pharmaceutics Article Purification and Characterization of a Novel Thermostable Papain Inhibitor from Moringa oleifera with Antimicrobial and Anticoagulant Properties Juliana Cotabarren 1, *, Santiago Claver 1 , Juan Abreu Payrol 2,3 , Javier Garcia-Pardo 4, * and Walter David Obregón 1, * 1 Centro de Investigación de Proteínas Vegetales (CIPROVE), Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de la Plata, Buenos Aires B1900, Argentina; sclaver@hotmail.com 2 Escuela Latinoamericana de Medicina, La Habana 19108, Cuba; jabreu@bionaturasm.cu 3 ECTI “Sierra Maestra”, Complejo Barlovento Ave 5ta. y 246, Santa Fé, Playa, La Habana 19108, Cuba 4 Institut de Biotecnologia i Biomedicina and Departament de Bioquimica i Biologia Molecular, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain * Correspondence: cotabarren.juliana@biol.unlp.edu.ar (J.C.); javiergarciapardo@msn.com (J.G.-P.); davidobregon@biol.unlp.edu.ar (W.D.O.); Tel.: +54-221-423-5333 (ext. 57) (J.C. & W.D.O.); +34-93-581-1233 (J.G.-P.)  Abstract: Plant cystatins (or phytocystatins) comprise a large superfamily of natural bioactive small  proteins that typically act as protein inhibitors of papain-like cysteine proteases. In this report, Citation: Cotabarren, J.; Claver, S.; we present the purification and characterization of the first phytocystatin isolated from Moringa Payrol, J.A.; Garcia-Pardo, J.; oleifera (MoPI). MoPI has a molecular mass of 19 kDa and showed an extraordinary physicochemical Obregón, W.D. Purification and stability against acidic pHs and high temperatures. Our findings also revealed that MoPI is one Characterization of a Novel of the most potent cysteine protease inhibitors reported to date, with Ki and IC50 values of 2.1 nM Thermostable Papain Inhibitor from and 5.7 nM, respectively. More interestingly, MoPI presents a strong antimicrobial activity against Moringa oleifera with Antimicrobial and Anticoagulant Properties. human pathogens such as Enterococcus faecalis and Staphylococcus aureus. In addition, MoPI also Pharmaceutics 2021, 13, 512. showed important anticoagulant activity, which is an unprecedented property for this family of https://doi.org/10.3390/ protease inhibitors. These results highlight the pharmaceutical potential of this plant and its derived pharmaceutics13040512 bioactive molecules. Academic Editors: Ian S. Blagbrough Keywords: Moringa oleifera; protease; plant protease inhibitor; phytocystatin; papain inhibitor; and Thierry Vandamme antibacterial; anticoagulant; bioactive compound Received: 23 February 2021 Accepted: 1 April 2021 Published: 8 April 2021 1. Introduction Moringa oleifera (M. oleifera) is a plant native to India that grows in the tropical and Publisher’s Note: MDPI stays neutral subtropical regions of the world. Due to its high nutritional properties, the whole plant is with regard to jurisdictional claims in published maps and institutional affil- typically used for nutritional or commercial purposes. It is worth mentioning that different iations. parts of this tree are applied as food to combat malnutrition, especially among infants and breastfeeding woman in many developing countries, including India, Pakistan, the Philippines, Hawaii and many regions of Africa [1]. In addition to its nutritional value, M. oleifera has an important medicinal and phar- macological potential. Recent studies have shown that extracts of this plant possess strong Copyright: © 2021 by the authors. antioxidant, anticancer, anti-inflammatory and antidiabetic properties [2]. Furthermore, Licensee MDPI, Basel, Switzerland. evidence suggests that almost all parts of the plant exhibit antibacterial activity [3]. In many This article is an open access article distributed under the terms and cases, such antibacterial activity has been associated with the presence of small chemical conditions of the Creative Commons compounds such as glucosinolates (β-thioglucoside N-hydroxysulfates), isothiocyanates, Attribution (CC BY) license (https:// organic carbamates, chalcone-oxazolidinone hybrids and thiocarbamate [4]. Although creativecommons.org/licenses/by/ extensive research has been carried out with M. oleifera extracts, little is known regarding 4.0/). the presence of protease inhibitors in this plant and their role as antibacterial agents. Pharmaceutics 2021, 13, 512. https://doi.org/10.3390/pharmaceutics13040512 https://www.mdpi.com/journal/pharmaceutics Pharmaceutics 2021, 13, 512 2 of 11 Cysteine proteases (CPs) are among the most widely distributed and relevant pro- teases. They are present in almost all living organisms and participate in a variety of biological processes [5]. It has been reported that the imbalance in the activity of endoge- nous CPs can lead to numerous pathologies such as rheumatoid arthritis, multiple sclerosis, neurological disorders, tumours, and osteoporosis [6]. CPs produced by bacteria, viruses, and parasites are also considered important factors in the development of many human diseases such as paradontosis [7], malaria, Chagas disease and schistosomiasis [8,9], among others. Special mention deserves the role of the papain-like cysteine protease (PCPs) from the SARS-CoV-2, which is required for the processing of viral polyproteins during viral spread and to evade the innate immunity of the host [10]. From a more general point of view, PCPs play essential roles in growth, cell differentiation, signalling and host in- vasion in pathogenic organisms and frequently act as a virulence factor by attacking the host’s immune system [10–13]. Thus, fine control of the proteolytic activity is essential for the proper functioning of whole cells and organisms. In fact, this is achieved at many levels, from the regulation of protein expression, secretion and maturation (through the specific cleavage of the proenzyme) to the blocking of its activity by inhibition by specific protease inhibitors [14]. Cystatins (or phytocystatins) are a family of structurally related proteins with molec- ular masses in the range of 12–23 kDa that function as CPs inhibitors [15]. Previous studies suggested that phytocystatins isolated from corn, barley, tomato, rice, papaya, etc., [16–18] can inhibit insect CPs in vitro [19–21]. On the other hand, phytocystatins iso- lated from chestnut, sugar cane, carnation, barley, taro, strawberry, wheat, cocoa, amaranth and sesame showed strong inhibitory activity against broad-range fungal pathogens [22]. However, despite all these previous studies, there is only one known cystatin that specif- ically inhibits the growth of bacteria. This protein has been isolated from kiwi and is capable of blocking the growth of Agrobacterium tumefasciens, Burkholderia cepacia, and Erwinia carotovora [23]. Over the past years, M. oleifera has shown great nutritional and pharmacological potential. However, previous research has been focused on evaluating the activity of crude extracts and the identity and biological role of its bioactive compounds remains elusive. In the present study, we report the isolation, purification and biochemical characterization of a novel papain inhibitor obtained from M. oleifera seeds. Herein, we also studied the antibacterial properties of the isolated bioactive inhibitor, which was assayed against a panel with nine different bacterial strains. More importantly, anticoagulant activity studies results indicated that MoPI is a pharmacologically active anticoagulant molecule. Taken together, the activities and distinctive properties of this novel inhibitor represent the first report carried out for M. oleifera phytocystatins and suggests direct applicability of this bioactive molecule for pharmaceutical and/or biotechnological applications. 2. Materials and Methods 2.1. Materials The seeds of M. oleifera were hand-collected from local trees near La Coronela neighbor- hood, La Habana, Cuba. The seeds were washed and stored at −80 ◦ C until use. Coomassie Blue G-250, N,N,N 0 ,N 0 -tetramethylethylenediamine (TEMED), sodium chloride, tris (hy- droxymethyl) aminomethane, sodium dodecyl sulphate (SDS), β-mercaptoethanol (βME), bovine serum albumin (BSA), L-pyroglutamyl-L-phenylalanyl-L-leucine-p-nitroanilide (PFLNA), Trypsin, 4-nitrophenol-α-D-glucopyranoside (PNPG) and α-glucosidase were purchased from Sigma-Aldrich (San Luis, MO, USA). Glyoxyl-agarose was delivered from FlukaTM. All other chemicals used in this work were of analytical grade and purchased from Sigma-Aldrich (San Luis, MO, USA) (unless otherwise specified). 2.2. Crude Extract Preparation M. oleifera seeds (30 g) were washed with distilled water and dried at room temperature during 16–20 h. The seeds were ground using a blender with the addition of 450 mL of Pharmaceutics 2021, 13, 512 3 of 11 0.01 M phosphate buffer, pH 7.4 in an ice bath to avoid possible protein denaturation. After incubation for 2 h at room temperature, the mixture was filtered with gauze, and the insoluble material was removed by centrifugation for 30 min at 7000× g at 4 ◦ C. The clarified supernatant (from now on: MoCE) was collected and immediately frozen at −20 ◦ C until analysis. The total protein content was determined by the Bradford’s assay [24], as described by Cotabarren et al. [25] using bovine serum albumin (BSA) as standard (0.1 mg/mL). Papain inhibitory activity was determined as described in Section 2.4. 2.3. Identification and Purification of MoPI 2.3.1. Partial Purification by Heat Treatment According to our previous studies, protease inhibitors present high physicochemical stability with minimal loss of inhibitory activity [26]. Accordingly, in the first purification step, the crude extract was subjected to 100 ◦ C for 15, 60, and 120 min. Afterward, the samples were cooled at room temperature, and the thermally denatured proteins were removed by centrifugation for 30 min at 7000× g and 4 ◦ C. The total protein content and the inhibitory activity of the non-treated crude extract and heat-treated samples were determined. Each obtained sample was called MoHT15, MoHT60 and MoHT120, in accordance to the incubation time. 2.3.2. Affinity Chromatography A sample of 150 mL of MoHT15 was loaded onto a papain-glyoxyl-agarose column prepared in-house following the method of Obregón and colleagues [27] (1.5 × 12 cm) connected to an Äkta-Purifier (GE Healthcare, Chicago, IL, USA) previously equilibrated with 0.01M phosphate buffer, pH 5.5–considering the optimal pH for the papain-inhibitor interaction–. After complete removal of the non-retained proteins with equilibration buffer, affiliated proteins were eluted generating a sudden pH change by the addition of 0.01 M HCl, pH 2.5 at a flow rate of 0.7 mL/min. The eluted fractions were immediately neutralized by adding 0.1 M NaOH. The purified papain inhibitor was named MoPI (M. oleifera papain inhibitor). Papain inhibitory activity and protein quantification were determined as previously described. Samples were analysed by SDS-PAGE as described by Cotabarren et al. [25] 2.4. Enzymatic Analysis of the Inhibitory Activity against Papain Papain inhibitory activity was determined by using the substrate L-pyroglutamyl-L- phenylalanyl-L-leucine-p-nitroanilide (PFLNA). The hydrolysis of PFLNA was monitored by measuring the increase of the absorbance at 410 nm at 37 ◦ C every minute for 10 min [27]. Reaction volumes were adapted to a 96-well plate with a final volume of 200 µL. The inhibition of papain activity caused a decrease in the hydrolysis rate of the substrate, which resulted in the attenuation of the hydrolysis slope. The inhibitory activity was estimated as the residual proteolytic activity in the presence of the inhibitor and expressed as a percentage of inhibition in comparison with the control assay (in the absence of the inhibitor). In the case of heat-treated samples, the volume used in each assay was 20 µL. One papain inhibitory unit (1 PIU) was defined as the decrease in 0.01 unit of ab- sorbance at 410 nm per 10 min assay, at 37 ◦ C. The inhibitory constant (Ki) and the IC50 (defined as the inhibitor concentration required for half-activity of the enzyme) were calcu- lated according to the protocol described by Tellechea and colleagues [28], modified for papain inhibition. All measurements were performed in triplicate. 2.5. Biological Assays 2.5.1. Anticoagulant Activity Anticoagulant activity of MoPI was evaluated by determining prothrombin time (PT) and the time of activated partial thromboplastin (aPTT) using a Coatron M1 coagulometer (TECO, Neufahrn, Germany). In both cases, a pool of blood plasmas from the mixture, in Pharmaceutics 2021, 13, 512 4 of 11 equal parts, of 5 healthy individuals, maintained at 37 ◦ C with 3.8% sodium citrate (ratio sample: anticoagulant 9:1) was used as a sample (from now on: PBP). For the PT test, the commercial Soluplastin reactive (Wiener Lab.) was employed. Initially, equal parts of the PBP sample and the papain inhibitor (12.5 µg/mL) were incu- bated for 2 min at 37 ◦ C, then 50 µL of Soluplastin were added to 25 µL of this mixture and checked for the coagulation time. For the aPTT test, 25 µL of aPTT (Wiener Lab.) were added to an equal volume of PBP-inhibitor mixture (previously incubated for 2 min at 37 ◦ C). After 2 min incubation at 37 ◦ C, 25 µL of 50 mM CaCl2 was added to initiate the coagulation time determination. For both assays, measurements were carried out in triplicate and appropriate controls were achieved. 2.5.2. Antimicrobial Activity In order to determine the inhibitory capacity on the growth of various bacterial strains by the inhibitor, the agar diffusion assay based on the Kirby-Bauer test was per- formed with slight modifications [29]. Initially, the pre-inoculums of different pathogenic microorganisms (i.e., Citrobacter amalonaticus, Enterobacter cloacae, Enterococcus faecalis, Es- cherichia coli, Klebsiella pneumoniae, Proteus vulgaris, Pseudomonas aeruginosa, Salmonella typhimurium and Staphylococcus aureus) were grown at 37 ◦ C for 18–24 h and the turbidity of the bacterial suspension was adjusted with a physiological solution to 0.5 McFarland scale (1.5 × 108 UFC/mL). The inoculums were plated on Müller Hinton agar plates, in which a drop of 10 µL inhibitor was placed. After the drop placed in the plate was dry, the plates were incubated at 37 ◦ C for 24 h. Then, the diameters of the bacterial growth inhibition halos (in millimetres) were measured. The tests were conducted by triplicate, including the respective controls. 2.6. Statistical Analysis Statistical analyses (ANOVA) were performed with GraphPad Prism (v6.03, GraphPad Sofware Inc.: San Diego, CA, USA, 2012). Significant differences between the means of the parameters were determined by Tukey’s post-hoc test (p < 0.05). 3. Results and Discussion 3.1. Isolation and Purification of MoPI Moringa seeds are known to be rich sources of lipids (31%), and their elimination leads to a high protein expeller (19%), generating an interesting product for human nutrition. As described in Section 2.2, we prepared a crude extract of M. oleifera seeds (MoCE). The protein concentration of the sample was 3.51 ± 0.03 mg/mL (see Table 1), which is in agreement with previous reports [30]. Next, we aimed to investigate the papain inhibitory activity of the crude extract. This initial examination showed that small amounts of MoCE to the reaction tube produce a drastic decrease in the papain activity, which is consistent with the presence of inhibitory molecules in the sample. A more detailed dose-response analysis showed that MoCE strongly inhibits papain with an IC50 value of 0.025 µg/mL. Table 1. Concentration for M. oleifera crude extract and heat-treated samples. Sample Total Protein Content (mg/mL) a M. oleifera crude extract (MoCE) 3.51 ± 0.03 M. oleifera crude extract after 100 ◦ C heat treatment for 15 min (MoHT15) 1.73 ± 0.05 M. oleifera crude extract after 100 ◦ C heat treatment for 60 min (MoHT60) 1.66 ± 0.06 M. oleifera crude extract after 100 ◦ C heat treatment for 120 min (MoHT120) 1.5 ± 0.04 a Values are the mean ± standard deviation (n = 3). A number of plant protease inhibitors (PIs) have been purified and found to be stable and active at high temperatures [26,31]. For this reason, we incubated the MoCE at 100 ◦ C for 15, 60 and 120 min as an initial purification step. As observed in Table 1, the heat treatment at 100 ◦ C produced a decrease in protein content of 50–60% with respect to the ment at 100 °C produced a decrease in protein content of 50–60% with respect to the raw starting crude extract. Papain inhibitory activity was then determined in the samples cor- responding to each heat treatment (MoHT15, MoHT60 and MoHT120). As shown in Figure 1A, the inhibitory activity against papain was maintained even after 120 min of incubation, suggesting an extraordinary thermostability of the inhibitor. It is worth men- Pharmaceutics 2021, 13, 512 5 of 11 tioning that this property is very interesting for most biotechnological applications, be- hind an added value for their commercial exploitation [32]. In addition to the thermal stability, we also evaluated the effect of the temperature on the stability raw starting of the crude inhibitor extract. at two Papain extremeactivity inhibitory pH values was(pH then2determined and 9). Surprisingly, after in the samples 60 min of incubation, corresponding to eachweheat were able to recover treatment 100%MoHT60 (MoHT15, of the inhibitory activity against and MoHT120). As shownpa- pain. This 1A, in Figure unusual physicochemical the inhibitory activity stability is a remarkable against papain feature even was maintained for phytocystatins. after 120 min So of far, there is only incubation, one previous suggesting report describing an extraordinary a papain inhibitor thermostability isolated from of the inhibitor. Vigna It is worth unguiculata mentioningseeds thatproperty that this presentedis both very thermal stability interesting andbiotechnological for most a similar tolerance to a wide applications, range behindofanpHs [33].value for their commercial exploitation [32]. added Figure Figure 1. 1. Isolation Isolationandandpurification purification ofM. oleifera of M. papain oleifera papaininhibitor (MoPI) inhibitor fromfrom (MoPI) the crude extract the crude (MoCE). extract (A) Papain (MoCE). inhib- (A) Papain itory activity (%) in the presence of the different M. oleifera heat-treated samples. MoCE: M. oleifera crude inhibitory activity (%) in the presence of the different M. oleifera heat-treated samples. MoCE: M. oleifera crude extract, extract, MoHT15- 120: M. oleifera M. MoHT15-120: crude extract oleifera crudeafter 100 °C extract heat after treatment 100 for 15, 60 for ◦ C heat treatment and15, 12060min. and *120 p