Research Article

Novel Oligopeptides with Selective Cytotoxicity Against Human and Mouse Melanoma Cells

Basil M. Hantash*

Escape Therapeutics, Inc, 3800 Geer Road #200, Turlock, CA, USA

*Corresponding author: Basil M. Hantash, Escape Therapeutics, Inc, 3800 Geer Road #200, Turlock, CA, USA. E-mail: basil@escapetherapeutics.com

Citation: Hantash BM (2020) Novel Oligopeptides with Selective Cytotoxicity Against Human and Mouse Melanoma Cells. J Dermatol Surg Res Ther 2020: 03-13.

Received Date: 22 December, 2020; Accepted Date: 09 February, 2020; Published Date: 16 February, 2020

Abstract

Background: Melanoma continues to be a devastating and costly cancer with very few effective therapeutic or preventive options.

Aim: To assess cytotoxicity effects of two novel oligopeptides on mouse and human melanoma cells.

Methods: Melanoma, melanocyte, epidermal keratinocyte, and fibroblast cells were exposed to various concentrations of oligopeptides or hydroquinone (HQ) for various times and cellular viability and proliferation were measured.

Results: Oligopeptides were found to be cytotoxic to mouse and human melanoma cells but not to primary human melanocytes, keratinocytes, or fibroblasts while HQ was broadly cytotoxic.

Conclusion: Oligopeptides P5 and P14, but not HQ, hold promise as selective cytotoxic agents against human melanoma.

Keywords: Hydroquinone; Melanoma; Oligopeptide; PLX4032; RAF; Sorafenib

Introduction

The incidence of malignant melanoma has been increasing at an alarming rate worldwide with over 96,000 new cases and 7,230 deaths expected in the US alone in 2019 [1], leading to direct costs of over $8 billion [2]. This does not include its incalculable psychosocial impact. Melanoma originates in melanocytes, most commonly in the skin. Ultraviolet radiation (UVR) exposure appears to be the greatest melanoma risk factor through pleiotropic mechanisms including suppression of the skin immune system, induction of melanocyte cell division, free radical production, and damage of melanocyte DNA [3]. Other risk factors include history of sunburn, xeroderma pigmentosum, and tanning bed us [4-8]. Twenty-five percent of melanoma occurs in conjunction with a preexisting nevus [9-11]. Treatment of melanoma depends on early recognition of superficial tumors, highlighting the importance of early detection and the need more efficacious therapeutic options.

Over the past decade, improved knowledge of the mitogen-activated protein kinase (MAPK) pathway and its implication in cancer development has led to attempts to target mutated forms of oncogenic kinases such as B-RAF, extracellular-signal-regulated kinases (ERK), and MAPK/ERK kinase (MEK) [12]. B-RAF is a member of the RAF kinase family, which acts in the MAPK/ERK signaling pathway responsible for regulating cellular proliferation, differentiation, and survival. Of the three RAF kinases (A-RAF, B-RAF and C-RAF), only B-RAF is frequently mutated in various cancers [12]. A single base missense substitution (T to A at nucleotide 1,799), that changes valine to glutamic acid at codon 600 (V600E) in exon 15, accounts for 90% of all BRAF mutations within melanoma tumors [13]. The consequence of this mutation is a 500-fold increase in kinase activity relative to the wildtype form, leading to constitutive upregulation of ERK signaling and subsequent unregulated cell growth, cell transformation, and eventual tumor initiation [14,15]. More recently, the novel serine threonine kinase STK19 was characterized and implicated in melanomagenesis [16].

To date, there are no preventative treatments offered for high risk groups other than sun avoidance, application of physical sunscreens with SPF ≥ 30, and routine skin surveillance by a dermatologist. To meet this urgent unmet medical need, we screened a series of novel oligopeptides with high affinity for both kinases B-RAF^V600E and C-RAF using molecular modeling in silico and compared them to the known kinase inhibitors sorafenib and PLX4032 [17-19]. Herein, we describe the identification of two oligopeptides, P5 and P14, with selective cytotoxic activity towards multiple melanoma cell lines in vitro.

Materials and methods

Oligopeptide synthesis

Oligopeptides P5, P14, and P15 (Figure 1) were synthesized by Bio Basic, Inc. (Ontario, Canada) using solid-phase FMOC chemistry. Hydroquinone (HQ) was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Figure 1. Chemical structure of hydroquinone and oligopeptides P5, P14, and P15 with amino acid composition noted parenthetically.

Molecular modeling of BRAF^V600E and CRAF inhibitors

Enzyme 3-D X-ray crystallography structures of wild type B-RAF (PDB ID: 1UWH, chain A), mutated B-RAF^V600E (PDB ID: 3C4D, chain A) and C-RAF (PDB ID: 3LB7, chain A) were obtained from PDB. Oligopeptides were docked into the ATP binding pocket of each prospective enzyme using AutoDock version 4.2.2.1 and AutoDock Vina version 1.0.3 [20-22]. Enzymes were prepared for docking by replacing missing hydrogens, removing water, NO³⁺, and Ca²⁺ ions, adding Kollman charges, and computing Gasteiger charges using Swiss-PdbViewer version 4.0.1 and AutoDockTools version 1.5.4. Sorafenib and PLX4032 structures were obtained from the PubChem Compound Database [23]. Peptide ligands were created using PyMOL version 1.1 beta3 or Avogadro version 1.0.0 and saved as “.pdb” files. The ligands were then processed using the Dundee PRODRG2 server [24] and AutoDockTools to determine charges and torsions. Each peptide was allowed all possible torsions. The binding modes of these inhibitors to B-RAF^V600E and C-RAF were analyzed through molecular docking to derive structure-activity relationships.

Cell culture

Human and mouse melanoma cell lines as wells as fibroblasts were cultured using DMEM with L-glutamine, 10% fetal bovine serum, and 1% penicillin/streptomycin (Thermo Fisher Scientific, NY, USA). Human melanocytes were cultured using medium 254 (Thermo Fisher Scientific) and incubated at 37 °C in a humidified 5% CO₂chamber. Human neonatal epidermal keratinocytes were cultured in Epilife media containing 60 µM of calcium chloride (Thermo Fisher Scientific). All cell cultures were incubated in a humidified chamber at 37 °C and 5% CO₂.

Viability/proliferation and cytotoxicity assays

Proliferation rates were determined using a TACS^® MTT Cell Proliferation Kit (R&D systems, Minneapolis, MN). Cells were seeded at 2.5 × 10⁴/well in 96-well plates in a humidified atmosphere with 5% CO₂ at 37 ºC. Twenty-four hours later, oligopeptides or HQ were added to the corresponding wells at varying concentrations (0.03, 0.1, 0.3,1 and 3 mM), and cultures were incubated for 72h. The remainder of the procedure was performed following the manufacturer’s protocol.

Cellular toxicity

Cellular toxicity was measured using a trypan blue dye exclusion assay. Cells were cultured in 6-well plates at a density of 4 x 10⁵ cells/well. Each well received a different concentration of oligopeptide or HQ (0.03, 0.1, 0.3, 1, and 3 mM). Plates were incubated at 37 °C in a humidified 5% CO₂chamber. After 24 h, an aliquot was taken, and cells counted using a hemocytometer. Cytotoxicity was measured according to the following formula:

[1- (# of cells in control) - (# of live cells in test sample)] X 100%

# of cells in control well

The same procedure was repeated after 3 and 6 d of incubation.

Results

Molecular docking of oligopeptide P14 and BRAF^V600Einhibitors

P14 PLX4032 and sorafenib served as benchmarks for in silico screening of our internally generated oligopeptide library. The region (activation loop) encompassing the V600E mutation in B-RAF, which consequently induces the conformational change and hyperactivation of the kinase, is highlighted in green (Figure 2A & Figure 2B). Figure 2 also shows that despite the significant interaction similarities between oligopeptide P14 (Figure 2A) and PLX4032 (Figure 2B) with the ATP binding pocket, P14 demonstrated a closer interaction with the activation loop. P14 formed several hydrogen bonds, which in combination with hydrophobic interactions pulled it to within 2-3 Å from important residues in the ATP binding pocket (Figure 2C) versus 3-4 Å for PLX4032 (Figure 2D).

Figure 2. Molecular docking of oligopeptide P14 (A & C) and PLX4032 (B & D) in the ATP binding pocket of B-RAFV600E. The phosphorylation loop (P-loop) is shown in yellow, catalytic loop (Cat. loop) in pink, and activation loop (Act. loop) in green.

Molecular docking of oligopeptide P14 and CRAFinhibitors

(Figure 3) illustrates the molecular docking interaction of P14 and sorafenib with the ATP binding pocket of C-RAF. P14 exhibited favorable conformations and enhanced binding to the catalytic pocket (Figure 3B) relative to that demonstrated by sorafenib (Figure 3A). This advantage is facilitated by favorable hydrogen bonding and hydrophobic interactions. Our preliminary molecular modeling data suggest that, relative to the 2 benchmarks sorafenib and PLX4032, the proposed oligopeptides possess comparable or improved affinity to both mutated B-RAF and wildtype C-RAF, justifying their further study as potential development candidates.

Figure 3. Interaction of sorafenib (A) and P14 (B) with the C-RAF ATP binding pocket.

Cytotoxic effects of oligopeptides on human and mouse melanoma cells

We next performed MTT assays to test for oligopeptide effects on the viability and proliferation of mouse melanoma cells and human metastatic melanoma line previously shown to harbor the V600E BRAF mutation. Figure 4 shows that incubation for 3 d with 3 mM P5 or P14 resulted in 44% or 49% cell death, respectively, compared to 90% cytotoxicity for HQ after same period of incubation. Oligopeptide P15 showed only minor cytotoxic effects after a 3-d incubation.

Figure 4. MTT cell proliferation and viability assay showing the effects of oligopeptides P5, P14, and P15 on mouse melanoma cells compared to hydroquinone (HQ). Cells were incubated with variable concentrations for 72 h at 37 °C and 5% CO₂. Data are presented as % toxicity relative to the control as a function of concentration. P15 is used as a negative control to show the potent cytotoxic effects of P5 and P14, whereas HQ was used as a positive control with known cytotoxic effects on many cell lines.

(Figure 4) also shows that the cytotoxic effect was dose dependent. (Figure 5) shows the cytotoxic effects on human melanoma cells. At a concentration of 1 mM, P5 and P14 showed nearly similar cytotoxicity of 36%, whereas HQ demonstrated cytotoxicity of nearly 80%. (Supplemental Figures 1-6) confirm the cytotoxic effects that P5, P14, and HQ exert on mouse and human melanoma cells.

Figure 5. MTT cell proliferation and viability assay showing the effects of oligopeptides P5, P14, and P15 on human melanoma cells compared to hydroquinone (HQ). Cells were incubated with variable concentrations for 72 h at 37 °C and 5% CO₂. Data are presented as % toxicity relative to the control as a function of concentration. P15 is used as a negative control to show the potent cytotoxic effects of P5 and P14, whereas HQ was used as a positive control with known cytotoxic effects on many cell lines.

Cytotoxicity effects of oligopeptides on human melanocytes, fibroblasts, and keratinocytes

(Table 1) (Table 2) and (Table 3) show the effects of P5 and P14 on the vitality and viability of human melanocytes and fibroblasts compared to HQ. After 72 h of incubation with 1 mM P5 and P14, cell death was 9% and 9% for melanocytes, respectively, and 3% and 6% for fibroblasts, respectively, whereas 1 mM HQ resulted in 100% cell death in both melanocytes and fibroblasts. Moreover, (supplemental figures 7-9) demonstrate the selective cytotoxicity of P5 and P14 compared to the indiscriminate cytotoxicity of HQ towards healthy normal skin cells such as epidermal keratinocytes (Sup. Figure 7), melanocytes (Sup. Figure 8), and fibroblasts (Sup. Figure 9).

P5
Day 1	[µM]	%toxicity	SEM
	30	0.1	0
	100	0.1	0
	300	2.2	0.91
	1000	7.8	0.91
	3000	13.3	1.57

Day 3	[µM]	%toxicity	SEM
	30	0.1	0
	100	0.1	0
	300	2.4	0.33
	1000	8.7	0.56
	3000	13.5	1.06

Day 6	[µM]	%toxicity	SEM
	30	0.1	0
	100	0.1	0
	300	2.7	1.09
	1000	9.3	1.03
	3000	14.5	1.26

P14
Day 1	[µM]	%toxicity	SEM
	30	0.1	0
	100	0.1	0
	300	0.1	0
	1000	6.3	0.26
	3000	7.9	0.32

Day 3	[µM]	%toxicity	SEM
	30	0.1	0
	100	0.1	0
	300	0.1	0
	1000	5	0.37
	3000	8.4	0.57

Day 6	[µM]	%toxicity	SEM
	30	0.1	0
	100	0.1	0
	300	0.1	0
	1000	7.9	0.41
	3000	11.6	0.87

Table 1: Oligopeptide cytotoxicity on human primary melanocytes. Cultured melanocytes were treated with various concentrations of P5 and
P14 and their viability examined using trypan blue on days 1, 3, and 6.

P5
Day 1	[µM]	%toxicity	SEM
	10	0.1	0
	30	0.1	0
	100	0.1	0
	300	0.1	0
	1000	2.1	0.32

Day 3	[µM]	%toxicity	SEM
	10	0.1	0
	30	0.1	0
	100	0.1	0
	300	0.5	0
	1000	2.6	1.02

Day 6	[µM]	%toxicity	SEM
	10	0.1	0
	30	0.1	0
	100	0.1	0
	300	1.1	0
	1000	3.9	0.32

Day 6	[µM]	%toxicity	SEM
	10	0.1	0
	30	0.1	0
	100	0.1	0
	300	1.1	0
	1000	3.9	0.32

Day 3	[µM]	%toxicity	SEM
	10	0.1	0
	30	0.1	0
	100	0.1	0
	300	3.8	0.27
	1000	5.9	0.17

Day 6	[µM]	%toxicity	SEM
	10	0.1	0
	30	0.1	0
	100	0.1	0
	300	4	0.13
	1000	6.9	0.41

Table 2: Oligopeptide cytotoxicity on human fibroblasts. Cultured melanocytes were treated with various concentrations of P5 and P14 and their
viability examined using trypan blue on days 1, 3, and 6.

Human Primary Melanocytes
Day 1	[µM]	%toxicity	SEM
	10	27	3.3
HQ	30	41	2.8
	100	98	1.1
	300	100	0
	1000	100	0

Day 3	[µM]	%toxicity	SEM
	10	29	4.6
HQ	30	53	5.2
	100	100	0
	300	100	0
	1000	100	0

Day 3	[µM]	%toxicity	SEM
	10	29	4.6
HQ	30	53	5.2
	100	100	0
	300	100	0
	1000	100	0

Day 3	[µM]	%toxicity	SEM
	10	29	4.6
HQ	30	53	5.2
	100	100	0
	300	100	0
	1000	100	0

Day 3	[µM]	%toxicity	SEM
	30	9	0
HQ	100	20	1.2
	300	44	2.4
	1000	100	0
	3000	100	0

Day 6	[µM]	%toxicity	SEM
	30	15	1.2
HQ	100	30	1.1
	300	54	3.1
	1000	100	0
	3000	100	0

Table 3: HQ cytotoxicity on human fibroblasts and primary melanocytes. Cultured fibroblasts and melanocytes were treated with various
concentrations of HQ and their viability examined using trypan blue on days 1, 3, and 6.

The results observed in the MTT assay appeared consistent with our preliminary in silico modeling data. For example, P15 showed weak in silico binding when docked in the ATP pocket of either B-RAF^V600E or C-RAF, and weak effects on the proliferation and viability of melanoma cells (Figure 4 & Figure 5). On the other hand, oligopeptides P5 and P14 showed free binding energies that were comparable with those of sorafenib and PLX4032.

Discussion

Oligopeptides P5 and P14 are capable of selectively killing melanoma cells while exhibiting very little to no cytotoxic effects towards normal human melanocytes, keratinocytes, and fibroblasts which are the major constituents of skin. HQ on the other hand is a potent nonselective cytotoxic agent against many cell lines. We used HQ as a positive control because of its known cytotoxic effects against some cancer cell lines [25]. Oligopeptide P15 was used as a negative control to highlight the selectivity of P5 and P14 against melanoma cells and to validate the output of the in silico molecular modeling. P15 showed weak in silico binding when docked in the ATP pocket of either B-RAF^V600E or C-RAF, and weak cytotoxicity for P15 (Figure 4 & Figure 5). On the other hand, oligopeptides P5 and P14 showed free binding energies that were comparable with those of sorafenib and PLX4032.

Our findings support the use of molecular modeling as a screening tool to identify oligopeptides with potential cytotoxic effects towards human metastatic melanoma lines in vitro. Unlike HQ, P5 and P14 exhibited selective cytotoxicity towards melanoma cells with minimal or no cytotoxic effects towards normal skin cells, specifically melanocytes, keratinocytes, and fibroblasts. MAPK inhibition has been shown to result in dephosphorylation of the pro-apoptotic Bcl-2 family members, Bad, and Bim. This, in turn, leads to activation of the caspase pathway, and ultimately, the demise of melanoma cells through the induction of apoptosis [26].

Although expectations were high when sorafenib, the first RAF kinase-targeting drug, reached clinical trials [27], initial studies in melanoma patients were disappointing and did not show the anticipated single agent efficacy. The problem with sorafenib and similar development candidates is their lack of specificity [28]. Furthermore, melanoma tumors harboring the BRAF^V600E mutation were found to be resistant to sorafenib treatment, implying only tumors with low B-RAF activity were sensitive to the drug. This phenomenon has been attributed to dependence of these tumors on C-RAF for MEK activity and B-cell lymphoma 2-mediated cell survival [29]. Thus, since >90% of melanomas exhibit B-RAF hyperactivity, only a small percentage of tumors will respond to sorafenib. Interestingly, sorafenib was later shown to more potently inhibit C-RAF kinase.

More recently, the resolution of the B-RAF crystal structure has led to the development of more specific B-RAF inhibitors such as PLX4032 [19]. This drug induced a dramatic response rate in phase I trials, validating B-RAF as a clinically relevant target. Unfortunately, it has recently been shown that elevated CRAF gene expression mediates resistance to B-RAF inhibitors in BRAF^V600E-mutant melanoma cells [30]. Furthermore, in RAS-mutant melanoma cells, C-RAF is essential because oncogenic RAS signals through this kinase rather than B-RAF [31]. These findings suggest that highly targeted and specific B-RAF inhibitors may be ineffective against both RAS-mutant melanomas and BRAF-mutant melanomas with high C-RAF activity. In other words, the above findings point to the need for pan-RAF inhibition.

Of equal significance is the discovery that inhibition of BRAF^V600E increased levels of melanocyte differentiation antigens (MDAs), resulting in improved recognition by antigen-specific T lymphocytes [32]. This is supported by the finding that loss of antigen expression led to relapse after immunotherapy [33].Melanocytic cells express a set of genes specific to that lineage. However, with respect to immunotherapy, several melanocytic gene products, including MART1 and gp100, have been directly shown to function as target antigens for cytotoxic T lymphocytes [34-36]. This has led some investigators to hypothesize that reduced MDA expression by melanoma cells facilitates evasion of tumor surveillance in vivo [37, 38]. Thus, blockade of MAPK signaling via a specific BRAF^V600E inhibitor with subsequent elevation of MDA expression may hold great potential as an immunotherapeutic approach for advanced melanoma.

Based on our data and previous studies, we hypothesize that oligopeptide treatment enhances melanoma killing by inhibiting B-RAF^V600E- and C-RAF-dependent MAPK signaling, leading to unopposed caspase activity and subsequent apoptosis. Additional effects include increasing MDA expression, resulting in the potential for improved recognition by MART1-specific and gp100-specific autologous T lymphocytes.

Moreover, and to help address the urgent need for an effective preventative treatment for melanoma, our approach would be to target high risk groups with a non-invasive and non-surgical prophylactic topical treatment formulated with P5 and/or P14. These oligopeptides may prove to be revolutionary, cost effective, and efficacious treatments for lesions spanning from precancerous (actinic keratosis, dysplastic nevi) to pre-malignant (melanoma in situ, squamous cell carcinoma in situ) to malignant or even metastatic stages. Prophylactic efficacy would be achieved through cytotoxic elimination of any damaged skin cells resulting from sunscreen slippage prior to transformation into dysplastic cells.

Acknowledgements

Anan Abu Ubeid assisted with manuscript preparation and technical execution of the experiments.

Financial disclosure

This research was funded by Escape Therapeutics, Inc. Authors are consultants and/or stockholders of Escape Therapeutics, Inc. which may pursue patenting and/or commercialization of these oligopeptides in the future.

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Supplemental figure 1. P5 is selectively cytotoxic to mouse B16 melanoma cells. Cytotoxicity is evident in cells incubated for 48 h with 3 mM P5 (B) but not vehicle (A).

Supplemental figure 2. P5 is selectively cytotoxic to human A375 melanoma cells. Cytotoxicity is evident in cells incubated for 48 h with 3 mM P5 (B) but not vehicle (A).

Supplemental figure 3. P14 is selectively cytotoxic to mouse B16 melanoma cells. Cytotoxicity is evident in cells incubated for 48 h with 3 mM P14 (B) but not vehicle (A).

Supplemental figure 4. P14 is selectively cytotoxic to human A375 melanoma cells. Cytotoxicity is evident in cells incubated for 48 h with 3 mM P14 (B) but not vehicle (A).

Supplemental figure 5. HQ is a potent nonselective cytotoxic agent against mouse B16 melanoma cells. Cytotoxicity is evident in cells incubated for 48 h with 1 mM HQ (B) but not vehicle (A).

Supplemental figure 6. HQ is a potent nonselective cytotoxic agent against human A375 melanoma cells. Cytotoxicity is evident in cells incubated for 48 h with 3 mM HQ (B) but not vehicle (A).

Supplemental figure 7. Effects of P5, P14, and HQ on human epidermal keratinocytes. Cytotoxicity is evident in cells incubated for 48 h with 3 mM HQ (C) but minimal to none with P5 (A) and P14 (B).

Supplemental figure 8. Effects of P5, P14, and HQ on human melanocytes. Cytotoxicity is evident in cells incubated for 48 h with 3 mM HQ (C) but minimal to none with P5 (A) and P14 (B).

Supplemental figure 9. Effects of P5, P14, and HQ on human fibroblasts. Cytotoxicity is evident in cells incubated for 48 h with 3 mM HQ (C) but minimal to none with P5 (A) and P14 (B).

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