**About the Editor**

**Hang Fai (Henry) Kwok** (Associate Professor, Biomedical Sciences/Consultant, Histopathology at the Faculty of Health Sciences University of Macau): Prof. Kwok obtained his first-class BSc (Hons) degree and PhD in Biomedical Sciences in the UK in 2000 and 2003, respectively. He then became a Knowledge Transfer Partnerships Fellow as a postdoctoral researcher at Queen's University Belfast, a Top UK Pharmacy School. After 4 years of postdoctoral training, he moved to the pharmaceutical industry as a Senior Scientist from 2007 to 2011. Then, Prof. Kwok returned to academia as a Senior Research Fellow in the Department of Oncology at the University of Cambridge, bringing together his interests in protease biochemistry research with biologics (antibody/peptide drug) development to pursue novel therapeutic and prognostic approaches in the treatment of cancer and other chronic diseases. Apart from protease and antibody/peptide drug research, Prof. Kwok is also interested in the isolation and characterization of novel bioactive molecules from sources in nature, including amphibian defensive skin secretions and reptile, scorpion and insect venoms, for their anticancer and/or antimicrobial therapeutic potential.

## *Editorial* **Novel Natural-based Biomolecules Discovery for Tackling Chronic Diseases**

#### **Hang Fai Kwok 1,2**

Received: 20 October 2020; Accepted: 19 November 2020; Published: 15 December 2020

In the last decade, natural-derived/-based biomolecules have continuously played an important role in novel drug discovery (as a prototype drug template) for potential chronic disease treatment. Many recent research studies have demonstrated that the development of natural peptide/protein-based, toxin-based, and antibody-based drugs can significantly improve the biomedical efficiency of disease-specific therapy.

The focus of this Special Issue of Biomolecules includes eleven papers: ten original research articles and one communication article from nine different countries/regions dealing with a broad range of the discovery and development of the natural biomolecules as potential medical therapy for tackling chronic diseases (e.g., cancer, diabetes, cardiovascular diseases, rheumatoid arthritis, and pain treatment)

Four cancer-related research articles by Wang Q. et al. [1], Miao Y. et al. [2], Martínez-García D. et al. [3], Thangaraj K. et al. [4], and Lai Y. et al. [5] demonstrate that several natural-based compounds/proteins could effectively influence the cancer formation and progression. These study findings unveil the relationship of the SUMO pathway and DAPK1 protein degradation, demonstrate the target modifications of novel protease could effectively and efficiently alter its anticancer bioactivity, study the survivin levels through potent STAT3 Inhibition in lung cancer, investigate the cell cycle arrest and mitochondria-mediated intrinsic apoptosis in colorectal carcinoma, and develop a Fucoidan-based drug delivery system by using hydrophilic anticancer polysaccharides to simultaneously deliver hydrophobic anticancer drugs, respectively.

For potential diabetes therapy, the fascinating study by Lee D. et al. [6] evaluates two isoflavonoids and a nucleoside which were isolated from the roots of *Astragalus membranaceus*. These bioactive compounds can improve insulin secretion in β-cells, representing the first step towards the development of potent antidiabetic drugs. Besides, the paper by Martínez-Navarro I. et al. [7] concludes that the anionic lipid environment and degree of solvation are critical conditions for the stability of segments with the propensity to form β-sheet structures. This situation will eventually affect the structural characteristics and stability of IAPP within insulin granules, thus modifying the insulin secretion.

Two articles deals with inflammatory-related diseases. The Lee J. S. et al. [8] study speculates on two characterized compounds, petasitesin A (1) and cimicifugic acid D (3), which are worthy of further pharmacological evaluation for their potential as anti-inflammatory drugs. In addition, Celiksoy V. et al. [9] aimed to examine punicalagin in combination with Zn (II), and demonstrate that this novel combination promotes anti-inflammatory and fibroblast responses to aid oral healing.

The article by Sz ˝ucs E. et al. [10] explored the biological effect of novel opioid peptide analogs incorporating L-kynurenine (L-kyn) and kynurenic acid (kyna) in place of native amino acids. This novel oligopeptide exhibits a strong antinociceptive effect after i.c.v. and s.c. administrations in in vivo tests, according to good stability in human plasma which has a potential for tackling pain syndromes.

Finally, the short communication paper by Maheshwari G. et al. [11] tested the hypothesis that monomethyl branched-chain fatty acids (BCFAs) and a lipid extract of Conidiobolus heterosporus (CHLE) can activate the nuclear transcription factor peroxisome proliferator-activated receptor alpha (PPARalpha). In conclusion, they showed that the monomethyl BCFA isopalmitic acid (IPA) IPA is a potent PPARalpha activator. CHLE activates PPARalpha-dependent gene expression in Fao cells, an effect that is possibly mediated by IPA.

Overall, this Special Issue describes important findings related to natural-derived/-based biomolecules for potential chronic diseases treatment by dysregulating several biological pathways/receptors. It also highlights the most recent progress on the knowledge and the clinical and pharmacological applications related to the most relevant areas of healthcare.

**Acknowledgments:** The editor is grateful to all the important authors who contributed to this Special Issue "Novel Natural-based Biomolecules Discovery for Tackling Chronic Diseases". They are also mindful that without the rigorous and selfless evaluation of the submitted manuscripts by external peer reviewers/expertise, this Special Issue could not have happened. Moreover, the editor (Kwok H. F.) thanks for the support from the Science and Technology Development Fund (FDCT) of Macau SAR [File no. 0055/2019/A1 and 019/2017/A1] and the Faculty of Health Sciences (FHS) University of Macau [File no. MYRG2015-00025-FHS]. Finally, the valuable contributions, organization, and editorial support of the MDPI management team and staff are greatly appreciated.

**Conflicts of Interest:** The author declares no conflict of interest.

#### **References**

11. Maheshwari, G.; Ringseis, R.; Wen, G.; Gessner, D.K.; Rost, J.; Fraatz, M.A.; Zorn, H.; Eder, K. Branched-Chain Fatty Acids as Mediators of the Activation of Hepatic Peroxisome Proliferator-Activated Receptor Alpha by a Fungal Lipid Extract. *Biomolecules* **2020**, *10*, 1259. [CrossRef] [PubMed]

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

© 2020 by the author. 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/).

## *Article* **Regulation of the Expression of DAPK1 by SUMO Pathway**

**Qingshui Wang 1,**†**, Xiuli Zhang 1,**†**, Ling Chen 1,**†**, Shuyun Weng 1, Yun Xia 1, Yan Ye 1, Ke Li 1, Ziqiang Liao 1, Pengchen Chen 1, Khaldoon Alsamman 2, Chen Meng 1, Craig Stevens 3, Ted R. Hupp 4 and Yao Lin 1,\***

Received: 15 March 2019; Accepted: 15 April 2019; Published: 17 April 2019

**Abstract:** Death Associated Protein Kinase 1 (DAPK1) is an important signaling kinase mediating the biological effect of multiple natural biomolecules such as IFN-γ, TNF-α, curcumin, etc. DAPK1 is degraded through both ubiquitin-proteasomal and lysosomal degradation pathways. To investigate the crosstalk between these two DAPK1 degradation pathways, we carried out a screen using a set of ubiquitin E2 siRNAs at the presence of Tuberous Sclerous 2 (TSC2) and identified that the small ubiquitin-like molecule (SUMO) pathway is able to regulate the protein levels of DAPK1. Inhibition of the SUMO pathway enhanced DAPK1 protein levels and the minimum domain of DAPK1 protein required for this regulation is the kinase domain, suggesting that the SUMO pathway regulates DAPK1 protein levels independent of TSC2. Suppression of the SUMO pathway did not enhance DAPK1 protein stability. In addition, mutation of the potential SUMO conjugation sites on DAPK1 kinase domain did not alter its protein stability or response to SUMO pathway inhibition. These data suggested that the SUMO pathway does not regulate DAPK1 protein degradation. The exact molecular mechanism underlying this regulation is yet to be discovered.

**Keywords:** DAPK1; SUMO; SENP; protein degradation; post-translational modification

#### **1. Introduction**

Death-Associated Protein Kinase 1 (DAPK1) is an important serine/threonine kinase that is involved in multiple cellular processes such as apoptosis, autophagy, inflammation [1]. DAPK1 Plays a vital role in the anti-carcinogenic effects of many natural-based biomolecules, such as IFN-γ, TNF-α, curcumin, etc. [2,3]. Decreased expression of DAPK1 has been proven to be an unfavorable prognostic factor in bladder cancer, liver cancer and non-small cell lung cancer, etc. [4,5].

DAPK1 protein is composed of multiple functional domains including a catalytic kinase domain, a Ca2+/CaM auto-regulatory domain, eight ankyrin repeats, a Ras of Complex proteins (ROC)-C-terminus of ROC (COR) domain, a death domain and a serine-rich tail [6]. Dysregulation of DAPK1 expression or activity is often related to multiple diseases including cancer and stroke [7]. Recently, DAPK1 was also found to inhibit Hypoxia-inducible factor 1α (HIF-1α) in T cells [8], maintain epidermal tissue integrity through regulation of the microtubule cytoskeleton in C.elegans [9], and mediate pegylated Interferon-α (IFN-α)-induced suppression of hepatitis C virus (HCV) replication [10].

Expression of DAPK1 is often lost in cancers due to DNA methylation of the DAPK1 gene [11]. Due to DNA methylation, the expression of DAPK1 is lost in primary tumor samples of 26% of rectal cancer patients.  Previous studies revealed that the expression of the DAPK1 protein does not match its expression of mRNA in some cancers, indicating that the regulation of DAPK1 expression is a complex process. The degradation of DAPK1 protein is controlled by both proteasomal and lysosomal degradation pathways [15]. Three ubiquitin E3s, Mind Bomb 1(Mib1) [16], C-terminus of Hsc70-interacting protein (CHIP) [17] and KLHL20-Cullin3-RBX1 complex [18], target DAPK1 for ubiquitin-proteasome system (UPS)-mediated degradation. The lysosomal degradation pathway of DAPK was found to be late compared to proteasome degradation. Proteins known to be involved in the DAPK1 protein of lysosomal degradation include s-DAPK, Tuberin (TSC2) and cathepsin B. The TSC complex, formed by two proteins (TSC1 (hamartin) and TSC2), is a major regulator of the mTORC1 signaling pathway [19]. In our previous work, we discovered that TSC2 and a splice variant of DAPK1 (s-DAPK1) induced the lysosomal degradation of DAPK1 [20,21]. Moreover, a lysosomal protease, cathepsin B, is able to cleave DAPK1 in response to Tumor Necrosis Factor Receptor 1 (TNFR-1) over-expression [22].

Growing evidence demonstrates that there is intense crosstalk between UPS and lysosome [23]. Ubiquitination on proteins such as p62 can lead to their degradation by both UPS and lysosome [23]. Although DAPK1 has been shown to be degraded by both degradation pathways, it is not clear whether the ubiquitin related signaling pathways contribute to its lysosomal degradation. To investigate the crosstalk between these two DAPK1 degradation pathways, we carried out a screen using a set of ubiquitin E2 siRNAs and identified that the small ubiquitin like molecule (SUMO) pathway regulates DAPK1 protein levels.

SUMO is a family of ubiquitin-related modifiers that can be post-translationally conjugated to various substrates [24]. Intracellular proteins can be modified by SUMO, which affects substrate protein localization, stability, protein modification, and protein-protein interactions [25]. Five different SUMO paralogues have been reported in vertebrates, named SUMO-1 to SUMO-5 [24]. SUMO-1 shares 45% homology with SUMO-2/3, and there are only two amino acids difference between SUMO-2 and SUMO-3 [26]. SUMO-4 encodes a 95-amino acid protein having an 86% amino acid homology with SUMO-2 [27]. SUMO5 is a novel SUMO variant and contains a protein-coding sequence of 306 nucleotides [28]. The covalent modification reaction of SUMO is catalyzed by a series of enzymes including E1 activating enzyme (SAE1/SAE2), E2 binding enzyme (UBC9) and E3 ligase enzyme [29]. The process of SUMOylation is dynamic and reversible. A family of SUMO specific proteases (SENPs) are capable of removing SUMO from attached substrates and responsible for SUMO maturation [24]. Family members of SENPs include SENP-1, SENP-2, SENP-3, SENP-5, SENP-6 and SENP-7. The SENP family can be divided into three subfamilies based on the degree of amino acid sequence homology, cell localization, and substrate preference. The first subfamily comprises SENP-1 and SENP-2, which are located on the nuclear membrane and have the widest selection of substrates, which participate in the deubiquitination of proteins modified by SUMO-1 and SUMO-2/3 [30,31]. The second subfamily is SENP-3 and SENP-5. They are mainly found in nucleoli and involved in the synthesis of ribosomes and the regulation of cell mitosis [32]. The third subfamily is SENP-6 and SENP-7. They are located in the nucleus and are essential for the formation of multi-cluster ubiquitin chains [33,34].

In summary, the degradation of DAPK protein is regulated by both proteasome and lysosome. The research of E2 on degradation of DAPK1 protein will help to better understand the regulation of DAPK protein degradation by ubiquitin and ubiquitin-like small molecule signaling pathways and to discover the link between ubiquitin-related small molecule and lysosomal degradation signaling pathways.

#### **2. Materials and Methods**

#### *2.1. Cell Culture and Transfection*

HEK293 (human embryonic kidney cell line) and HCT116 (Human colon carcinoma) cells were obtained from ATCC (Manassas, MD, USA). Cell lines were examined for mycoplasma contamination using Mycoplasma Detection Kit (Vazyme, Nanjing, Jiangsu, China). Both cells were cultured in DMEM medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) and a penicillin and streptomycin mixture at 37 ◦C with 5% CO2 in a humidified atmosphere. Before harvesting, cells were first washed twice with PBS and then scraped into 1 mL of PBS. PCDNA3-HA-DAPK1 was a gift from Ted R. Hupp (University of Edinburgh). Flag-SENP1 (Plasmid #17357, deposited by Edward Yeh), FLAG-SENP2 (Plasmid #18047, deposited by Edward Yeh), RGS-SENP3 (Plasmid #18048, deposited by Edward Yeh), RGS-SENP5 (Plasmid #18053, deposited by Edward Yeh), FLAG-SENP6 (Plasmid #18065, deposited by Edward Yeh), 3xFLAG-SENP7 (Plasmid #42886, deposited by Edward Yeh) and Flag TSC2 wt (Plasmid #12132, deposited by Cheryl Walker) were obtained from Addgene (Cambridge, UK). The DAPK1 mutant constructs were generated using the QuikChange Lightning Site-Directed Mutagenesis Kit from Vazyme (Nanjing, China). All plasmids were sequenced to verify the integrity of the constructs. UBE2A siRNA (E-009424-00-0005), UBE2B siRNA(E-009930-00-0005), UBE2C siRNA(E-004693-00-0005), UBE2D1 siRNA (E-009387-00-0005), UBE2D2 siRNA (E-010383-00-0005), UBE2D3 siRNA (E-008478-00-0005), UBE2E1 siRNA(E-008850-00-0005), UBE2E2 siRNA (E-031782-00-0005), UBE2E3 siRNA (E-008845-00-0005), UBE2G1 siRNA (E-010154-00-0005), UBE2G2 siRNA (E-009095-00-0005), UBE2H siRNA (E-009134-00-0005), UBE2I siRNA (E-004910-00-0005), UBE2J1 siRNA (E-007266-00-0005), UBE2J2 siRNA (E-008614-00-0005), UBE2L3 siRNA (E-010384-00-0005), UBE2M siRNA (E-004348-00-0005), UBE2N siRNA (E-003920-00-0005), UBE2NL siRNA (E-031625-00-0005), UBE2Q1 siRNA (E-008631-00-0005), UBE2R2 siRNA (E-009700-00-0005), UBE2S siRNA (E-009707-00-0005) and UBE2V2 siRNA (E-008823-00-0005) were purchased from Dharmacon (Lafayette, MA, USA). The transfection was performed using lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's guidelines. To confirm that the difference in different lanes is due to the biological effect of the transfected plasmids, rather than technical differences, equal amounts of lacz in each plate were transfected as a co-transfection plasmid to balance the difference in transfection efficiency.

#### *2.2. Western Blot*

After harvesting, the cells were lysed in ice-cold lysis buffer (50 mM Tris (pH 8.0), 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 1% SDS, 1× protease inhibitor mixture (Roche, Basel, Switzerland)) for 30 min and centrifuged at 4 ◦C, 13,000 rpm for 10 min to remove insoluble material. The soluble protein concentration was determined by Bradford assay. Protein samples (60 μg) were separated by SDS-PAGE and transferred to nitrocellulose blotting membranes (Bio-Rad, Hercules, CA, USA). The membranes were treated with block buffer (5% non-fat milk in 0.1% TBST (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% Tween-20)) at room temperature for 1 h. The membranes were then incubated with primary antibodies overnight at 4 ◦C, then washing (3 × 12 min) in PBS/Tween 20, followed by incubating with secondary antibodies in blocking buffer at room temperature for 2 h. Finally, washing (3 × 12 min) in PBS/Tween 20 again. The signals were detected and measured using LICOR Odyssey system (LI-COR, Lincoln, NE, USA). All the western blots were repeated at least three times.

#### *2.3. Statistical Analysis*

Data were analyzed using Prism 5.0 software (Graphpad Software, Inc., La Jolla, CA, USA). Results are presented as the mean ± standard deviation of three independent experiments. The difference between the means were tested by the One-way ANOVA testing or Student's *t*-test, *p* < 0.05 was considered to indicate a statistically significant difference.

#### *2.4. Prediction of SUMOylation Sites*

In this study, prediction of the SUMOylation sites on the kinase domain on DAPK1 was performed using GPS-SUMO, which is a novel web server that can be used to predict potential SUMOylation sites (http://sumosp.biocuckoo.org/) [35].

#### *2.5. Antibodies and Chemicals*

Anti-GAPDH Antibody (2118) and anti-DAPK1 Antibody (3008) were purchased from Cell Signaling (Boston, MA, USA), anti-HA-Tag Antibody (902301) was purchased from Biolegend (San Diego, CA, USA), anti-flag Antibody (M20008) was purchased from Abmart (Shanghai, China), anti-SENP2 antibody (ab96865) was purchased from ABCAM (Cambridge, MA, USA), anti-Beta Galactosidase (β-gal) (*E. coli*) antibody (28449) was purchased from Rockland (Limerick, PA, USA). IRDye 800CW Goat-anti-Mouse (C60405-05), IRDye 680RD Goat-anti-Mouse (C60405-08), IRDye 800CW Goat-anti-Rabbit (C60607-15) and IRDye 680RD Goat-anti-Rabbit (C60329-15) were purchased from LI-COR (Lincoln, NE, USA).

MG132 was purchased from Calbiochem (LaJolla, NJ, USA) and used at 10 μM. Chloroquine and Cycloheximide (CHX) were purchased from Sigma (Louis, MO, USA) and used at 100 μM and 10 μg/mL, respectively. EST (E-64D) and Leupeptin were purchased from EMD Millipore Crop (Billerica, MA, USA) and used at 10 μg/mL and 200 μM respectively.

#### **3. Results**

To search the potential ubiquitin or ubiquitin-like regulatory pathways involved in TSC2-mediated DAPK1 protein degradation, a screen using an E2 siRNA library was carried out. Co-transfection of the siRNAs targeting three E2s (UBE2B, UBE2D1 and UBE2I) up-regulated HA-DAPK1 protein levels upon co-transfection of TSC2 (Figure 1A). Of these three E2s, UBE2I, also named UBC9, is the E2 for SUMO, which has been shown to participate in protein degradation [36]. Therefore, we co-transfected four different SENPs with TSC2 and HA-DAPK1. Co-transfection of SENPs enhanced the level of HA-DAPK1 protein (Figure 1B,C), but not to the level without TSC2 co-transfection, suggesting inhibition of SUMO pathway is not able to abrogate the effect of TSC2 towards DAPK1 protein levels.

Next, we co-transfected individual SUMO construct with HA-DAPK1. Co-transfection of neither SUMO construct resulted in significant down-regulation of HA-DAPK1 protein levels (Figure 2A). However, when SUMO-1 was co-transfected with SUMO-2 or SUMO-3, it significantly stimulated the reduction of the HA-DAPK1 protein levels, whereas co-transfection of SUMO-2 and SUMO-3 did not seem to pose additive effect (Figure 2B). Furthermore, all six known SENPs significantly enhanced the levels of HA-DAPK1 protein when co-transfected with HA-DAPK1 in HEK293T cells (Figure 2C) and HCT-116 cells (Figure 2D). Moreover, consistent with the exogenous expression data, when endogenous UBC9 was knocked down using siRNA, the endogenous DAPK1 protein also significantly increased (Figure 2E), suggesting SUMO pathway is able to regulate DAPK1 protein levels without simultaneously manipulation of TSC2-related pathway.

**Figure 1.** Inhibition of SUMO pathway partially restored DAPK1 protein levels at the presence of TSC2. (**A**) HEK293T was transfected with HA-DAPK1, Flag-TSC2, and different E2 siRNAs. (**B**,**C**) HEK293T cells were transfected with HA-DAPK1, LacZ, TSC2 and four different SENPs as indicated. Cell lysates were extracted 48 h post-transfection and immunoblotted with respective antibodies. The intensity of the bands was quantified using LICOR Odyssey software and represented by bar graphs. The experiments were repeated three times (n = 3), and representative images are presented. The representative western blot images are from different gels and each lane was loaded with the same amount of proteins. Data of triplicate assays are presented as mean ± S.D. \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001; NS, no significance. "+" indicated that the plasmid was transfected, "−" indicated that the plasmid was not transfected.

To further elucidate the underlying molecular mechanisms, a deletion series of DAPK1 constructs was created (Figure 3A) and co-transfected with three different SENPs. SENP1 (Figure 3B), SEP2 (Figure 3C) and SENP6 (Figure 3D) significantly enhanced the expression levels of all the deletion mutants, suggesting SUMO pathway regulates DAPK1 protein levels via the kinase domain. This is further confirmed when three SENPs displayed no effect towards the level of HA-DAPK1 (275–1430) lacking the kinase domain (Figure 3E).

Next, the HA-DAPK1 (1–364) was exposed to both proteasome and lysosome inhibitors. Only the proteasome inhibitor MG132 significantly enhanced the protein levels of HA-DAPK1 (1–364) in both HEK293T (Figure 4A) and HCT116 cells (Figure 4B), suggesting this kinase domain mutant HA-DAPK1 (1–364) is predominantly degraded via proteasome. In the protein stability assays

using cycloheximide (CHX), MG132 significantly enhanced HA-DAPK1 (1–364) protein stability (Figure 4C,D,F), whereas co-transfection of SENP6 was unable to enhance the stability of HA-DAPK1 (1–364) protein (Figure 4C,E,F), indicating that the SUMO pathway does not regulate DAPK1 protein levels via protein degradation. This also suggested that the SUMO pathway was unlikely to control DAPK1 protein levels through direct conjugation.

Using the GPS-SUMO system, we identified two potential SUMO conjugation sites on HA-DAPK1 (1–364). Therefore, we mutated these two sites separately or simultaneously (Figure 5A). As expected, the mutation did not influence the effect of SENP6 on DAPK1 (1–364) (Figure 5B), supporting that the SUMO pathway does not regulate DAPK1 protein levels via direct conjugation. Next, we compared the protein stability of HA-DAPK1 (1–364) and the mutants with potential SUMO conjugation sties mutated. No mutants were able to enhance the protein stability of HA-DAPK1 (1–364) (Figure 5C–E).

**Figure 2.** The SUMO pathway regulated the protein levels of DAPK1. HEK293T were transfected with (**A**) HA-DAPK1, LacZ, V5-UBC9, His-SUMO-1, His-SUMO-2 or His-SUMO-3, or (**B**) HA-DAPK1, LacZ, V5-UBC9, His-SUMO-1, His-SUMO-2 or His-SUMO-3, or (**C**) HA-DAPK1, LacZ and six different SENPs, or (**E**) control and UBC9 siRNA as indicated. HCT116 were transfected with (**D**) HA-DAPK1, LacZ and six different SENPs as indicated. Cell lysates were extracted 48 h post-transfection and immunoblotted with respective antibodies. The intensity of the bands was quantified using LICOR Odyssey software and represented by bar graphs. The experiments were repeated three times (n = 3) and representative images are presented. The representative western blot images are from different gels and each lane was loaded with the same amount of proteins. Data of triplicate assays are presented as mean ± S.D. \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001; NS, no significance. "+" indicated that the plasmid was transfected, "−" indicated that the plasmid was not transfected.

**Figure 3.** SENPs up-regulated DAPK1 protein levels via the 1-364 kinase domain. (**A**) A diagram illustrating the panel of DAPK1 deletion mutants. (**B–D**) The DAPK1 deletion mutants were co-transfected with LacZ and either Flag-SENP1 (**B**), Flag-SENP2 (**C**) or Flag-SENP6 (**D**), as indicated. (**E**) HA-DAPK (275–1430) mutant was co-transfected with LacZ and either Flag-SENP1, Flag-SENP2 or Flag-SENP6. Cell lysates were extracted 48 h post-transfection and immunoblotted with respective antibodies. The intensity of the bands was quantified using LICOR Odyssey software and represented by bar graphs. The experiments were repeated three times (n = 3) and representative images are presented. The representative western blot images are from different gels and each lane was loaded with the same amount of proteins. Data of triplicate assays are presented as mean ± S.D. \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001; NS, no significance. "+" indicated that the plasmid was transfected, "−" indicated that the plasmid was not transfected.

**Figure 4.** SUMO pathway did not regulate the protein degradation of HA-DAPK1 (1–364). (**A**) HEK293T cells or (**B**) HCT116 cells transfected with DAPK1 (1–364) and LacZ were exposed to MG132 (10 μM, 6 h) or leupeptin (200 μM), Est (10 μg/mL) and chloroquine (100 μM) for 24 h as indicated. (**C**) HEK293T transfected with LacZ, DAPK1(1–364) were exposed to 20 μg/mL CHX for 0–8 h as indicated. (**D**) HEK293T transfected with HA-DAPK1 (1–364) and LacZ were exposed to 10 μM MG132 and 20 μg/mL CHX for 0–8 h as indicated. (**E**) HEK293T transfected with LacZ, HA-DAPK1 (1–364) and Flag-SENP6 were exposed to 20 μg/mL CHX for 0–8 h as indicated. The statistical results of (**C**–**E**) are summarized in (**E**). Cell lysates were extracted and immunoblotted with respective antibodies. The intensity of the bands was quantified using LICOR Odyssey software and represented by trend lines. The experiments were repeated three times (n = 3) and representative images are presented. The representative western blot images are from different gels and each lane was loaded with the same amount of proteins. Data of triplicate assays are presented as mean ± S.D. \* *p* <0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001; NS, no significance. "+" indicated that the plasmid was transfected, "−" indicated that the plasmid was not transfected.

**Figure 5.** SUMO pathway did not regulate DAPK1 protein levels via direct conjugation. (**A**) A diagram illustrating the panel of DAPK1 (1–364) point mutants. (**B**) HEK293T cells were transfected with LacZ, SENP6 and the DAPK1 point mutants as indicated. (**C**–**E**) HEK293T transfected with LacZ, DAPK1 (1–364) point mutants were exposed to 20 μg/mL CHX for 0–8 h as indicated and the statistical results were summarized in (**F**). The intensity of the bands was quantified using LICOR Odyssey software and represented by bar graphs. The experiments were repeated three times (n = 3) and representative images are presented. The representative western blot images are from different gels and each lane was loaded with the same amount of proteins. Data of triplicate assays are presented as mean ± S.D. \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001; NS, no significance. "+" indicated that the plasmid was transfected, "−" indicated that the plasmid was not transfected.

#### **4. Discussion**

In our previous work, we discovered that TSC2 mediated the lysosomal degradation of DAPK1 via binding to the death domain of DAPK1 [20]. In this study, we discovered that inhibition of the SUMO pathway was able to enhance HA-DAPK1 protein levels at the presence of TSC2 co-transfection (Figure 1). However, the minimum domain of DAPK1 protein that the SUMO pathway is able to regulate is the kinase domain (Figure 3). The protein degradation of HA-DAPK1 (1–364) mutant is via proteasome (Figure 4). All these data suggest that the SUMO pathway regulates DAPK1 protein levels independent of TSC2. In this research, we co-transfected Lacz plasmid to balance the transfection variation and found that every single exogenous protein co-expressed with TSC2 shown decreased expression (Figure 1B,C). TSC2 is an important suppressor of mammalian target of rapamycin (mTORC1), which is a key regulator of translation and may be critical for exogenous protein levels. Therefore, when TSC2 was co-transfected, the expression of all other plasmids might be reduced due to inhibition of mTORC1 activity. The reason Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was stable could be because its translation is not mTORC1 dependent or its long half-life [37].

In two high-throughput mass spectrometry SUMOylation assays [38,39], DAPK1 protein was not found to be SUMOylated, suggesting DAPK1 may not be able to be SUMOylated, despite the presence of its potential SUMO conjugation sites. Our study is consistent with these two reports, but did not fully rule out the possibility that DAPK1 protein can be SUMOylated. It is only clear that even if DAPK1 protein can be SUMOylated, the SUMO conjugation is unlikely to affect the protein levels of DAPK1.

Although the SUMO pathway regulates DAPK1 protein levels, inhibition of the SUMO pathway does not change the protein stability of DAPK1 (Figure 4E). Therefore, the SUMO pathway probably regulates DAPK1 protein levels upstream of protein degradation. Considering that inhibition of the SUMO pathway enhanced the expression of exogenous HA-DAPK1, which only contains the coding region of DAPK1 mRNA, it is unlikely that SUMO pathway regulates DAPK1 protein levels through transcription. Recently, long noncoding RNAs (lncRNAs) have been shown to regulate gene expression at various levels, including chromatin modification, transcription and post-transcriptional processing [40]. Moreover, a new regulatory mechanism has been identified in which crosstalk between lncRNAs and mRNAs occurs by competing for shared microRNAs (miRNAs) response elements [41]. It is possible that the SUMO pathway regulates the expression of the miRNAs or lncRNAs targeting DAPK1, thus affect the levels of DAPK1 via these additional post-transcriptional regulatory pathways. It is also possible that the SUMO pathway regulates the expression levels or activity of proteins that are responsible for DAPK1 mRNA stability or translation.

In summary, our study discovered that the protein levels of DAPK1 can be regulated by the SUMO pathway. However, this regulation is not mediated via manipulation of DAPK1 protein degradation. The molecular mechanisms underlying this SUMO-mediated regulation of DAPK1 expression is still unclear. Further investigation is needed to elucidate this observation.

**Author Contributions:** Conceptualization, Q.W. and Y.L.; Data curation, X.Z. and L.C.; Methodology, X.Z., L.C., S.W., Y.X., Y.Y., K.L., Z.L., P.C. and C.M.; Resources, K.A., C.S. and T.R.H.; Writing–original draft, Q.W. and Y.L.; Writing–review & editing, Y.L.

**Funding:** This work was funded by the International S & T Cooperation Program of China (ISTCP, 2016YFE0121900), the Educational and Scientific Research Project for Young Scholars in Fujian Province (JAT170136), Natural Science Foundation of Fujian Province (2018J01723), scientific research innovation team construction program of Fujian Normal University (IRTL1702), United Fujian Provincial Health and Education Project for Tackling the Key Research (WKJ2016-2-27) and Fujian normal university (FZSKG2018008).

**Conflicts of Interest:** The authors have no conflicts of interest to declare.

#### **References**

© 2019 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/).

## *Article* **Discovery and Rational Design of a Novel Bowman-Birk Related Protease Inhibitor**

**Yuxi Miao 1,**†**, Guanzhu Chen 1,**†**, Xinping Xi 1, Chengbang Ma 1,\*, Lei Wang 1, James F. Burrows 1, Jinao Duan 2, Mei Zhou 1,\* and Tianbao Chen 1**

Received: 31 May 2019; Accepted: 12 July 2019; Published: 14 July 2019

**Abstract:** Anuran amphibian skin secretions are a rich source of peptides, many of which represent novel protease inhibitors and can potentially act as a source for protease inhibitor drug discovery. In this study, a novel bioactive Bowman-Birk type inhibitory hexadecapeptide of the Ranacyclin family from the defensive skin secretion of the Fukien gold-striped pond frog, *Pelophlax plancyi fukienesis*, was successfully isolated and identified, named PPF-BBI. The primary structure of the biosynthetic precursor was deduced from a cDNA sequence cloned from a skin-derived cDNA library, which contains a consensus motif representative of the Bowman-Birk type inhibitor. The peptide was chemically synthesized and displayed a potent inhibitory activity against trypsin (Ki of 0.17 μM), as well as an inhibitory activity against tryptase (Ki of 30.73 μM). A number of analogues of this peptide were produced by rational design. An analogue, which substituted the lysine (K) at the predicted P1 position with phenylalanine (F), exhibited a potent chymotrypsin inhibitory activity (Ki of 0.851 μM). Alternatively, a more potent protease inhibitory activity, as well as antimicrobial activity, was observed when P16 was replaced by lysine, forming K16-PPF-BBI. The addition of the cell-penetrating peptide Tat with a trypsin inhibitory loop resulted in a peptide with a selective inhibitory activity toward trypsin, as well as a strong antifungal activity. This peptide also inhibited the growth of two lung cancer cells, H460 and H157, demonstrating that the targeted modifications of this peptide could effectively and efficiently alter its bioactivity.

**Keywords:** amphibian Bowman-Birk inhibitor; Tat peptide; molecular cloning; antifungal; drug design; protease inhibitor

#### **1. Introduction**

Serine proteases are a widely studied group of proteins as they play various roles in healthy and diseased tissues. Serine protease inhibitors can also modulate a series of important biological processes, such as coagulation and inflammation, making them a focus for biomedical studies [1,2].

Plants are remarkable sources of the serine protease inhibitor, which can be grouped into at least 10 families. The Bowman-Birk family inhibitors (BBIs) are the best studied and most widely known among them. Identified in and isolated from soybean, they were the first to often be referred to as "classical BBI". However, subsequently multiple BBIs have been isolated from plants such as legumes and Gramineae [3–5].

The skin of frogs is the main organ involved in their defense system, which manufactures diverse bioactive peptides that possess cytolytic pharmacological activities [6], and as a result it is also an excellent source of protease inhibitors. To date, many BBIs isolated from amphibians have been reported, such as peptide leucine arginine (pLR) [7], peptide tyrosine arginine (pYR) [8], the Bowman-Birk-like trypsin inhibitor from *Huia versabilis* (HV-BBI) [9], Hejiang trypsin inhibitor (HJTI) [10], the Bowman-Birk-type inhibitor from *Odorrana schmackeri* (OSTI) [11], *Hylarana erythraea* chymotrypsin inhibitor (HECI) [12] and *Pelophyla esculentus Bowman-Birk proteinase inhibitor*(PE-BBI) [13]. Generally, the BBI peptides from amphibians possess a highly-conserved 11-residue canonical disulfide loop, which is different from plant BBIs. The structure of this peptide follows the consensus sequence, CWTP1SXPPXPC, with P1 representing the inhibitory active site and X indicating that various amino acids are found in these positions. This disulfide-bridged loop is considered a trypsin inhibitory loop (TIL), which has a significant trypsin inhibitory activity [14].

Based on previous studies, these amphibian BBIs not only have potent protease inhibitory activities, but also exhibit an antimicrobial activity. Antimicrobial peptides (AMPs) are attractive alternatives to producing novel antibiotics. However, their susceptibility to proteases appreciably limits the potential applications of most AMPs. Therefore, a bifunctional peptide possessing both antimicrobial and protease inhibitory activities, with a low cytotoxicity, could represent an ideal template for future clinical use [15].

In this report, a novel peptide from the defensive skin secretion of the Fukien gold-striped pond frog, *Pelophlax plancyi fukienesis*, was successfully isolated and identified, named PPF-BBI. It was shown to possess a potent trypsin and tryptase inhibitory activity with a high specificity. Several analogues were created by rational design, and a P1 site F substituted analogue displayed a considerable and specific chymotrypsin inhibitory activity. A better antimicrobial activity was observed when P16 was replaced by Lys, and the addition of the cell-penetrating peptide Tat48–56 resulted in a peptide with a strong antifungal activity. Moreover, anti-proliferative effects on H157 and H460 were also observed.

#### **2. Materials and Methods**

#### *2.1. Specimen Biodata and Secretion Harvesting*

Specimens of the Fukien gold-striped pond frog, *Pelophelax plancyi fukienensis* (n = 3, snout-to-vent length 7 cm) were captured in Fuzhou City, Fujian Province, China. All frogs were adults, and skin secretion was obtained by a mild electrical stimulation on the dorsal skin surface of the frogs [16]. The secretion was collected by washing the skin using deionized water and was lyophilized after the liquid nitrogen freezing. The obtained secretion was stored at −20 ◦C. This study is approved by the Nanjing University of Chinese Medicine Ethical Review Board-Approval Code: SYXK (SU) 2018-0048.

#### *2.2. Identification of Precursor-Encoding cDNA from the Skin Secretion*

The precursor encoding cDNA from the skin secretion was obtained as described previously [17]. Briefly, the 3 -RACE reactions employed a nested universal (NUP) primer (supplied with the kit) and a sense primer (S: 5 -GCIYTIMGIGGITGYTGGACIAA-3 ) that was complementary to the amino acid sequence, ALRGCWTK-, of PPF-BBI. The RACE reactions were purified and cloned using a pGEM-T vector system (Promega Corporation, Madison, WI, USA) and sequenced using an ABI 3100 automated sequencer. The nucleotide sequence of PPF-BBI has been deposited in the GenBank database under the accession number MK965542.

#### *2.3. Isolation and Identification of PPF-BBI from Skin Secretion*

The isolation and identification of the mature peptide from the skin secretion using the RP-HPLC and LC-MS analyses were performed as outlined previously [18]. A molecular mass analysis of the contents contained in the HPLC fraction was achieved by use of a matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometer (Voyager DE, Perseptive Biosystems, Framingham, MA, USA). The major peptide within this fraction was subjected to MS/MS fragmentation sequencing using an LCQ-Fleet mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA).

#### *2.4. Peptide Design and Solid Phase Peptide Synthesis of PPF-BBI, F8-PPF-BBI, K16-PPF-BBI, Tat-loop, Tat and Trypsin Inhibitory Loop*

There are three design strategies based on the obtained parent peptide, one to alter the inhibitory specificity, one to enhance the antimicrobial activity, and one to enhance the drug delivery and cell targeting. In the first case, since synthetic work on the BBI-like peptides has focused mostly on the P1 site, the lysine at the P1 site was replaced by a phenylalanine (F8-PPF-BBI; ALRGCWTFSIPPKPCP-NH2) which confers chymotrypsin inhibitory specificity. In the second case, the last amino acid residue, proline, was substituted with a lysine to give a positive charge and achieve a more structural similarity with the members of the Ranacyclin family, which have an antimicrobial activity (K16-PPF-BBI; ALRGCWTKSIPPKPCK-NH2). The trypsin inhibitory loop (TIL, CWTKSIPPKPC), derived from the amphibian Bowman-Birk-type protease inhibitor, is found to have a potent trypsin inhibitory activity. Thus, in the last case, a short cell-penetrating peptide Tat48-56 (RKKRRQRRR), which has been considered one of the most promising tools to improve the cellular delivery of therapeutic molecules [19–21], was added to the N-terminal of the TIL (Tat-loop; RKKRRQRRRCWTKSIPPKPC) by solid phase peptide synthesis. The Tat peptide and TIL were used in the antimicrobial assays for a comparison with the activity of Tat-loop. The TIL peptide was also involved in protease inhibitory assays to determine the influence on the inhibitory activity of the extended amino acid residues at both termini.

The novel-cloned cDNA-encoded peptide, wild-type PPF-BBI, and its analogues were synthesized by chemical synthesis using a Tribute peptide solid-phase synthesizer (Protein Technologies, Inc, Tucson, AZ, USA), as outlined previously [11]. The synthetic peptides were analyzed both by reverse phase HPLC and MALDI-TOF mass spectrometry to establish the degree of purity and the identity of the structure.

#### *2.5. Trypsin, Chymotrypsin and Tryptase Inhibition Assay*

The trypsin, chymotrypsin inhibition tests were performed as described previously [22]. 10 μL tryptase (1 mg/mL, Calbiochem, UK) was added to the wells of a micro-titer plate containing 180 μL substrate (Boc-Phe-Ser-Arg-NHMec, obtained from Bachem, UK) (50 μM) and 20 μL synthetic replicates (0.1–100 μM) in a tryptase buffer, pH7.6, containing 0.05 M Tris, 0.15 M NaCl, and 0.2% (*w*/*v*) polyethylene glycol 6000 (final volume 210 μL).

The rate of hydrolysis of the substrate was monitored by measuring the rate of increase of fluorescence due to the release of 7–amino–4–methylcoumarin (AMC) at 460 nm (excitation 360 nm) in a FLUOstar OPTIMA multi-well plate reader. The inhibition curves of the trypsin/chymotrypsin inhibition assay and tryptase inhibition assay were formed as outlined before [11,12].

#### *2.6. Minimal Inhibitory Concentration (MIC) Assay and Minimal Batericidal Concentration (MBC) Assay*

The MIC and MBC of the synthetic peptides were determined as previously described [17], using *S. aureus* (NCTC 10788), *E. coli* (NCTC 10418) and *C. albicans* (NCYC 1467), together with two species of resistant micro-organisms, methicillin-resistant *S. aureus* (MRSA) (ATCC 12493) and *P. aeruginosa* (ATCC 27853).

#### *2.7. Membrane Permeability Assay*

The membrane permeability assay was performed as described previously [20]. The peptides at concentrations of 1-fold MIC, 2-fold MIC and 4-fold MIC were mixed with a bacterial cell suspension. The membrane permeability rate was measured via the monitor of the fluorescent intensity of SYTOX Green Nucleic Acid Stain (Life Technologies, Glasgow, UK) by a Synergy HT plate reader with excitation at 485 nm and emission at 528 nm.

#### *2.8. Secondary Structure Analysis through Circular Dichroism (CD)*

The sample peptide solutions (50 μM) were prepared in a 1 mm high precision quartz cell (Hellma Analytics, Essex, UK) with 10 mM ammonium acetate and 50% TFE in 10 mM ammonium acetate buffer respectively. CD measurements were performed at 20 ◦C by a JASCO J-815 CD spectrometer (Jasco, Essex, UK) across the wavelength range of 190–250 nm. The scanning speed was 100 nm/min, the bandwidth was one nm, and the data pitch was 0.5 nm. The CD spectra were further analysed using the online software, BeStSel [23], and the proportion of different secondary structures were predicted.

#### *2.9.  The anti-metabolite 5-fluorouracil (5-FU) was utilized as the positive control group.

#### *2.10. Haemolysis Test*

The haemolytic activity of each peptide was measured by incubating a range of final peptide concentrations from 512 to 1 μM in a two-fold dilution in a 2% suspension of horse erythrocytes, as described in a previous study [20].

#### *2.11. Statitical Analysis*

The data of all the bioactive assays were statistical analyzed using Prism 6 (GraphPad Prism Software, GraphPad, San Diego, CA. USA). The data points represent the average of three independent experiments with the error bars representing the standard error of the mean (SEM).

#### **3. Results**

#### *3.1. Identification and Structural Determination of PPF-BBI*

From a skin-derived cDNA library, a cDNA encoding a biosynthetic precursor of PPF-BBI was consistently and repeatedly cloned (Figure S1). The crude skin secretion of *Pelophlax plancyi fukienesis* was analysed by LC-MS. The retention time of the fragmentation indicated in Figure S2 showed a corresponding molecular weight (Figure S3A) to the prediction for PPF-BBI according to the putative peptide from the cloned cDNA. The elution fraction was analysed by an electrospray mass spectrometer, and the primary structure sequence was confirmed (Figure S3B,C). The open-reading frame consisted of 65 amino acid residues. The alignment of this peptide with other members of the Ranacyclin family of Bowman-Birk-type protease inhibitors indicates that it is structurally related as its sequence shows a high degree of conservation, as well as including a typical inhibition loop (Figure 1), which started with a 22-residue putative signal peptide at the N-terminus. After the acidic spacer of 24-amino acids, the deduced mature peptide of 16 residues at the C-terminus is present in a single copy (Figure 1). The sequence was preceded by two consecutive basic amino acids, Lys-Arg (KR), representing a typical processing site for endoproteolytic cleavage, and was immediately followed by a glycine residue amide donor.

**Figure 1.** Multiple sequence alignment test results from Clustal Omega. Fully conserved residue indicated by asterisks.

#### *3.2. Peptide Design*

Three analogues were designed based on the natural peptide PPF-BBI (Table 1). Briefly, a substitution of phenylalanine at the P1 site, F8-PPF-BBI, was aimed to produce a chymotrypsin inhibitory peptide. The substitution of a lysine at the position 16 of the native peptide (K16-PPF-BBI) enhanced the net positive charge, which might improve the ability to interact with the cell membrane. The Tat sequence was added at the N-terminus of the typical 11-mer trypsin inhibitory loop structure to increase the membrane penetration effect. All the analogues were chemically synthesized, purified by RP-HPLC and analysed by MALDI-TOF.

**Table 1.** The sequence and positive charge of *Pelophlax plancyi fukienesis* Bowman-Birk-type inhibitor (PPF-BBI) and its rational design analogues.

The highly conserved loop is shaded, and the substituted sites are in bold.

#### *3.3. Synthesis and Secondary Structure Analysis of PPF-BBI and its Analogues*

PPF-BBI and the analogue peptides were successfully synthesized, impurities were removed by HPLC, and their identity was confirmed by MALDI-TOF. The secondary structures of all peptides were determined by circular dichroism spectroscopy (Figure 2). PPF-BBI, F8-PPF-BBI and K16-PPF-BBI revealed a broad negative band with the minimum around 200 nm, typical of an unfolded peptide in equilibrium with a β-sheet structure [24], except that Tat-loop exhibited the negative band close to 197 nm, which is considered to be a random coil. With the presence of 50% TFE, which is a secondary structure promoting the reagent, the negative minimum of PPF-BBI shifted from 200–203 nm to 206–210 nm and displayed a broad negative band. Additionally, F8-PPF-BBI and K16-PPF-BBI displayed the same red shift trend of the negative minimum. Since the spectra displayed a broad minimum spanning the region 200–210 nm and did not show positive bands above 210 nm, this suggests that the conformation of peptides is likely to consist of a mixture of secondary structures of β-sheet structure and random coil, which is consistent with previous studies [11,24–26]. Furthermore, the Tat peptide possess a random coil structure, the presence of which would not increase the helicity of the peptide [26].

**Figure 2.** Secondary structures of PPF-BBI and the analogues. The CD spectra of peptides were measured in their free form (aqueous 10 mM NH4AC buffer) and membrane-mimic 10 mM NH4AC/50% TFE buffer, respectively (PPF-BBI, red; F8-PPF-BBI, green; K16-PPF-BBI, blue; Tat-loop, black).

#### *3.4. Trypsin, Chymotrypsin and Tryptase Inhibitory Activity of PPF-BBI and its Analogues*

PPF-BBI and its analogues were tested for inhibitory activity against trypsin, chymotrypsin and tryptase, respectively. The progress curves for the hydrolysis of the fluorogenic substrate were used to estimate an initial rate (Vi) to generate the Morrison plots. All of the progress curves and corresponding Morrison plots in the presence of each peptide of different concentrations are shown in Figure S4. Among these, both wild type PPF-BBI and K16-PPF-BBI exhibited a potent trypsin inhibitory activity. However, K16-PPF-BBI had a more potent inhibitory effect against tryptase than the parent peptide did. In addition, F8-PPF-BBI only displayed a strong chymotrypsin inhibitory activity and lost the trypsin inhibitory activity. Interestingly, the trypsin inhibitory loop (TIL) and Tat-loop kept the trypsin inhibition activity but did not exhibit a tryptase inhibitory activity (Table 2).

**Table 2.** PPF-BBI and its analogues against trypsin, chymotrypsin and tryptase.

The highly conserved loop is shaded, and the substituted sites are in bold. N.I.\* means that no inhibition was observed.

#### *3.5. Antimicrobial Activity*

The antimicrobial activity of PPF and its analogues was tested against a representative set of microorganisms (Table 3). Both PPF-BBI and K16-PPF-BBI displayed a mild activity against the tested microorganisms, although K16-PPF-BBI exhibited a better bioactivity than the parent peptide against *S. aureus* and *C. albicans*. However, although its activity against the other microorganisms was similar to the parental peptide, Tat-loop showed a much more potent activity against *C. albicans*. The component parts of Tat-loop (separate Tat peptide and the TIL) were also tested, but had little activity on their own. To sum up, Tat-loop showed a potent activity against *C. albicans*, as well as exhibiting slightly more activity against MRSA and *P. aeruginosa*. K16-PPF-BBI exhibited the best activity against *S. aureus* and was slightly better than PPF-BBI against all of the others except *E. coli*.

**Table 3.** The minimal inhibitory concentrations (μM) and minimal bactericide concentrations (μM) of PPF-BBI and the synthetic analogue peptides against microorganisms.

#### *3.6. Membrane Permeability*

The MIC and MBC results indicated that only Tat-loop had a potent activity on any of the tested microorganisms. Therefore, it was tested to determine if its impact against *C. albicans* was due to it impacting its membrane permeability. However, Tat-loop did not cause any membrane permeabilization, even at high concentrations (4-fold of its MIC versus *C. albicans*, Figure 3).

**Figure 3.** The cell permeability of *C. albicans* treated for 2 h by Tat-loop at 1-fold, 2-fold and 4-fold of MIC. The membrane permeabilized cells by 70% isopropanol were used as the positive control (100% permeability).

#### *3.7. Anti-Cancer and Haemolytic Activity*

PPF-BBI and its analogues were subjected to an MTT assay using a series of lung cancer cell lines (H460, H157, H23 and H838) and other cancer cell lines (HT29, PC-3, U251MG), and were also tested on a human normal cell (HMEC-1) (Figure 4a). Among these, only Tat-loop inhibited the growth of H460 and H157 at a concentration of 100 μM. All of the peptides showed a slight inhibition on HMEC-1. Furthermore, they also exhibited a low degree of haemolytic activity on horse erythrocytes (Figure 4b).

(**a**)

**Figure 4.** *Cont*.

**Figure 4.** (**a**) The cell viability of the cancer cell lines H460, H157, H23, H838, HT-29, PC-3, U251MG and HMEC-1 at 4 mM 5-FU (stripe bar), 100 μM and 10 μM PPF-BBI, F8-PPF-BBI, K16-PPF-BBI and Tat-loop. The control represents the cell viability without any treatments. The statistical significance of difference was analyzed by a one-way ANOVA (\* *p* < 0.05, \*\*\*\* *p* < 0.0001). (**b**) The haemolysis rates of PPF-BBI, F8-PPF-BBI, K16-PPF-BBI and Tat-loop on erythrocytes after being incubated for 4 h. The incubation of erythrocytes with 2% (*v*/*v*) Triton X-100 was designated as a positive control (100% haemolysis).

#### **4. Discussion**

In this study, we report the identification and bioactivity evaluations of PPF-BBI, a novel Bowman-Birk type protease inhibitor from the skin secretion of the Fukien gold-striped pond frog, *Pelophylax plancyi fukienensis*. In addition, we also examine the bioactivity of three rationally designed analogues of PPF-BBI, F9-PPF-BBI, K16-PPF-BBI and Tat-loop.

Like other BBI-type peptides, PPF-BBI has potent protease inhibitory activities. The specificity of inhibition is determined by whether the P1 position residue can fit into the S1 pocket of protease. Based on previous reports, Lys as the P1 position is optimal for trypsin inhibition, and Phe is optimal for chymotrypsin [11,27]. Similarly, PPF-BBI, which has a Lys at the P1 site, showed a strong trypsin inhibitory activity and substitution of the Lys at the P1 position, with Phe leading to the elimination of the trypsin inhibition and giving rise to a chymotrypsin inhibitory activity. In the tryptase activity assay, PPF-BBI displayed a mild potency toward tryptase with a Ki value of 30.52 μM. K16-PPF-BBI shows a three-fold better inhibition with a Ki value of 9.67 μM, but intriguingly, Tat-loop and TIL lost their inhibitory activity against tryptase, even though they retained their trypsin inhibitory activity. Indeed, this contradicts a previous study [28], which demonstrated that short BBI-derived cyclic peptides had an inhibitory activity against tryptase, even though tryptase is seen as unique due to its resistance to all known endogenous proteinase inhibitors [29]. As the data of K16-PPF-BBI showed here, the substitution of Lys at the C-terminus improved the tryptase inhibitory activity, indicating that the C-terminal extensive residue might contribute to the binding between the BBI peptide and tryptase. Therefore, the lack of such an extension of TIL and Tat-loop could eliminate the affinity to the reactive pocket of tryptase, so that both cannot produce any inhibitory activity.

The mode of action of Ranacyclins is different from most known positively charged antimicrobial peptides. They bind and insert into both zwitterionic and negatively charged membranes, and they presumably form transmembrane pores without bacteria wall damage. Indeed, it has been reported that Ranacyclins E and T have a great potential as antimicrobials [30]. PPF-BBI and K16-PPF-BBI shared a high sequence similarity with Ranacyclin members, and they were also found to have moderate effects on the tested microorganisms, as was expected. However, K16-PPF-BBI was shown to have a

better effect, which is possibly because one more lysine increases its positive charges, and it is easier to get it close to the negative groups of the cell membrane.

Tat, a cationic-rich cell-penetrating peptide derived from the HIV protein, has been used to conjugate with other compounds to enhance the cell penetrating activity. In the previous study, the design of Tat-fusion biopeptides demonstrated a remarkable improvement on their biological activities [20,21]. In the meantime, the BBI trypsin inhibitory loop was also considered as a drug template that was applied in some studies [14,31]. Therefore, a combination of the Tat peptide with TIL was conducted here, one that could attempt to introduce the cell-penetrating effect and that possessed an inhibition against trypsin-like activity intracellularly.

Interestingly, this is reflected in the significant increase of potency against the tested strains (especially *C. albicans* with MIC of 4 μM) of Tat-loop. However, this does not appear to be due to its impact upon the membrane, as treatment with this peptide does not induce changes in membrane permeability even at high concentrations (4-fold of its MIC versus *C. albicans*). Using the principle of plant protease inhibitors, it would be pertinent to evaluate the antifungal activity of Tat-loop, possibly because it might interfere with the trypsin-mediated activation of the chitin synthase zymogen and further affect the process of cell wall development [32].

In the report by Zhang et al. [12], a Bowman-Birk type chymotrypsin inhibitory peptide, HECI, and its analogue, K9-HECI, exhibited great anti-proliferative potency against H157, PC-3 and MCF-7. Also, PE-BBI was reported to have an inhibition effect on several colon cancer cell lines [13]. In our study, Tat-loop suppresses the growth of the lung cancer cells H460 and H157. It was not clear whether Tat-loop could enter the nucleus and bind the target receptor in vitro; compared to the native peptide, Tat-loop exhibited a slight improvement of the anti-proliferative activity. Moreover, a low haemolytic activity was observed when Tat-loop was assessed, which further supports the data which indicates that it does not exhibit cytotoxicity. Similarly, no significant haemolytic activity was observed with the parental peptide and the other synthetic analogues. This indicates that the Tat-loop will not have toxicity issues and lays a solid foundation for further in vivo research.

In addition, PPF-BBI, F9-PPF-BBI and K16-PPF-BBI initiated a slight conversion from a random coil into a β-structure in the TFE buffer due to assisting the folding of the secondary structure [33]. However, Tat-loop did not show the band shift to 210 nm, but around 197 nm, which indicated that it mainly formed a random coil in both aqueous and TFE buffer [26]. We assume that BBI-related peptides might exhibit a certain degree of β-sheet structure involved in binding with enzyme; therefore, Tat-loop demonstrated a lower inhibitory activity against trypsin compared with the others. Although the spectra data showed slightly different results, the major part of the secondary structure of the BBI-related peptides is random coil and β-sheet, which is consistent with previous studies [11,25,34].

#### **5. Conclusions**

In summary, PPF-BBI is a naturally occurring peptide with a remarkable trypsin and tryptase inhibitory activity, as well as a moderate antimicrobial activity. This provided the basis for the rational design of further multifunctional protease inhibitors. Moreover, this is the first report investigating the addition of a cell-penetrating peptide to an amphibian skin-derived protease inhibitor. Tat-loop has high potency against *C. albicans*, which also results from its inhibition of trypsin, which might have potential towards fungal diseases. However, the mechanism by which Tat-loop impacts upon *C. albicans* is unclear, and further research is required to determine how this peptide exerts its impact upon this fungus.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2218-273X/9/7/280/s1, Figure S1: The nucleotide and translated open-reading frame amino acid sequence of cloned cDNA encoding the biosynthetic precursor of PPF-BBI from a skin secretion of *Pelophylax plancyi fukienensis*. Figure S2. Region of reverse-phase HPLC chromatogram of the skin secretions of *Pelopholax plancyi fukienesis*. Figure S3. The identification of PPF-BBI from the skin secretion of *Pelopholax plancyi fukienesis*. Figure S4. The identification of other peptides used in this study. Figure S5. The inhibitory activity of PPF-BBI and the analogues on trypsin, chymotrypsin and tryptase.

**Author Contributions:** Conceptualization, L.W., J.F.B., M.Z. and T.C.; Formal analysis, C.M.; Investigation, Y.M. and G.C.; Methodology, X.X., C.M. and J.D.; Project administration, T.C.; Resources, J.D.; Supervision, J.F.B. and M.Z.; Validation, X.X., C.M. and M.Z.; Visualization, Y.M. and G.C.; Writing–original draft, Y.M.; Writing–review & editing, X.X. and J.F.B.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

© 2019 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/).

*Article*


**Author details**

Zahra Naeef Ayoub Department of Plant Protection, College of Agriculture, University of Duhok, Kurdistan Region, Iraq

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© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


**2.1 Sources of drugs**

290 Complementary Pediatrics

appropriate despite strict control programs and such effort (Akan, 2006; Çetinkaya et al, 2010). In Turkey, antibiotic treatment is prescribed too frequently (Bal, 2005; Celen et al, 2005; Çetinkaya et al, 2010; Özgüneş, 2005;). In a study it is stated that antibiotics are being prescribed to significant portion of the patients who apply to clinics (15-48%) and only 2- 2.5% of those prescribed medication was based on culture results (Çetinkaya et al, 2010;

Since the prevalence of drug errors are high, it is that imperative that nurses understand the

Without considering by whom it was prescribed, nurses are responsible for the every each medication that is administered personally on legal grounds, moral grounds, and ethical

All professional nurses should take these issues seriously. Safe and correct medication is one of the principal responsibilities of a nurse during patient care. For a nurse to make free decisions over right medication, correct administration, and providing appropriate means for measurement and monitoring is important for the assessment of the side effects of medication and the patient reactions against it. Reliable medication requires knowledge

Comparing to that on an adult, medication administration greatly differs on children, while bringing along responsibilities. It is of the physician's responsibility to write down the doses into drug master file. Nurse, on the other hand, is responsible for administering the medication at the correct amount and on the correct time. Nurse has to know when this medication would start to be effective, for how long it would be effective, what side-effects it might cause, any

In a study carried out to analyze the knowledge and behavior of pediatricians regarding to the rational use of antibiotics, as well as the socio-demographic factors that might be affecting, 89.8% of the participants reported that, when prescribing antibiotics, they need to see the patient first, whereas, 78.1% indicated that they were prescribing antibiotics according to the patient's clinical condition, 71.1% reported that they had paid attention to the appropriateness of the symptoms, and 67.2% told that they were going to take microbiological culture samples for examination. As for the question '*who should give education about antibiotics*' 32.1% replied as the physician who had prescribed the medication, 23.4% said the junior doctors, 21.9% said pharmacists, 17.2% said the pediatric nurse, 9.4%

The pediatrics nurses auditing the medication use, evaluation by them if the importance of regular use of the medication is understood or not, to obtain the suitable feedback from the patent and the patient's family by the prescribing physician, adverse effects and what they should do under such circumstances etc., providing training and consultancy on all these

If taken by a living organism, drug is an agent that brings changes in body functions. Administration of drugs is of the principal practices in nursing. Safety of the patient is the

factors leading to errors, and avoid them to the best of their ability (Dinc, 2011).

synthesis, experience, critical approach and intellectual norms (Dinç, 2011).

toxic indications, and counter-measures in respect thereof (Kavakli et al, 1998).

are among their duties (Çetinkaya et al, 2010; Çetinkaya &Tengir, 2006).

Özgüneş, 2005).

grounds (Dinç, 2011).

said nurses (Çetinkaya et al, 2010).

**2. Pharmacological concepts**

These are the basic sources of drugs: