Biological evaluation of naproxen–dehydrodipeptide conjugates with self-hydrogelation capacity as dual LOX/COX inhibitors

pharmaceutics Article Biological Evaluation of Naproxen–Dehydrodipeptide Conjugates with Self-Hydrogelation Capacity as Dual LOX/COX Inhibitors Rute Moreira 1,†, Peter J. Jervis 2,*,†, AndréCarvalho 2, Paula M. T. Ferreira 2, JoséA. Martins 2, Patrícia Valentão1, Paula B. Andrade 1and David M. Pereira 1,* 1REQUIMTE/LAQV, Laboratório de Farmacognosia, Departamento de Química, Faculdade de Farmácia, Universidade do Porto, R. Jorge Viterbo Ferreira, n 228, 4050-313 Porto, Portugal; rutemartinsmor[email protected] (R.M.); [email protected] (P.V.); [email protected] (P.B.A.) 2Centre of Chemistry, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal; [email protected] (A.C.); [email protected] (P.M.T.F.); [email protected] (J.A.M.) *Correspondence: [email protected] (P.J.J.); [email protected] (D.M.P.) †These authors contributed equally to this work. Received: 31 December 2019; Accepted: 29 January 2020; Published: 3 February 2020   Abstract: The use of peptide–drug conjugates is emerging as a powerful strategy for targeted drug delivery. Previously, we have found that peptides conjugated to a non-steroidal anti-inflammatory drug(NSAID), morespecificallynaproxen–dehydrodipeptideconjugates, readily formnanostructured fibrilar supramolecular hydrogels. These hydrogels were revealed as efficacious nano-carriers for drug delivery applications. Moreover, the incorporation of superparamagnetic iron oxide nanoparticles (SPIONs) rendered the hydrogels responsive to external magnetic fields, undergoing gel-to-solution phase transition upon remote magnetic excitation. Thus, magnetic dehydrodipeptide-based hydrogels may find interesting applications as responsive Magnetic Resonance Imaging (MRI) contrast agents and for magnetic hyperthermia-triggered drug-release applications. Supramolecular hydrogels where the hydrogelator molecule is endowed with intrinsic pharmacological properties can potentially fulfill a dual function in drug delivery systems as (passive) nanocariers for incorporated drugs and as active drugs themselves. In this present study, we investigated the pharmacological activities of a panel of naproxen–dehydrodipeptide conjugates, previously studied for their hydrogelation ability and as nanocarriers for drug-delivery applications. A focused library of dehydrodipeptides, containing N-terminal canonical amino acids (Phe, Tyr, Trp, Ala, Asp, Lys, Met) N-capped with naproxen and linked to a C-terminal dehydroaminoacid ( ∆ Phe, ∆ Abu), were evaluated for their anti-inflammatory and anti-cancer activities, as well as for their cytotoxicity to non-cancer cells, using a variety of enzymatic and cellular assays. All compounds except one were able to significantly inhibit lipoxygenase (LOX) enzyme at a similar level to naproxen. One of the compounds 4 was able to inhibit the cyclooxygenase-2 (COX-2) to a greater extent than naproxen, without inhibiting cyclooxygenase-1 (COX-1), and therefore is a potential lead in the search for selective COX-2 inhibitors. This hydrogelator is a potential candidate for dual COX/LOX inhibition as an optimised strategy for treating inflammatory conditions. Keywords: anti-inflammatory; hydrogel; dehydrodipeptide; cyclooxygenase; lipoxygenase; cancer; proteasome Pharmaceutics 2020,12, 122; doi:10.3390/pharmaceutics12020122 www.mdpi.com/journal/pharmaceutics Pharmaceutics 2020,12, 122 2 of 18 1. Introduction Inflammatory diseases affect millions of people all over the world, having severe consequences on their quality of life. Nowadays, there are two types of anti-inflammatory drugs used in therapeutics: steroidal anti-inflammatory drugs and non-steroidal anti-inflammatory drugs (NSAIDs). The challenge of drug delivery is to transport enough drug molecules to the target sites, whilst minimizing adverse effects in healthy and non-target tissues. Two main approaches in this field have been exploited: (1) the use of a delivery vehicle, such as nanoparticles [ 1 – 6 ], and (2) the covalent modification of a drug with a small moiety, such as peptide–drug conjugates [ 7 – 9 ]. Peptide–drug conjugates are a conventional class of therapeutic agent that are formed through covalent attachment of specific peptide sequences to established drugs via suitable linker and linkage strategies. The conjugation of peptide epitopes of cell receptors to drug molecules allows the targeted delivery of drugs to specific cells and tissues. The vehicle peptide ideally should not detrimentally affect the pharmacological properties of the conjugate. Alternatively, when the conjugate does not retain the pharmacological properties of the drug, it is necessary to ensure that the drug molecule is released at the therapeutic site via a specific stimulus, generally the enzymatic cleavage of the connecting linker. This release step adds an extra level of specificity and safety to the targeted drug-delivery system. In other cases, the peptide endows the conjugate with self-assembly properties that result in improved drug efficacy, e.g., enhanced enzymatic stability of the aggregates or drug nanostructures [7]. The local administration of drugs that can act directly at the disease site presents many advantages over systemic delivery, such as increased bioavailability, reduced off-target and adverse effects, and lower cost. The COX enzymes initiate the arachidonic acid metabolic cascade, leading to the formation of pro-inflammatory prostaglandins and thromboxanes. COX inhibitors are good candidates for treating acute and chronic pain through topical application, since it is well known that their prolonged systemic use can produce several side effects, e.g., gastrointestinal, blood clotting and kidney issues, arising from COX-1 inhibition, and cardiovascular problems, arising from COX-2 inhibition [ 10 , 11 ]. Thus, there is a need to modulate the selectivity of these drugs according to their target and to minimize the use of anti-inflammatory drugs through systemic delivery [12]. Hydrogels have emerged in recent years as promising carriers for drug delivery applications owing totheireasypreparation, compliancebythepatient, andtheirbiocompatibilityandbiodegradability[ 13 ]. Hydrogels made of biodegradable polymers have frequently served as carriers to encapsulate therapeutic agents, allowing their controlled release [ 14 ]. Drug release can be tuned by adjustment of the pore sizes, incorporation of microand nanoparticles, or through the cleavage of covalent or non-covalent bonds [ 15 ]. Drug molecules can also be incorporated into supramolecular nanostructured hydrogelsthroughnon-covalentinteractions. Thereversiblenatureoftheweaknoncovalentinteractions ensures sustained drug delivery and hydrogel’s responsiveness to environmental stimuli [16]. The main limitation of supramolecular hydrogels is the proteolytic sensitivity of the peptide hydrogelator molecules by endogenous proteases [ 17 ]. One of the strategies reported to overcome this limitation is to replace canonical amino acids with non-proteinogenic analogues, such as D-amino acids, β -amino acids or dehydroamino acids [ 18 ]. Conjugates of naproxen with peptides containing D-amino acids are efficacious hydrogelators with resistance to proteolysis. Importantly, the D-amino acid conjugates display enhanced selectivity for the COX-2 isozyme in relation to the unconjugated drug naproxen, a non-selective inhibitor of both COX isozymes [12]. Dehydroamino acid residues are commonly encountered in drug discovery, with plinabulin, thiostrepton, imipenem and cilastatin being medicinally important molecules [ 19 – 21 ]. In our laboratory, we have investigated peptides containing dehydroamino acids (dehydrophenylalanine- ∆ Phe, dehydroalanine- ∆ Ala, dehydroaminobutyric acid- ∆ Abu) as alternatives to D-amino acids, not only to provide proteolytic stability but also for the reduced conformational flexibility of the peptide backbone. We have recently reported that dehydrodipeptides capped on the N-terminus with naproxen self-assemble into nanostructured hydrogels [ 18 , 22 ]. These peptide conjugates were found to be resistant to proteolysis by chymotrypsin, whereas the corresponding canonical dipeptide–naproxen Pharmaceutics 2020,12, 122 3 of 18 conjugates undergo proteolysis readily under the same conditions. We have also shown that naproxen N-capped dehydrodipeptides, further conjugated with known peptide bioepitopes, such as GRDGD, can also produce hydrogels [ 17 ]. Furthermore, we have been able to incorporate drug molecules into dehydropeptide-based hydrogels and demonstrated their sustained drug-delivery properties, thus identifying this type of supramolecular hydrogel as potential nano-carriers in drug delivery systems [ 17 ]. Superparamagnetic iron oxide nanoparticles (SPIONs) can also be incorporated into the dehydrodipeptide-based hydrogel networks, providing concentration-dependent T2-MRI contrast enhancement. Upon magnetic excitation, the SPIONs generate heat, which causes the hydrogel to undergo a gel-to-solution phase transition. This means that magnetic hyperthermia can potentially be used as a remote trigger for the temporally and spatially controlled release of contrast agents [23]. While our previous work has focused on the ability of the hydrogels to act as delivery agents for incorporated drugs or diagnostic agents, we have also been keen to investigate the potential of naproxen–dehydrodipeptide conjugates as therapeutic agents by themselves, with obvious added potential for highly targeted topical applications as hydrogels (Figure 1). Usually, drug–peptide conjugates require enzymatic proteolysis to release the active drug from the peptide vehicle. Interestingly, the proteolytic stability imparted by the dehydroamino acid residue presumably allows the naproxen–dehydrodipeptide conjugates to combine the anti-inflammatory properties of naproxen with theadministrative benefits ofagel, thus merging structuralfunction with intrinsicpharmacological activity. Alternatively, the conjugates could display novel pharmacological properties not associated with the naproxen moiety. In a preliminary study [ 23 ], compounds 1 and 2 (Figure 2) showed a modest ability to inhibit lipopolysaccharide (LPS)-induced production of nitric oxide ( • NO) radicals (a biomarker for inflammation) in RAW 264.7 cells. These compounds were also tested in enzymatic assays for their ability to inhibit key enzymes in the inflammatory cascade. Compounds 1 and 2 displayed modest levels of COX inhibition (with a low selectivity towards COX-2 inhibition) at 25 µ M and high levels of lipoxygenase (LOX) inhibition at 100 µM. Pharmaceutics 2020, 12, 3 of 18 naproxen conjugates undergo proteolysis readily under the same conditions. We have also shown that naproxen N-capped dehydrodipeptides, further conjugated with known peptide bioepitopes, such as GRDGD, can also produce hydrogels [17]. Furthermore, we have been able to incorporate drug molecules into dehydropeptide-based hydrogels and demonstrated their sustained drugdelivery properties, thus identifying this type of supramolecular hydrogel as potential nano-carriers in drug delivery systems [17]. Superparamagnetic iron oxide nanoparticles (SPIONs) can also be incorporated into the dehydrodipeptide-based hydrogel networks, providing concentrationdependent T2-MRI contrast enhancement. Upon magnetic excitation, the SPIONs generate heat, which causes the hydrogel to undergo a gel-to-solution phase transition. This means that magnetic hyperthermia can potentially be used as a remote trigger for the temporally and spatially controlled release of contrast agents [23]. While our previous work has focused on the ability of the hydrogels to act as delivery agents for incorporated drugs or diagnostic agents, we have also been keen to investigate the potential of naproxen–dehydrodipeptide conjugates as therapeutic agents by themselves, with obvious added potential for highly targeted topical applications as hydrogels. Usually, drug–peptide conjugates require enzymatic proteolysis to release the active drug from the peptide vehicle. Interestingly, the proteolytic stability imparted by the dehydroamino acid residue presumably allows the naproxen– dehydrodipeptide conjugates to combine the anti-inflammatory properties of naproxen with the administrative benefits of a gel, thus merging structural function with intrinsic pharmacological activity. Alternatively, the conjugates could display novel pharmacological properties not associated with the naproxen moiety. In a preliminary study [23], compounds 1 and 2 (Figure 2) showed a modest ability to inhibit lipopolysaccharide (LPS)-induced production of nitric oxide (•NO) radicals (a biomarker for inflammation) in RAW 264.7 cells. These compounds were also tested in enzymatic assays for their ability to inhibit key enzymes in the inflammatory cascade. Compounds 1 and 2 displayed modest levels of COX inhibition (with a low selectivity towards COX-2 inhibition) at 25 µM and high levels of lipoxygenase (LOX) inhibition at 100 µM. These initial results prompted us to further explore the structure–activity relationship (SAR) around the naproxen–dehydrodipeptide molecular scaffold (Figure 1) and conduct a detailed investigation of the anti-inflammatory, anti-cancer and cytotoxicity properties of a focused library of naproxen–dehydrodipeptides (Figure 2). Figure 1. General structure of the hydrogelators to be studied. 2. Materials and Methods 2.1. Standards and Reagents Dimethyl sulfoxide (DMSO), ethanol and 2-propanol were obtained from Fischer Scientific (Loughborough, UK). Naproxen, quercetin, sulphanilamide, N-(1-naphthyl)ethylenediamine, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), LPS of Salmonella enterica, linoleic acid, LOX from glycine max (soybean) and trypan blue were obtained from Sigma-Aldrich (St. Louis, MO, USA). COX fluorescent inhibitor screening assay kit was purchased from Cayman chemicals (Ann Arbor, MI, USA). Dulbecco’s Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), heat inactivated foetal bovine serum (FBS), Pen-Strep solution (penicillin 5000 units mL−1 and streptomycin 5000 mg mL−1) and trypsin were obtained from Gibco Invitrogen TM (Grand Island, NY, USA). Proteasome 20S, lactacystin and Suc-Leu-Leu-Val-Tyr-AMC were purchased from Enzo Life Sciences. Proteasome 26S was obtained from AGS cells, as described below. Figure 1. General structure of the hydrogelators to be studied. These initial results prompted us to further explore the structure–activity relationship (SAR) around the naproxen–dehydrodipeptide molecular scaffold (Figure 1) and conduct a detailed investigation of the anti-inflammatory, anti-cancer and cytotoxicity properties of a focused library of naproxen–dehydrodipeptides (Figure 2). 2. Materials and Methods 2.1. Standards and Reagents Dimethyl sulfoxide (DMSO), ethanol and 2-propanol were obtained from Fischer Scientific (Loughborough, UK). Naproxen, quercetin, sulphanilamide, N-(1-naphthyl)ethylenediamine, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), LPS of Salmonella enterica, linoleic acid, LOX from glycine max (soybean) and trypan blue were obtained from Sigma-Aldrich (St. Louis, MO, USA). COX fluorescent inhibitor screening assay kit was purchased from Cayman chemicals (Ann Arbor, MI, USA). Dulbecco’s Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), heat inactivated foetal bovine serum (FBS), Pen-Strep solution (penicillin 5000 units mL −1 and streptomycin 5000 mg mL −1 ) and trypsin were obtained from Gibco Invitrogen TM (Grand Island, NY, Pharmaceutics 2020,12, 122 4 of 18 USA). Proteasome 20S, lactacystin and Suc-Leu-Leu-Val-Tyr-AMC were purchased from Enzo Life Sciences. Proteasome 26S was obtained from AGS cells, as described below. 2.2. Compounds Tested The chemical synthesis and characterisation data for the compounds 1 – 4 , 7 and 8 have been described previously [ 17 , 18 , 23 ]. The synthesis and characterisation data for compounds 5 and 6 are described in articles submitted for publication. The partition coefficient between water and n-octanol (Log P) of each compound was estimated using Molinspiration Cheminformatics software (Molinspiration, Slovensky Grob, Slovak Republic, 2017, http://www.molinspiration.com), as a sum of fragment-based contributions and correction factors, and it is used as quantitative descriptor of compound lipophilicity [24]. 2.3. Lipoxygenase Glycine Max (Soybean) Assay The inhibitory effect on LOX was assessed in 96-well plates, using a modified version of a previously reported procedure from Pereira et al. [ 25 ]. The compounds were tested in a reaction mixture of each compound (20 µ L), phosphate buffer (200 µ L, pH 9.0) and soybean LOX (20 µ L, ~100 U). After 5 min pre-incubation at room temperature, the reaction was started by addition of linoleic acid substrate (20 µ L of a 4.18 mM solution in ethanol). The reaction was monitored at 234 nm using a multiplate reader (Multiskan Thermo Fisher Scientific Oy, Vantaa, Finland), for 3 min. 2.4. Cyclooxygenase-1 (COX-1) and Cyclooxygenase-2 (COX-2) Inhibition Assay The assay was performed using the COX fluorescent inhibitor screening assay kit (Cayman chemical, MI, USA), with some modifications. Briefly, 60 µ L of assay buffer (100 mM Tris-HCl, pH 8.0), 5 µ L of hemin, 5 µ L of enzyme (either COX-1 or COX-2) and 5 µ L of compound (25 µ M) were added to a black 96-well plate. After 5 min of incubation at room temperature, 5 µ L of 10-acetyl-3,7-dihydroxyphenoxazine (ADHP) and 20 µ L of a solution containing arachidonic acid (0.5 mM) and KOH (2.5 mM) were added to each well. After a further 2 min at room temperature, the fluorescence of resorufin was monitored with an excitation wavelength between 530–540 nm and an emission wavelength between 585–595 nm, using a multiplate reader (Synergy H1, Biotek Instruments Winooski, USA). SC-560 and DuP-697 inhibitors were used as positive controls to COX-1 and COX-2 assay, respectively. 2.5. 20S Proteasome Inhibition Proteasome activity was measured by adapting a method described by Silva et al. [ 26 ]. The substrate used to study chymotrypsin-like enzyme activity was Suc-Leu-Leu-Val-Tyr-AMC and the enzyme used was purified 20S proteasome isolated from erythrocytes (Enzo Life Sciences). Briefly, Suc-Leu-Leu-Val-Tyr-AMC (25 µ L of a 160 µ M solution in Tris-HCl) was added to a solution containing naproxen–peptide conjugate (required amount) and 20S proteasome (70 ng) in Tris-HCl assay buffer (75 L), in a black 96-well plate. The plate was incubated at 37 ◦ C in the dark for 2 h. The inhibition of 20S proteasome was then measured at 340 nm absorption and 460 nm emission in a microplate reader (Synergy H1, Biotek Instruments Winooski, USA). Lactacystin was used as positive control. 2.6. 26S Proteasome Inhibition This assay was carried out in the same way as described for 20S proteasome (vide supra), except using purified 26S proteasome isolated from AGS cells [ 27 ]. To extract 26S proteasome from cells, they were centrifuged at 1300 rpm for 3 min at 37 ◦ C and the supernatant was rejected. The pellet was then resuspended in 5 mL of HBSS. After this step, the cells were centrifuged again under the conditions referred to above and the supernatant was rejected. Then, 1 mL of cell lysis buffer was added, before Pharmaceutics 2020,12, 122 5 of 18 being placed in ice (4 ◦ C) for 30 min. Subsequently, the mixture was centrifuged at 14,000 g during 30 min at 4 ◦ C. The pellet was rejected, and the supernatant was kept in an Eppendorf tube. The quantity of protein isolated was determined by Bradford Assay. 2.7. Bradford Assay A stock solution of bovine serum albumin (BSA) (1.0 mg/mL) was prepared in H 2 O. From the stock solution, six concentrations were prepared: 5%, 4%, 3%, 2%, 1% and 0% stock solution in H 2 O. An aliquot of each sample (40 µ L) was added to a solution of the Bradford reagent (200 µ L). After 5 min, the absorbance was measured at 595 nm and the calibration curve was plotted (correlation > 0.99). Proteasome 26S (40 mL of a sample of unknown concentration) was added to a solution of the Bradford reagent (200 mL). After 5 min, the absorbance was measured at 595 nm and the result was compared with the correlation curve in order to calculate the concentration of protein. 2.8. Cell Culture Adenocarcinoma gastric cells (AGS; Sigma-Aldrich, St. Louis, MO, USA), murine-macrophage cell line (RAW 264.7; American Type Culture Collection, LGC Standards S.L.U., Barcelona, Spain), and human foetal lung fibroblasts (MRC-5; ECACC, Porton Down Salisbury, UK) were cultured as a monolayer at 37 ◦ C in a humidified incubator with 5% carbon dioxide. AGS and RAW 264.7 cells were grown in DMEM, supplemented with 1% streptomycin/penicillin and 10% FBS (Gibco ® ). MRC-5 cells were grown in MEM, supplemented with 1% streptomycin/penicillin and 10% FBS. 2.8.1. 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) Reduction Assay Cell viability was evaluated by the MTT reduction assay [ 28 ]. Cells were cultured in 96-well plates (15,000 cells/well for AGS, 25,000 cells/well for RAW and 20,000 cells/well for MRC-5) and allowed to attach for 24 h. After incubation with compounds for 24 h, MTT (0.5 mg/mL final concentration) was added to each well and the plate was incubated for 75 min at 37 ◦ C. Formazan crystals were dissolved by the addition of a DMSO: isopropanol mixture (3:1) and then quantified spectrophotometrically at 570 nm using a microplate reader (Multiskan Thermo Fisher Scientific Oy, Vantaa, Finland). 2.8.2. Evaluation of NO Levels RAW 264.7 cells were cultured in 96-well plates (35,000 cells/well) for 24 h and then pre-treated with different concentrations of each compound. After 2 h, LPS was added (final concentration 1µg/mL ) and the plates were incubated at 37 ◦ C, in a humidified atmosphere of 5% CO 2 . After 22 h, 75 µ L of cell supernatant was transferred to a 96-well plate and mixed with 75 µ L of Griess reagent (1% sulfanilamide and 0.1% naphthylethylenediamine dihydrochloride in 2% H 3 PO 4 ). The plate was incubated at room temperature for 10 min in the dark and then the absorbance was read at 540 nm using a microplate reader (Multiskan Thermo Fisher Scientific Oy, Vantaa, Finland). 2.9. Statistical Analysis Statistical analysis was performed using GraphPad Prism 6 software (San Diego, CA, USA). A Shapiro–Wilk normality test was conducted to evaluate the distribution of the data, and a Grubb’s test was used to determine the presence of outliers. One-way analysis of variance (ANOVA) and Sidak’s multiple comparison test were used to determine the statistical significance between treated and untreated cells. All experiments were performed in duplicate or triplicate with at least three independent assays. Data are expressed as the mean values ± standard deviation. In all cases, values of p≤0.05 were considered statistically significant. Additionally, in order to compare the results of this library of compounds and these results with other previously published, the compound dose causing 50% of enzyme/cell growth inhibition Pharmaceutics 2020,12, 122 6 of 18 (IC 50 —half maximal inhibitory concentration) was calculated. This value allows the comparison of the effectiveness of the substances. 2.10. Docking Studies The crystal structure of Ovis aries COX-1, expressed in Spodoptera frugiperda (PDB code: 3N8Z) [ 29 ], Mus musculus COX-2, expressed in Spodoptera frugiperda (PDB code: 3NT1) [ 30 ], and Saccharomyces cerevisiae yeast 20S open gate proteasome (PDB code: 3MG8) [ 31 ] were used as the protein receptor model. The optimized geometries of the hydrogelators ground state were obtained from ab initio molecular quantum chemistry calculations, with Gaussian 09 software and use of a 6-31+G(d,p) basis set at the DFT B3LYP level of theory. Docking of the receptor protein with the hydrogelators was performed using AutoDock4.2 suite of programs with Lamarckian Genetic Algorithm. The calculation was set up to 150 runs, 270,000 maximum number of generations, 2,500,000 maximum number of energy evaluations, and 50 50 50 grid points for proteins with 0.375 Å spacing. The macromolecule was kept rigid and ligand molecules were flexible. Visualization of the complex protein–ligand interactions was analysed with PyMOL software. 3. Results To follow-up on the initial anti-inflammatory results obtained for dehydrodipeptide-naproxen conjugates 1 and 2 , a more detailed biological study of a focused library of naproxen-dehydrodipeptides ( 1 – 8 ) (Figure 2) was conducted, regarding their anti-inflammatory and anti-cancer activity, as well as their potential toxicity towards non-cancer cells. The synthesis of hydrogelators 1 – 8 and the rheological and physical-chemical characterization of their hydrogels was reported elsewhere [ 18 ]. Here, we report for the first time the anti-inflammatory and anti-cancer properties of conjugates 3 – 8 (Figure 2). We had previously reported the anti-inflammatory properties of compounds 1 and 2 , but no results related to their effect on cancer cells or proteasome inhibitory activity [ 23 ]. Some previous biological results for compounds 1 and 2 will be presented alongside the results for compounds 3 – 8 , where they are considered useful for comparison purposes. The importance of the N-terminal canonical amino acid residue was investigated by variation of the amino acid side chain. Studying the biological activities of 1 and 3 – 6 would allow the SAR at this position to be determined. Additionally, a comparison of compound 8 with compound 3 , where the ∆ Phe residue is swapped for a ∆ Ala residue, would give an insight into the effect of the dehydroamino acid residue on the biological activity. A comparison of the biological results obtained for compound 7 with the results previously published for compound 1 , where the free carboxylic acid group is replaced by an ester, would give an insight into the SAR of modifications at the C-terminus. Pharmaceutics 2020,12, 122 7 of 18 Pharmaceutics 2020, 12, 7 of 18 Figure 2. Library of compounds 1–8, evaluated for anti-inflammatory and anti-cancer activity. The molecular weight and cLogP data are shown (values in parenthesis refer to the carboxylate salt form). 3.1. Anti-inflammatory Activity The anti-inflammatory activity of naproxen–dehydrodipeptide conjugates was investigated using a murine–macrophage cell line (RAW 264.7). Macrophages play a key role in inflammation, by initiating the immune response against invaders. Therefore, when activated by LPS, the chosen cell line can release pro-inflammatory cytokines and chemokines that lead to the production and secretion of reactive oxygen species and reactive nitrogen species, such as •NO. •NO levels can be measured to determine the potential of the compounds in regulating an inflammatory response. The choice of this specific macrophage cell line is based on the fact that this is one of the most used cell lines for anti-inflammatory activity studies [32–34], which allows us to compare our results with other available results. Figure 2. Library of compounds 1 – 8, evaluated for anti-inflammatory and anti-cancer activity. The molecular weight and cLogP data are shown (values in parenthesis refer to the carboxylate salt form). 3.1. Anti-Inflammatory Activity The anti-inflammatory activity of naproxen–dehydrodipeptide conjugates was investigated using a murine–macrophage cell line (RAW 264.7). Macrophages play a key role in inflammation, by initiating the immune response against invaders. Therefore, when activated by LPS, the chosen cell line can release pro-inflammatory cytokines and chemokines that lead to the production and secretion of reactive oxygen species and reactive nitrogen species, such as • NO. • NO levels can be measured to determine the potential of the compounds in regulating an inflammatory response. The choice of this specific macrophage cell line is based on the fact that this is one of the most used cell lines for anti-inflammatory activity studies [ 32 – 34 ], which allows us to compare our results with other available results. Pharmaceutics 2020,12, 122 8 of 18 3.1.1. Effect of the Compounds on the Viability of RAW 264.7 Macrophages The library of naproxen–dehydrodipeptide conjugates shown in Figure 2was tested for their effect on the viability of rat macrophages (RAW 264.7 cells) (Figure 3). Compound 5 elicited a small cytotoxic effect at 100 µ M. Compounds 3 , 4 , 6 and 8 did not show any statistically significant cytotoxic effects on RAW cells up to 100 µ M, as was the case with the previously studied compounds 1 and 2 [ 23 ]. Compound 7 , containing a methyl ester at the C-terminus, was found to be cytotoxic to some degree, with an IC 50 value of 23.1 µ M, suggesting that it could cross the cell membrane and elicit cellular toxicity, presumably following ester hydrolysis by intracellular esterases [35]. Pharmaceutics 2020, 12, 8 of 18 3.1.1. Effect of the Compounds on the Viability of RAW 264.7 Macrophages The library of naproxen–dehydrodipeptide conjugates shown in Figure 2 was tested for their effect on the viability of rat macrophages (RAW 264.7 cells) (Figure 3). Compound 5 elicited a small cytotoxic effect at 100 µM. Compounds 3, 4, 6 and 8 did not show any statistically significant cytotoxic effects on RAW cells up to 100 µM, as was the case with the previously studied compounds 1 and 2 [23]. Compound 7, containing a methyl ester at the C-terminus, was found to be cytotoxic to some degree, with an IC50 value of 23.1 µM, suggesting that it could cross the cell membrane and elicit cellular toxicity, presumably following ester hydrolysis by intracellular esterases [35]. Figure 3. Cell viability of RAW 264.7, AGS and MRC-5 in the presence of compounds 1–8 at 100 µM for 24 h. Values are shown with mean ± SD. ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. The results for compounds 1 and 2 on the viability of RAW 264.7 have been reported previously but are included here for comparison purposes. 3.1.2. Effect of the Compounds on the Production of •NO in RAW 264.7 Macrophages The compounds which were shown to be non-toxic to rat macrophages (3, 4, 6 and 8) were tested for their ability to inhibit LPS-dependent •NO production in rat macrophages (Figure 4). •NO is an important mediator of the inflammatory response, which is synthesized by inducible nitric oxide synthase (iNOS) from oxygen and L-arginine [36]. Its excessive production is associated with inflammatory diseases [37]. The dehydrodipeptides generally elicited only modest effects on the production of •NO. IC50 values of 64.7 µM and 84.4 µM were determined for the most active compounds, 3 and 8, respectively, in line with the IC50 value of 79.3 µM, previously reported for compound 1 [20]. Figure 3. Cell viability of RAW 264.7, AGS and MRC-5 in the presence of compounds 1 – 8 at 100 µ M for 24 h. Values are shown with mean ± SD. ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. The results for compounds 1 and 2 on the viability of RAW 264.7 have been reported previously but are included here for comparison purposes. 3.1.2. Effect of the Compounds on the Production of •NO in RAW 264.7 Macrophages The compounds which were shown to be non-toxic to rat macrophages ( 3 , 4 , 6 and 8 ) were tested for their ability to inhibit LPS-dependent • NO production in rat macrophages (Figure 4). • NO is an important mediator of the inflammatory response, which is synthesized by inducible nitric oxide synthase (iNOS) from oxygen and L-arginine [ 36 ]. Its excessive production is associated with inflammatory diseases [ 37 ]. The dehydrodipeptides generally elicited only modest effects on the production of • NO. IC 50 values of 64.7 µ M and 84.4 µ M were determined for the most active compounds, 3 and 8 , respectively, in line with the IC 50 value of 79.3 µ M, previously reported for compound 1[20]. Pharmaceutics 2020,12, 122 9 of 18 Pharmaceutics 2020, 12, 9 of 18 Figure 4. LPS-induced •NO production in rat macrophages in the presence of the compounds 1–4, 6 and 8 for 24 h. Values are shown with mean ± SD. * p ≤ 0.05; *** p ≤ 0.001; **** p ≤ 0.0001. The results for compound 1 and 2, reported previously, are included for comparison purposes. 3.1.3. Effect of the Compounds on LOX Activity The compounds 1–6 and compound 8 were tested for their ability to inhibit the arachidonicpathway-related 5-LOX enzyme (Figure 5). Compound 7 could not be tested, owing to insolubility in the assay buffer solution. The LOX enzyme is responsible for the production of inflammatory leukotrienes, which are a major cause of inflammation in asthma, allergic rhinitis and osteoarthritis [38]. The compounds were tested at single concentrations of 100 µM in the first instance. As reported previously for dehydrodipeptides 1 and 2 [23], compounds 3, 4, 6 and 8 at 100 µM concentration were also able to significantly inhibit the LOX enzyme to a similar level to naproxen. Compound 5 was not able to inhibit LOX activity. Aside from compound 5, the activity seems relatively insensitive to modifications at both the Nterminal canonical amino acid residue and the C-terminal dehydroamino acid residue. Compound 5, containing an N-terminal charged lysine residue, is the most polar compound of the set (cLogP = 3.89) and therefore might not be able to mimic the hydrophobic interactions of the fatty acid chain of the natural substrate (arachidonic acid) with the LOX enzyme active site. The other compounds in this series contain either non-polar (Ala for 4, Met for 6) or amphipathic (Tyr for 1, Trp for 3) amino acid residues. Figure 4. LPS-induced • NO production in rat macrophages in the presence of the compounds 1 – 4 , 6 and 8 for 24 h. Values are shown with mean ± SD. * p ≤ 0.05; *** p ≤ 0.001; **** p ≤ 0.0001. The results for compound 1and 2, reported previously, are included for comparison purposes. 3.1.3. Effect of the Compounds on LOX Activity The compounds 1 – 6 and compound 8 were tested for their ability to inhibit the arachidonic-pathway-related 5-LOX enzyme (Figure 5). Compound 7 could not be tested, owing to insolubility in the assay buffer solution. The LOX enzyme is responsible for the production of inflammatory leukotrienes, which are a major cause of inflammation in asthma, allergic rhinitis and osteoarthritis [ 38 ]. The compounds were tested at single concentrations of 100 µ M in the first instance. As reported previously for dehydrodipeptides 1 and 2 [ 23 ], compounds 3 , 4 , 6 and 8 at 100 µ M concentration were also able to significantly inhibit the LOX enzyme to a similar level to naproxen. Compound 5was not able to inhibit LOX activity. Pharmaceutics 2020,12, 122 16 of 18 Author Contributions: Conceptualization, D.M.P, J.A.M., P.M.T.F., P.V. and P.B.A., A.C.; investigation, R.M. and P.J.J.; writing—original draft preparation, R.M. and P.J.J.; writing—review and editing, D.M.P, J.A.M. and P.M.T.F.; funding acquisition, P.M.T.F., J.A.M., P.V., P.B.A., D.M.P. All authors have read and agreed to the published version of the manuscript. Funding: This work is funded by National Funds through FCT-Portuguese Foundation for Science and Technology under the Project PTDC/QUI-QOR/29015/2017 and CQ/UM UID/QUI/00686/2013 and UID/QUI/0686/2016. The NMR spectrometers are part of the National NMR Network (PTNMR) and are partially supported by Infrastructure Project No 022161 (co-financed by FEDER through COMPETE 2020, POCI and PORL and FCT through PIDDAC). Acknowledgments: This work is funded by FEDER funds through the COMPETE 2020 Programme and National Funds through FCT-Portuguese Foundation for Science and Technology under the Project UID/CTM/50025/2013 and UIDB/50006/2020. We thank Vera Alexandra de Macedo Ribeiro for isolation of the 26S proteasome subunit. We acknowledge the precious advice of Tarsila Castro on the docking studies. For computing resources:”Search-ON2: Revitalization of HPC infrastructure of UMinho, (NORTE-07-0162-FEDER-000086), co-funded by the North Portugal Regional Operational Programme (ON.2-O Novo Norte), under the National Strategic Reference Framework (NSRF), through the European Regional Development Fund (ERDF). Conflicts of Interest: The authors declare no conflict of interest. Abbreviations NSAIDs non-steroidal anti-inflammatory drugs SPIONs superparamagnetic iron oxide nanoparticles MRI magnetic resonance imaging COX-2 cyclooxygenase-2 COX-1 cyclooxygenase-1 COX cyclooxygenase LPS lipopolysaccharide LOX lipoxygenase •NO nitric oxide SAR structure-activity relationship DMSO dimethyl sulfoxide MTT N-(1-naphthyl)ethylenediamine, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide DMEM Dulbecco’s Modified Eagle Medium MEM Minimum Essential Medium FBS foetal bovine serum BSA bovine serum albumine PGG2 prostaglandin G2 ADHP 10-acetyl-3,7-dihydroxyphenoxazine References 1. Torchilin, V.P. Multifunctional, stimuli-sensitive nanoparticulate systems for drug delivery. Nat. Rev. Drug Discov. 2014,13, 813–827. [CrossRef] [PubMed] 2. Torchilin, V.P. Recent advances with liposomes as pharmaceutical carriers. Nat. Rev. Drug Discov. 2005 ,4, 145–160. [CrossRef] [PubMed] 3. Brannon-Peppas, L.; Blanchette, J.O. Nanoparticle and targeted systems for cancer therapy. Adv. Drug Deliv. Rev. 2012,64, 206–212. [CrossRef] 4. Rizvi, S.A.A.; Saleh, A.M. Applications of nanoparticle systems in drug delivery technology. Saudi Pharm. J. 2018,26, 64–70. [CrossRef] 5. Patra, J.K.; Das, G.; Fraceto, L.F.; Campos, E.V.R.; Rodriguez-Torres, M.d.P.; Acosta-Torres, L.S.; Diaz-Torres, L.A.; Grillo, R.; Swamy, M.K.; Sharma, S.; et al. Nano based drug delivery systems: Recent developments and future prospects. J. Nanobiotechnology 2018,16, 71. [CrossRef] 6. Xia, R.; Zheng, X.; Hu, X.; Liu, S.; Xie, Z. Photothermal-controlled generation of alkyl radical from organic nanoparticles for tumor treatment. Acs Appl. Mater. Interfaces 2019,11, 5782–5790. [CrossRef] 7. Wang, Y.; Cheetham, A.G.; Angacian, G.; Su, H.; Xie, L.; Cui, H. Peptide–drug conjugates as effective prodrug strategies for targeted delivery. Adv. Drug Deliv. Rev. 2017,110–111, 112–126. [CrossRef] Pharmaceutics 2020,12, 122 17 of 18 8. Li,S.; Zhao, L.; Chang,R.; Xing,R.; Yan,X.Spatiotemporally coupledphotoactivityof phthalocyanine–peptide conjugate self-assemblies for adaptive tumor theranostics. Chem.–A Eur. J. 2019 ,25, 13429–13435. [CrossRef] 9. Zou, Q.; Abbas, M.; Zhao, L.; Li, S.; Shen, G.; Yan, X. Biological photothermal nanodots based on self-assembly of peptide–porphyrin conjugates for antitumor therapy. J. Am. Chem. Soc. 2017 ,139, 1921–1927. [CrossRef] 10. Bresalier, R.S.; Sandler, R.S.; Quan, H.; Bolognese, J.A.; Oxenius, B.; Horgan, K.; Lines, C.; Riddell, R.; Morton, D.; Lanas, A.; et al. Cardiovascular events associated with rofecoxib in a colorectal adenoma chemoprevention trial. N. Engl. J. Med. 2005,352, 1092–1102. [CrossRef] 11. Catella-Lawson, F.; Reilly, M.P.; Kapoor, S.C.; Cucchiara, A.J.; DeMarco, S.; Tournier, B.; Vyas, S.N.; FitzGerald, G.A. Cyclooxygenase inhibitors and the antiplatelet effects of aspirin. N. Engl. J. Med. 2001 ,345, 1809–1817. [CrossRef] [PubMed] 12. Li, J.; Kuang, Y.; Gao, Y.; Du, X.; Shi, J.; Xu, B. D-Amino acids boost the selectivity and confer supramolecular hydrogels of a nonsteroidal anti-inflammatory drug (NSAID). J. Am. Chem. Soc. 2013 ,135, 542–545. [CrossRef] [PubMed] 13. Chen, Z.; Xing, L.; Fan, Q.; Cheetham, A.G.; Lin, R.; Holt, B.; Chen, L.; Xiao, Y.; Cui, H. Drug-bearing supramolecular filament hydrogels as anti-inflammatory agents. Theranostics 2017 ,7, 2003–2014. [CrossRef] [PubMed] 14. Uhrich, K.E.; Cannizzaro, S.M.; Langer, R.S.; Shakesheff, K.M. Polymeric systems for controlled drug release. Chem. Rev. 1999,99, 3181–3198. [CrossRef] [PubMed] 15. Rambhia, K.J.; Ma, P.X. Controlled drug release for tissue engineering. J. Control. Release 2015 ,219, 119–128. [CrossRef] [PubMed] 16. Kurbasic, M.; Romano, C.; Garcia, A.; Kralj, S.; Marchesan, S. Assembly of a tripeptide and anti-inflammatory drugs into supramolecular hydrogels for sustained release. Gels 2017,3, 29. [CrossRef] 17. Vilaça, H.; Castro, T.; Costa, F.M.G.; Melle-Franco, M.; Hilliou, L.; Hamley, I.W.; Castanheira, E.M.S.; Martins, J.A.; Ferreira, P.M.T. Self-assembled RGD dehydropeptide hydrogels for drug delivery applications. J. Mater. Chem. B 2017,5, 8607–8617. [CrossRef] 18. Vilaça, H.; Hortel ã o, A.C.L.; Castanheira, E.M.S.; Queiroz, M.-J.R.P.; Hilliou, L.; Hamley, I.W.; Martins, J.A.; Ferreira, P.M.T. Dehydrodipeptide hydrogelators containing naproxen N-capped tryptophan: Self-assembly, hydrogel characterization, and evaluation as potential drug nanocarriers. Biomacromolecules 2015 ,16, 3562–3573. [CrossRef] 19. Blayney, D.W.; Bazhenova, L.; Lloyd, G.K.; Huang, L.; Mohanlal, R. Plinabulin, a novel small molecule that ameliorates chemotherapy-induced neutropenia, is administered on the same day of chemotherapy and has anticancer efficacy. Blood 2016,128, 2508. [CrossRef] 20. Sandu, C.; Chandramouli, N.; Glickman, J.F.; Molina, H.; Kuo, C.-L.; Kukushkin, N.; Goldberg, A.L.; Steller, H. Thiostrepton interacts covalently with Rpt subunits of the 19S proteasome and proteasome substrates. J. Cell. Mol. Med. 2015,19, 2181–2192. [CrossRef] 21. Pastel, D.A. Imipenem-cilastatin sodium, a broad-spectrum carbapenem antibiotic combination. Clin. Pharm. 1986,5, 719–736. [CrossRef] [PubMed] 22. Vilaça, H.; Pereira, G.; Castro, T.G.; Hermenegildo, B.F.; Shi, J.; Faria, T.Q.; Mica ê lo, N.; Brito, R.M.M.; Xu, B.; Castanheira, E.M.S.; et al. New self-assembled supramolecular hydrogels based on dehydropeptides. J. Mater. Chem. B 2015,3, 6355–6367. [CrossRef] 23. Carvalho, A.; Gallo, J.; Pereira, D.; Valent ã o, P.; Andrade, P.; Hilliou, L.; Ferreira, P.; Bañobre-L ó pez, M.; Martins, J. Magnetic dehydrodipeptide-based self-assembled hydrogels for theragnostic applications. Nanomaterials 2019,9, 541. [CrossRef] [PubMed] 24. Cedr ó n, J.; Ravelo, Á .; Le ó n, L.; Padr ó n, J.; Est é vez-Braun, A. Antiproliferative and structure activity relationships of Amaryllidaceae alkaloids. Molecules 2015,20, 13854–13863. [CrossRef] 25. Pereira, R.B.; Taveira, M.; Valent ã o, P.; Sousa, C.; Andrade, P.B. Fatty acids from edible sea hares: Anti-inflammatory capacity in LPS-stimulated RAW 264.7 cells involves iNOS modulation. RSC Adv. 2015,5, 8981–8987. [CrossRef] 26. da Silva, D.C.; Andrade, P.B.; Valent ã o, P.; Pereira, D.M. Neurotoxicity of the steroidal alkaloids tomatine and tomatidine is RIP1 kinaseand caspase-independent and involves the eIF2 α branch of the endoplasmic reticulum. J. Steroid Biochem. Mol. Biol. 2017,171, 178–186. [CrossRef] Pharmaceutics 2020,12, 122 18 of 18 27. Ribeiro, V.; Andrade, P.B.; Valent ã o, P.; Pereira, D.M. Benzoquinones from Cyperus spp. trigger IRE1 α -independent and PERK-dependent ER stress in human stomach cancer cells and are novel proteasome inhibitors. Phytomedicine 2019,63, 153017. [CrossRef] 28. Pereira, D.; Correia-da-Silva, G.; Valent ã o, P.; Teixeira, N.; Andrade, P. Palmitic acid and ergosta-7,22-dien-3-ol contribute to the apoptotic effect and cell cycle arrest of an extract from Marthasterias glacialis L. in neuroblastoma cells. Mar. Drugs 2013,12, 54–68. [CrossRef] 29. Sidhu, R.S.; Lee, J.Y.; Yuan, C.; Smith, W.L. Comparison of cyclooxygenase-1 crystal structures: Cross-talk between monomers comprising cyclooxygenase-1 homodimers. Biochemistry 2010 ,49, 7069–7079. [CrossRef] 30. Duggan, K.C.; Walters, M.J.; Musee, J.; Harp, J.M.; Kiefer, J.R.; Oates, J.A.; Marnett, L.J. Molecular basis for cyclooxygenase inhibition by the non-steroidal anti-inflammatory drug naproxen. J. Biol. Chem. 2010 ,285, 34950–34959. [CrossRef] 31. Blackburn, C.; Gigstad, K.M.; Hales, P.; Garcia, K.; Jones, M.; Bruzzese, F.J.; Barrett, C.; Liu, J.X.; Soucy, T.A.; Sappal, D.S.; et al. Characterization of a new series of non-covalent proteasome inhibitors with exquisite potency and selectivity for the 20S β5-subunit. Biochem. J. 2010,430, 461–476. [CrossRef] [PubMed] 32. Wang, Y.; Wang,L.; Wise,J.T.F.; Shi, X.; Chen,Z.Verteporfin inhibitslipopolysaccharide-inducedinflammation by multiple functions in RAW 264.7 cells. Toxicol. Appl. Pharmacol. 2020,387, 114852. [CrossRef] [PubMed] 33. Nishanth, R.P.; Jyotsna, R.G.; Schlager, J.J.; Hussain, S.M.; Reddanna, P. Inflammatory responses of RAW 264.7 macrophages upon exposure to nanoparticles: Role of ROS-NF κ B signaling pathway. Nanotoxicology 2011,5, 502–516. [CrossRef] [PubMed] 34. Taciak, B.; Białasek, M.; Braniewska, A.; Sas, Z.; Sawicka, P.; Kiraga, Ł.; Rygiel, T.; Kr ó l, M. Evaluation of phenotypic and functional stability of RAW 264.7 cell line through serial passages. PLoS ONE 2018 ,13, e0198943. [CrossRef] [PubMed] 35. Li, J.; Bullara, D.; Du, X.; He, H.; Sofou, S.; Kevrekidis, I.G.; Epstein, I.R.; Xu, B. Kinetic analysis of nanostructures formed by enzyme-instructed intracellular assemblies against cancer cells. ACS Nano 2018 , 12, 3804–3815. [CrossRef] 36. Michel, T.; Feron, O. Nitric oxide synthases: Which, where, how, and why? J. Clin. Investig. 1997 ,100, 2146–2152. [CrossRef] 37. Guzik, T.J.; Korbut, R.; Adamek-Guzik, T. Nitric oxide and superoxide in inflammation and immune regulation. J. Physiol. Pharmacol. 2003,54, 469–487. 38. Werz, O. 5-Lipoxygenase: Cellular biology and molecular pharmacology. Curr. Drug Target -Inflamm. Allergy 2002,1, 23–44. 39. Zarghi, A.; Arfaei, S. Selective COX-2 inhibitors: A review of their structure-activity relationships. Iran. J. Pharm. Res. 2011,10, 655–683. 40. Carullo, G.; Galligano, F.; Aiello, F. Structure–activity relationships for the synthesis of selective cyclooxygenase 2 inhibitors: An overview (2009–2016). Med. Chem. Commun. 2017,8, 492–500. [CrossRef] 41. Asher, G.; Reuven, N.; Shaul, Y. 20S proteasomes and protein degradation “by default.”. BioEssays 2006 ,28, 844–849. [CrossRef] 42. Adams, J. The development of proteasome inhibitors as anticancer drugs. Cancer Cell 2004 ,5, 417–421. [CrossRef] 43. Jain, R.; Virander, S.C. Conformational characteristics of peptides containing α , β -dehydroamino acid residues. Pept. Sci. 1996,40, 105–119. [CrossRef] 44. Chen, D.; Frezza, M.; Schmitt, S.; Kanwar, J.; Dou, Q.P. Bortezomib as the first proteasome inhibitor anticancer drug: Current status and future perspectives. Curr. Cancer Drug Targets 2011 ,11, 239–253. [CrossRef] [PubMed] 45. Groll, M.; Berkers, C.R.; Ploegh, H.L.; Ovaa, H. Crystal structure of the boronic acid-based proteasome inhibitor bortezomib in complex with the yeast 20S proteasome. Structure 2006 ,14, 451–456. [CrossRef] [PubMed] © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).