Developing and Testing Methylated Nano-Structured Dipeptides that Inhibit Src Kinase Activity In Vitro for Anti-Cancer Applications

Summary

Presented here is a protocol for designing and testing dipeptides that can inhibit Src kinase enzyme activity using acellular and cellular assays for anti-cancer applications. The peptides formulated here (W-R_CH3, W_CH3-R_CH3, and W-R_(CH3)2) inhibited Src kinase with IC₅₀ values of 510 nM, 916 nM, and 1 µM, respectively.

Abstract

Here, with the aim of developing a novel anti-cancer treatment, seven dipeptides were designed that contained methylated tryptophan and/or methylated arginine and were produced using Fmoc solid-phase peptide synthesis. Overexpression of the Src tyrosine kinase enzyme has been implicated in the development of different cancers. Dipeptides containing unnatural amino acids such as methylated arginine (R_CH3), dimethylated arginine (R_(CH3)2), and/or methylated tryptophan (W_CH3) residues have earlier been shown to inhibit Src kinase. In this study, three such dipeptides, W-R_CH3, W_CH3-R_CH3, and W-R_(CH3)2, were tested using acellular assays and were found to have IC₅₀ values (the concentration at which 50% inhibition occurs) of 510 nM, 916 nM, and 1 µM, respectively. These values were comparable to those obtained for cyclic penta- to nano-W-R peptides ([W-R]₅-[W-R]₉) synthesized in previous studies. However, the unmethylated versions of the dipeptides did not show any inhibitory activity against Src kinase. All of these dipeptides (50 µM) did not show any cytotoxicity after incubation up to 72 h with three different cancer cell lines, including leukemia (CCRF-CEM), breast adenocarcinoma (MDA-MB-231), and ovarian adenocarcinoma (SK-OV-3) cell lines, indicating the limited permeability of the peptides through the cell membrane. Therefore, further study is needed to improve the permeability of these peptides for anticancer applications, such as by using a peptide carrier or additional peptide functionalization. In summary, this study provides a protocol to synthesize and test peptides that inhibit Src kinase activity, and thus possess promising anticancer ability, as demonstrated using acellular and cellular assays.

Introduction

Cancer is caused by the abnormal growth of normal cells and is one of the most lethal diseases around the world. These abnormal cells spread to different organs in the body by a process called metastasis. The most common form of cancer is breast cancer, which occurred in 2.26 million people in 2020. Moreover, there were around 1.80 million deaths due to lung cancer in 2020¹. According to the World Health Organization, around 10 million people died from cancer in 2020². Cancer cells differ from normal cells in that they overexpress certain enzymes, such as protein tyrosine kinases (PTKs). The National Cancer Institute defines kinases as enzymes able to phosphorylate other proteins or sugars³. Knowledge of the regulatory function of kinases can facilitate the design of effective anticancer drugs. For example, PTKs catalyze the phosphorylation of other proteins or sugars, and as a consequence, ATP is converted to ADP by the loss of a phosphate group. A total of 80% of oncogenes and protooncogenes encode PTKs⁴. Src kinases are a family of non-receptor tyrosine kinases, including Lck, Fyn, Hck, Blk, Yes, and Yrk, that are overexpressed in cancer cells, especially in breast cancer^5,6. Src tyrosine kinases are associated with mitogenesis, differentiation, T-cell activation, and cell transformation. Src helps cancer cell invasion and metastasis due to its ability to reduce cancer cell adhesion. There are five different domains in Src kinase, ordered from the N- to C-terminals as: fatty acid domain, Src homology 3 domain (SH3), Src homology 2 domain (SH2), tyrosine kinase domain (SH1), and C-terminal regulatory domain⁷.

Figure 1: The target domains in the Src kinase enzyme, including a SH3 domain, SH2 domain, kinase domain (SH1), and a short C-terminal regulatory segment. Please click here to view a larger version of this figure.

The kinase domain SH1 is most commonly targeted when designing Src kinase inhibitors, as it contains two conserved sites for ATP and substrate binding (Figure 1). If the amino acid sequence of the kinase domain is known, the substrate can also be used as a target to design a compound that mimics substrate binding to Src kinase⁸. In addition, other sites such as the SH3 and SH2 domains can be used as targets. Compared to other chemotherapy agents, kinase inhibitors exhibit less toxicity and higher efficacy⁹. As of September 2021, there are 73 small molecules that act as kinase inhibitors that have been approved by the FDA¹⁰. Imatinib is an example of an anticancer drug that selectively inhibits the activity of tyrosine kinase; however, some patients are resistant to the drug due to the appearance of a point mutation in the kinase domain¹¹. AstraZeneca released Saracatinib, which is a drug that inhibits the Src family of tyrosine kinases with an IC₅₀ value (the concentration at which 50% inhibition occurs) of 2.7 nM, but it was discounted in phase 2 trials¹². Of the 52 PTK inhibitors approved by the US FDA as of the beginning of 2020¹³, only 28 target receptor PTKs, 11 block the non-receptor PTK, 11 inhibit protein-serine/threonine protein kinases, and two block MEK_1/2¹³. The increasing research interest in oncology will continue to fuel the discovery of kinase inhibitors as potential anti-cancer drugs. However, only 50 out of 500 protein kinases have been targeted for treatment thus far; therefore, a greater number of kinases are expected to be studied for drug development in the near future¹⁴. In addition, there is a need to discover kinase inhibitors to explore as yet unidentified kinase mutations that lead to cancer.

Thus, this study aimed to develop peptides that could be used as inhibitors for the Src family and target the ATP binding site due to its ability to serve as a conserved site between different kinases. To this end, a series of dipeptides containing methylated tryptophan and/or methylated arginine were synthesized and tested for their synergistic ability to inhibit Src kinase. The indole ring of tryptophan mimics the adenine of ATP and competes with ATP from binding to the ATP-binding site. In addition, the methylated arginine in the ligand competes for the SH3 domain of Src. Researchers showed that a polypeptide containing demethylated arginine inhibits the SH3 domain, possibly due to a specific conserved sequence on the SH3 binding motif (i.e., PXXP), which has a binding affinity to a ligand containing two to three Arg residues in the N-terminal or one to two Arg residues on the C-terminal of the ligands^15,16,17. The guanidino group of Arg binds to the conserved Asp-99 residue of the SH3 domain^18,19, while the remaining portion of the Arg binds to the conserved Trp-118 of the enzyme, as confirmed from NMR analysis and the crystal structures of several SH3 domains¹⁹. Here, a protocol for the synthesis of seven methylated dipeptides and testing their inhibition ability against Src kinase is presented. Further, the ability of these peptides to kill several cancer cell lines in vitro was examined.

Protocol

1. Synthesis of peptides NOTE: The synthesis of W-R is described as a representative example (Figure 2). Weigh 566 mg (0.3 mmol) of H-Arg(Pbf)-2-chlorotriryl resin and add it to the peptide synthesis vessel (see Table of Materials), according to the procedure used by Mandal et al20. Perform peptide synthesis using the well-established solid phase peptide synthesis (SPPS) strategy<sup class="xref…

Representative Results

W-RCH3, WCH3-RCH3, W-R(CH3)2,WCH3-R, WCH3-R(CH3)2, and the control W-R peptides were synthesized using Fmoc solid-phase peptide synthesis (Figure 3), with 95%, 98.7%, 99%, 100%, 100%, and 99.5% purity, respectively. The chemical structures of these dipeptides were confirmed using ESI-MS. The m/z values of these dipeptides were 374.1624, 388.1949, 388.1794, 374.1815, 402.2022, and 361.1457, respectively.</…

Discussion

The peptides fabricated and tested here for the inhibition of Src kinase and consequent killing of cancer cells contained methylated tryptophan and/or methylated arginine, which are unnatural amino acids. Formation of the white precipitate upon adding diethyl ether is a critical step in the synthesis of these peptides. However, not all synthetic peptides can form a precipitate; therefore, even when a precipitate is not formed, successful peptide synthesis can be confirmed by the determination of the desired mass using li…

Materials

1,4-Dithiothreitol	Sigma-Aldrich	10708984001	Peptide synthesis
Aldrich fritted filter funnel for solid-phase synthesis	Sigma-Aldrich	Z283304	Peptide synthesis vessel
Alexa594 Tracer	Bell Brook Labs, Madison, WI	3013
Anisole	Sigma-Aldrich	8014520500
CellTiter 96 AQueous One Solution Cell Proliferation Assay reagents	Promega	G3582	Cell proliferation assay (MTS reagent)
Dichloromethane, 99.9%, Extra Dry	Fishersci	AC326850025
Fmoc-ADMA(Pbf)-OH	Sigma-Aldrich	8521070001
Fmoc-Arg(Me,Pbf)-OH	Sigma-Aldrich	8521050001
Fmoc-Arg(Pbf)-OH	Sigma-Aldrich	8520670025
Fmoc-Trp(Boc)-OH	Sigma-Aldrich	47561-25G-F
HPLC C18 column	Shimadzu  (RP-HPLC system)		water/acetonitrile gradient
IRDye QC-1 quencher	Bell Brook Labs, Madison, WI	3013
Microplate reader SpectraMax M2e	Molecular devices
Microsoft Excel	Microsoft		spreadsheet software
N,N-Diisopropylethylamine (DIPEA)	Sigma-Aldrich	496219
N,N-Dimethylformamide, anhydrous, 99.8%	Fishersci	AA43997M1
Piperidine 20%	Sigma-Aldrich	80645
Rink Amide resin (100-200 mesh)	Sigma-Aldrich	8550010025
Thioanisole	Sigma-Aldrich	92358
Transcreener ADP2 FI Assay	Bell Brook Labs, Madison, WI	3013	c-Src kinase activity assay kit