. Alicia Merlino: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Resources, Writing- Original draft preparation, Supervision, Project administration and Funding acquisition.
Acknowledgements This work was supported by Comisión Sectorial de Investigación Científica (CSICProyecto I+D 2014, ID 294) and PEDECIBA-Química, Uruguay. LC and GM are members of the National System of Researchers (SNI-ANII, Uruguay) whose support is also gratefully acknowledged. We also thank ANII for the scholarship to SC (FOR.INS.060). The authors are sincerely grateful to Dr. Williams Porcal for providing the nitrones evaluated throughout this study and to Dr. David Schubert for providing the HT-22 cell line. Finally, authors kindly thank to María Varela for English editing.
References Baker, N.A., Sept, D., Joseph, S., Holst, M.J., McCammon, J.A., 2001. Electrostatics of nanosystems: application to microtubules and the ribosome. PNAS. 98, 10037-10041. https://doi.org/10.1073/pnas.181342398 Barriga, G., Olea-Azar, C., Norambuena, E., Castro, A., Porcal, W., Gerpe, A., González, M., Cerecetto, H., 2010. New heteroaryl nitrones with spin trap properties: Identification of a 4-furoxanyl derivative with excellent properties to be used in biological systems, Bioorg. Med. Chem. 18, 795–802. https://doi.org/10.1016/j.bmc.2009.11.053 Berendsen, H.J.C., Postma, J.P.M., Van Gunsteren, W.F., DiNola, A., Haak, J.R., 1984. Molecular dynamics with coupling to an external bath. J. Chem. Phys. 81, 3584-3690. https://doi.org/10.1063/1.448118 Carney, J.M. and Floyd R.A., 1991. Protection against oxidative damage to CNS by alpha-phenyl-tert-butyl nitrone (PBN) and other spin-trapping agents: a novel series of nonlipid free radical scavengers. J Mol Neurosci. 3, 47-57. https://doi.org/10.1007/BF02896848 Case, D.A., Babin, V., Berryman, J.T., Betz, R.M., Cai, Q., Cerutti, D.S., Cheatham,T.E., III, Darden, T.A., Duke, R.E., Gohlke, H., Goetz, A.W., Gusarov, S., Homeyer, N., Janowski, P., Kaus, J., Kolossváry, I., Kovalenko, A., Lee, T.S., LeGrand, S., Luchko, T., Luo, R., Madej, B., Merz, K.M., Paesani, F., Roe, D.R., Roitberg, A., Sagui, C., Salomon-Ferrer, R., Seabra, G., Simmerling, C.L., Smith, W., Swails, J., Walker, R.C., Wang, J., Wolf, R.M., Wu, X., Kollman, P.A., 2014. AMBER 14, University of California, San Francisco.
Castro, A., Castaño, T., Encinas, A., Porcal, W., Gil, C., 2006. Advances in the synthesis and recent therapeutic applications of 1,2,4-thiadiazole heterocycles. Bioorg. Med. Chem. 14, 1644-1652. https://doi.org/10.1016/j.bmc.2005.10.012 Cerecetto, H., Porcal, W., 2005. Pharmacological Properties of Furoxans and Benzofuroxans: Recent Developments. Mini-Rev. Med. Chem. 5, 57-71. https://doi.org/10.2174/1389557053402864 Chai, J.D., Head-Gordon, M., 2008. Long-range corrected hybrid density functionals with dumped atom-atom dispersion corrections. Phys. Chem. Chem. Phys. 10, 66156620. https://doi.org/10.1039/b810189b Chavarría, C., Pérez, D.I., Pérez, C., Morales, J.A., Alonso, S., Pérez, A., Gil, C., Souza, J.M., Porcal, W., 2012. Microwave-assisted synthesis of hydroxyphenyl nitrones with protective action against oxidative stress, Eur. J. Med. Chem. 58, 44-49. https://doi.org/10.1016/j.ejmech.2012.09.044 Crawford, E.D., Wells, J.A., 2011. Caspase substrates and cellular remodeling. Annu. Rev. Biochem. 80, 1055-1087. https://doi.org/10.1146/annurev-biochem-061809121639 Dall’lgna, O.P., Porciúncula, L.O., Souza, D.O., Cunha, R.A., Lara, D.R., 2003. Neuroprotection by caffeine and adenosine A2A receptor blockade of β-amyloid neurotoxicity. Br. J. Pharmacol. 138, 1207-1209. https://doi.org/10.1038/sj.bjp.0705185 D’Amelio, M., Sheng, M., Cecconi, F., 2012. Caspase-3 in the central nervous system: beyond apoptosis, Trends Neurosci. 35, 700-709. https://doi.org/10.1016/j.tins.2012.06.004 Darden, T., York, D., Pedersen, L., 1993. Particle mesh Ewald: An N⋅ log (N) method for Ewald sums in large systems. J. Chem. Phys. 98, 10089-10092. https://doi.org/10.1063/1.464397 20
Das, A., Gopalakrishnan, B., Voss, O.H., Doseff, A.I., Villamena, F.A., 2012. Inhibition of ROS-induced apoptosis in endothelial cells by nitrone spin traps via induction of phase II enzymes and suppression of mitochondria-dependent pro-apoptotic signalling, Biochem. Pharmacol. 84, 486-497. https://doi.org/10.1016/j.bcp.2012.04.021 Duan, Y., Wu, C., Chowdhury, S., Lee, M.C., Xiong, G., Zhang, W., Yang, R., Cieplak, P., Luo, R., Lee, T., Caldwell, J., Wang, J., Kollman, P., 2003. A point-charge force field for molecular mechanics simulations of proteins based on condensed-phase quantum mechanical calculations. J. Comput. Chem. 24, 1999-2012. https://doi.org/10.1002/jcc.10349 Fang, B., Boross, P.I., Tozser, J., Weber, I.T., 2006. Structural and kinetic analysis of caspase-3 reveals role for S5 binding site in substrate recognition. J. Mol. Biol. 360, 654-666. https://doi.org/10.1016/j.jmb.2006.05.041 Feeney, B., Pop, C., Swartz, P., Mattos, C., Clark, A.C., 2006. Role of loop bundle hydrogen bonds in the maturation and activity of (Pro)caspase-3. Biochemistry. 45, 13249-13263. https://doi.org/10.1021/bi0611964 Frisch, M.J., Trucks, G.W., Schlegel, H.B., Scuseria, G.E., Robb, M.A., Cheeseman, J.R., Scalmani, G., Barone, V., Mennucci, V., Petersson, G.A., 2009. Gaussian 09, Revision A.02, Gaussian, Inc., Wallingford CT. Gasteiger, J., Marsili, M., 1980. Iterative partial equalization of orbital electronegativity – A rapid access to atomic charges. Tetrahedron. 36, 3219-3228. https://doi.org/10.1016/0040-4020(80)80168-2 Humphrey, W., Dalke, A., Schulten, K., 1996. VMD - Visual Molecular Dynamics. J. Mol. Graph. 14, 33-38. https://doi.org/10.1016/0263-7855(96)00018-5
Jorgensen, W.L.; Chandrasekhar, J., Madura, J.D., Impey, R.W., Klein, M.L., 1983. Comparison of simple potential functions for simulating liquid water, J. Chem. Phys. 79, 926-935. https://doi.org/10.1063/1.445869 Kelley, L.A., Gardner, S.P., Sutcliffe, M.J., 1996. An automated approach for clustering an ensemble of NMR-derived protein structures into conformationally related subfamilies. Protein Eng. 9, 1063-1065. https://doi.org/10.1093/protein/9.11.1063 Krishnan, R., Binkley, J.S., Seeger, R., Pople, J.A., 1980. Self-Consistent Molecular Orbital Methods. Basis set for correlated wave-functions. J. Chem. Phys. 72, 650-654. https://doi.org/10.1063/1.438955 Loğoğlu, E., Yilmaz, M., Katircioğlu, H., Yakut, M., Mercan, S., 2010. Synthesis and biological activity studies of furan derivatives. Med. Chem. Res. 19, 490-497. https://doi.org/10.1007/s00044-009-9206-8 MacKenzie, S.H., Schipper, J.L., Clark, A.C., 2010. The potential for caspases in drug discovery, Curr. Opin. Drug Discov. Devel. 13, 568-576. Minini, L., Ferraro, F., Cancela, S., Merlino, A., 2017. Insight into the mechanism of action and selectivity of caspase-3 reversible inhibitors through in silico studies. J. Molec. Struct. 1147, 558-568. https://doi.org/10.1016/j.molstruc.2017.06.118 Morimoto, B.H., Koshland, D.E., 1990. Induction and expression of long- and shortterm neurosecretory potentiation in a neural cell line, Neuron. 5, 875-880. https://doi.org/10.1016/0896-6273(90)90347-i Morris, G,M., Huey, R., Lindstrom, W., Sanner, M.F., Belew, R.K., Goodsell, D.S., Olson, A.J., 2009. AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility. J. Comput. Chem. 30, 2785-2791. https://doi.org/10.1002/jcc.21256.
Mosmann, T.J., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays, Immunol. Methods. 65, 55 - 63. https://doi.org/10.1016/0022-1759(83)90303-4 Pastor, R.W., Brooks, B.R., Szabo, A., 1988. An analysis of the accuracy of Langevin and molecular dynamics algorithms. Mol. Phys. 65, 1409-1419. https://doi.org/10.1080/00268978800101881 Poewe, W., Seppi, K., Tanner, C.M., Halliday, G.M., Brundin, P., Volkmann, J., Schrag, A.E., Lang, A.E., 2017. Parkinson disease. Nat Rev Dis Primers. 3, 17013. https://doi.org/10.1038/nrdp.2017.13. Porcal, W., Hernández, P., González, M., Ferreira, A., Olea-Azar, C., Cerecetto, H., Castro, A., 2008. Heteroarylnitrones as drugs for neurodegenerative diseases: synthesis, neuroprotective properties, and free radical scavenger properties, J. Med. Chem. 51, 6150-6159. https://doi.org/10.1021/jm8006432 Porȩba, M., Stróżyk, A. Salvesen, G.S., Drag, M., 2013. Caspase substrates and inhibitors, Cold Spring Harb. Perspect. Biol. 5, a008680. https://doi.org/10.1101/cshperspect.a008680 Rotonda, J., Nicholson, D.W., Fazil, K.M., Gallant, M., Gareau, Y., Labelle, M., Peterson, E.P., Rasper, D.M., Ruel, R., Vaillancourt, J.P., Thornberry, N.A., Becker J.W., 1996. The three-dimensional structure of apopain/CPP32, a key mediator of apoptosis. Nat. Struct. Biol. 3, 619-625. https://doi.org/10.1038/nsb0796-619 Ryckaert, J.P., Ciccotti, G., Berendsen, H.J.C., 1977. Numerical integration of cartesian equations of motion of a system with constraints -molecular-dynamics of N-alkanes. J. Comput. Phys. 23, 327-341. https://doi.org/10.1016/0021-9991(77)90098-5
Sowndhararajan, K., Deepa, P., Kim, M., Park, S.J., Kim, S., 2017. Baicalein as a potent neuroprotective agent: A review, Biomed. Pharmacoth. 95, 1021-1032. https://doi.org/10.1016/j.biopha.2017.08.135 Stefanis, L., Park, D.S., Friedman, W.J., Greene, L.A. 1999. Caspase-dependent and independent death of camptothecin-treated embryonic cortical neurons. J. Neurosci. 19, 6235-6247. https://doi.org/10.1523/JNEUROSCI.19-15-06235.1999 Tomasi, J., Mennucci, B., Cancès, E., 1999. The IEF version of the PCM solvation method: an overview of a new method addressed to study molecular solutes at the QM ab initio level. J. Molec. Struct. 464, 211-226. https://doi.org/10.1016/S01661280(98)00553-3 Vermes, I., Haanen, C., Steffens-Nakken, H., Reutelingsperger, C., 1995. A novel assay for apoptosis. flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J Immunol Methods. 184,39-51. https://doi.org/10.1016/0022-1759(95)00072-i World Alzheimer Report 2019. https://www.alz.co.uk/research/WorldAlzheimerReport2019.pdf, Accessed date: 31 October 2019). Wang, J., Cieplak, P., Kollman, P.A., 2000. How well does a restrained electrostatic potential (RESP) model perform in calculating conformational energies of organic and biological molecules? J. Comput. Chem. 21, 1049-1074. https://doi.org/10.1002/1096987X(200009)21:12<1049::AID-JCC3>3.0.CO;2-F Wang, J., Wolf, R.M., Caldwell, J.W., Kollman, P.A., Case, D.A., 2004. Development and Testing of a General Amber Force Field. J. Comput. Chem. 25, 1157-1174. https://doi.org/10.1002/jcc.20035
Wu, L., Lu, M., Yan, Z., Tang, X., Sun, B., Liu, W., Zhou, H., Yang, C., 2014. 1,2Benzisothiazol-3-one derivatives as a novel class of small-molecule caspase-3 inhibitors. Bioorg. Med. Chem. 22, 2416-2426. https://doi.org/10.1016/j.bmc.2014.03.002
Fig. 1. Chemical structure of the (Z)-α-aryl and heteroaryl N-alkyl or N-benzyl-nitrones under study.
Fig. 2. Inhibitory effects of nitrones (25 µM) on camtothecin-induced apoptotic HT22 cells stained with annexin V-FITC. Baicalein (BCL, 50 µM) was included as positive control. The results were normalized against the negative control DMSO and are expressed as the mean ± S.D., P ≤ 0.0001 ****, one-way ANOVA followed by Dunnet test compared to camptothecin. The graph represents three independent experiments.
Fig. 3. Effect of nitrones (25 µM) on active caspase-3 levels on camptothecin-induced apoptotic HT22 cells stained with anti-active caspase-3-FITC antibody. Baicalein (BCL, 50 µM) was included as positive control. The results were normalized against camptothecin and are expressed as the mean ± S.D., P ≤ 0.01 **, P ≤ 0.001 ***, P ≤ 0.0001 ****, one-way ANOVA followed by Dunnet test compared to camptothecin. The graph represents three independent experiments.
Fig. 4. Effect of nitrones on H2O2-induced reactive oxygen species in HT22 cells. Fluorescence intensity plot of cells (n=250) treated with H2O2 (250 µM) in absence or 25
presence of nitrones (25 µM) or baicalein (BCL, 50 µM) as positive control. Data are represented as the mean ± S.D., **** P ≤ 0.0001. Representative confocal images of DCF-fluorescence are shown.
Fig. 5. Superimposed structures of unliganded caspase-3 and caspase-3 in complex with nitrones (left). (A) caspase-3-4b, (B) capase-3-1a and (C) caspase-3-2 after 100 ns molecular dynamics simulations. Loops of unbound caspase-3 are shown as partially transparent. Active site loops L1 (orange), L2 (purple), L3 (green), L4 (red), L2’ (yellow). 2D interaction diagrams between caspase-3 and nitrones for each binding site (right). Residues accepting and donating hydrogen bonds are depicted in green colored. Hydrogen bonds are shown as green dash-lines, while curved lines indicate hydrophobic interactions. Nitrones are coloured by atom type.
Fig. 6. Surfaces of unliganded caspasase-3 (A) and caspase-3 in complex with nitrone 4b (B) colored according to the electrostatic potential. Red represents negative electrostatic potential and blue represents positive electrostatic potential.
Fig. 7. Caspase-3 (PDB ID 1RHM) structure. (A) Caspase-3 heterotetramer. Catalytic dyad is shown as balls (violet). Active site loops L1 (red), L2 (purple), L3 (orange), L4 (green), L2’ (cyan). (B) Amino acids belonging to S1-S5 sites formed by loops L1-L4. S1 (violet), S2 (blue), S3 (green), S4 (orange), S5 (yellow).
Table captions Table 1. Nitrones cytotoxicity (0-200 µM) determined by MTT assay.
Credit Author Statement
Saira Cancela: Methodology, Validation, Formal analysis, Writing- Original draft preparation and Visualization. Lucía Canclini: Methodology, Writing - Review & Editing and Visualization. Gustavo Mourglia-Ettlin: Methodology and Writing - Review & Editing. Paola Hernández: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Writing- Original draft preparation and Visualization. Alicia Merlino: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Resources, Writing- Original draft preparation, Supervision, Project administration and Funding acquisition.