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 def....

Protocol

1. Synthesis of peptides

NOTE: The synthesis of W-R is described as a representative example (Figure 2).

1. 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) strategy²¹.
   NOTE: Methylated or dimethylated dipeptides containing unnatural amino acids were assembled on a rink amide resin (loading capacity o....

Results

W-R_CH3, W_CH3-R_CH3, W-R_(CH3)2,_WCH3-R, W_CH3-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

List of materials used in this article

Name	Company	Catalog Number	Comments
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