Modification and Structure–Activity Relationship Study of Cyclodepsipeptide Trichodestruxin D Derivatives as Potential Antitumor Agents

Abstract Trichodestruxins A–D are cyclic peptides isolated from the plant endophyte fungus Trichoderma harzianum with inhibitory activities against the proliferation of tumor cells. This study aimed to modify the structure of trichodestruxin D ( TD- ( R )) to improve its antitumor activity and analyze the structure–activity relationship (SAR) to provide references for lead optimization. In this study, seven TD- ( R ) derivatives ( TD- ( S ), TD-1 , 2 , 3 , 4 , 5 , 6 ) were designed by different strategies, namely amino acid mutation, configuration switching, replacement of ester with amide, and N -methylation/demethylation. Those derivatives were prepared by a solid-phase peptide synthesis strategy, and structurally characterized by high-resolution mass spectra. The inhibitory activities of the peptides against the lung carcinoma A549 cells were assessed by determining cellular proliferation and migration using CCK-8 and a 24-well migration plate. Our data confirmed the inhibitory effect of those derivatives on A549 cell proliferation, among which TD -( S ), TD - 1 , and TD - 2 displayed higher inhibitory activity compared with the control (DMSO) group, but their inhibitory activity was slightly decreased than that of TD -( R ). The inhibitory activity of TD - 3 , TD - 4 , and TD - 6 on A549 cell migration was much better than that of TD -( R ). SAR studies demonstrated a pivotal role in the configuration of the residue of 2-hydroxy-4-methyl-pentenoic acid and some residues in the structure of TD -( R ). In conclusion, TD - 3 , TD - 4 , and TD - 6 may be potential agents for the treatment of cancer migration, and our modification methods will provide a reference for the development of anticancer drugs in the future.


Introduction
2][3][4][5] For example, the soybean-derived peptide Vglycin, isolated from soybean seeds, palm fern seeds (Cycas revoluta), significantly inhibits the proliferation of human epidermoid carcinoma (Hep2) and colon cancer cells (HCT15), and induces apoptosis by destroying ribosomal structures through direct binding to DNA. 7 In 2020, Pei and coworkers 8 isolated four new cyclodepsipeptides trichodestruxins A-D (►Fig. 1) from the plant endophytic fungus Trichoderma harzianum, and elucidated their structures using 1D/2D nuclear magnetic resonance (NMR), high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) spectroscopic analyses, the advanced Marfey's method, etc.In bioactivity assays, all cyclic peptides were cytotoxic to the tumor cell line A549 with IC 50 values ranging from 8.8 to 17.5 μmol/L.However, trichodestruxin D was not as effective as trichodestruxins A-C in its antiproliferative ability against A549 cells.
Structural optimization of natural peptides is one of the effective methods to develop potential lead compounds, which can improve peptide stability while increasing activity.0][11][12] Lokey's group reported more results regarding the effect of backbone N-methylation of the Sanguinamide A scaffold on biological activity.They found that the biological activity can be affected by the skeletal N-methylation, and can be greatly improved by the N-methylation at the Phe-NH site. 13Jin's group synthesized a variety of methylmodified and stereo-modified analogues of the marine cyclic peptide galaxamide with isoindolinone, 14 and demonstrated the benefit of monomethyl substitution in improving the biological activity of cyclic analogs with isoindolone fragments.Rekdal's group designed a series of derivatives by substituting amino acids of peptide L5 derived from the N-terminal α-helix region of bovine lactoferrin, 15 and disclosed a deep impact of the substitution of amino acids on the antitumor activity of the compound.The result revealed that the activity was enhanced by such modifications.Given the above, optimization of the natural peptides is one of the most effective ways to develop potential lead compounds.Therefore, we structurally modified trichodestruxin D in an attempt to improve its antiproliferative activity against tumor cells.
In this work, trichodestruxin D (TD-(R), as shown in ►Fig. 1) was used as the lead compound to explore a series of derivatives, which was composed of (R)-2-hydroxy-4-methylpentanoic acid ((R)-HMPA), a β-MePro unit, Val, NMe Val, NMe Ala, and β-Ala.Structure optimization of TD-(R) included the replacement of the unnatural amino acids with natural amino acids, the replacement of the ester group with the amide group, and the demethylation of the amide.In addition, (R)-HMPA was replaced with (S)-2hydroxy-4-methylpentanoic acid to generate TD-(S) to assess the effect of configuration on the antitumor activity.The cytotoxicity of the derivatives against the lung carcinoma A549 cells was assessed and the structure and activity relationships were discussed.Our data suggested that TD-(S), TD-1, and TD-2 displayed higher antiproliferative activity than the control (DMSO) group.However, TD-(S) and TD-1 showed slightly decreased antiproliferative activity compared with TD-(R), demonstrating the importance of the configuration of HMPA and some residues in the backbone of TD-(R).
CCK-8 cell viability assay was performed to evaluate the antitumor activity of the derivatives.The concentration was determined as 100 μmol/L based on our previous work. 16The cytotoxicity of TD-(S) and TD-1, 2, 3, 4, 5, 6 on A549 cells is  shown in ►Fig.3A.Our data suggested that TD-(S), TD-1, and TD-2 showed much stronger inhibitory activity than the control (DMSO) group.Unfortunately, they did not show improved antitumor activity compared with TD-(R), even up to the concentration of 100 μmol/L.The result suggested the influence of the configuration of HMPA on the antitumor activity of TD-(R), with its antitumor activity being reduced when the configuration was S, and also suggested that the ester bond and the N-methyl on the scaffold of TD-(R) played an important role for the activity.TD-3, 4, 5, and 6 even did not show any inhibitory activity, indicating the importance of residues Leu, Pro, and Val for the activity of the compounds.The antitumor activity of TD-3 was slightly decreased compared with TD-2, demonstrating that the Thr with polar but nonionized side chain was unfavorable.Pro residues have been proven helpful for the β-turn. 17When the Pro of TD-1 was substituted with His to generate TD-5, the cytotoxicity could decrease almost onefold, implying the importance of Pro in the activity.The cytotoxicity of TD-4 was lower than that of TD-2, implying that N-methylation may not be favorable to the antitumor activity.
In some cases, malignant tumor cells can penetrate the body's barriers and invade the surrounding environment, destroying the surrounding tissues. 18,19These cancer cells move to other parts of the body through the bloodstream or lymphatic system and invade other tissues of the body, growing and multiplying in the new organs to form metastatic tumors, which presents an additional challenge to clinical treatment. 20,21In this regard, we evaluated the inhibitory activity of TD-(R)-derived peptides against the migration of A549 cells.As shown in ►Fig.3B, TD-3, TD-4, and TD-6 inhibited the migration of A549 cells at the dose of 100 μmol/L.In ►Fig.3C, we could visually observe that TD-3, TD-4, and TD-6 inhibited the migration of A549 cells better than TD-(R).

Conclusion
In the present study, a series of TD-(R) derivatives were designed and synthesized, and most of them displayed antitumor activity when compared with the DMSO control group.Cyclic hexapeptide TD-(S), TD-1, and TD-2 showed much stronger antiproliferative activity in CCK-8 assays, but slightly decreased activity compared with TD-(R).It was also found that the configuration of TD-(R) affected the antiproliferative activity.When (R)-HMPA was replaced by (S)-HMPA, the antiproliferative activity of TD-(S) was decreased.The ester bond and N-methyl of Val also played an important role in the antitumor activity.It was concluded that most residues in TD-(R) including the configuration of HMPA are pivotal for the antitumor activity.However, TD-3, TD-4, and TD-6 showed inhibitory activity in cancer cell migration assays and may be used as potent lead peptides for the treatment of tumor migration.The results provide a valuable reference for the development of peptide analogs to treat cancer in the future.

Experimental Section Materials and General Procedures
Amino acids were purchased from CS Bio Ltd. (Shanghai, China).Wang resin and 2-chlorotriphenylmethyl chloride resin were provided by Tianjin Nankai Hecheng Science & Technology Co., Ltd.(Tianjin, China).All other reagents and solvents were supplied by Sinopharm Chemical Reagent Co. Ltd., Bide Pharmatech Ltd., and Shanghai Titan Scientific Co., Ltd.(Adamas-β) (Shanghai, China).Sterile Dulbecco's Modified Eagle's medium (DMEM), Trypsin-EDTA, fetal bovine serum (FBS), and penicillin-streptomycin used in biological assays were purchased from Gibco (Grand Island Biological Company, Grand Island, United States).The Cell Counting Kit-8 (CCK-8) was purchased from Beyotime Biotech Inc. (Shanghai, China).Phosphate-buffered saline (PBS), the Cell Culture Chamber (Falcon, 8.0 μm, PET membrane), and a 24-well migration plate were purchased from Shanghai Titan Scientific Co., Ltd.(Shanghai, China).A549 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China).The 1 H NMR and 13 C NMR spectra were determined using a Bruker AC-600P spectrometer (600 MHz for 1 H, and 151 MHz for 13 C).The solvent used for NMR spectra was chloroform-d with tetramethylsilane as internal standard.HRMS data were measured using an Agilent The migration of A549 cells was observed under a microscope when the cells were treated with 100 μmol/L TD-(R), TD-3, TD-4, and TD-6.Data were presented as mean AE standard error of the mean of at least three repeats.Data from Fig. 3A were analyzed using one-way ANOVA with GraphPad Prism software, and p 0.05 was considered statistically significant.Data from Fig. 3B was obtained by cell counting with Image J software and one-way ANOVA with GraphPad Prism software for Fig. 3C.p 0.05 was considered statistically significant.NC: negative controlled group (cells were treated with DMEM basal medium containing 1% DMSO and placed in the upper chamber).PC: positive controlled group (untreated cells with DMEM complete culture medium into the upper chamber).ÃÃ p < 0.01, ÃÃÃ p < 0.001 compared with the DMSO group.DMEM, Dulbecco's Modified Eagle's medium.
Technologies 6538 UHD accurate-Mass Q-TOF MS spectrometer with electrospray ionization.

Reversed-Phase Analytical HPLC
Analytical HPLC was run on an Agilent 1260 Infinity LC instrument using an analytical column (ZORBAX Eclipse XDB 80 Å C18, 4.6 mm Â 150 mm, 3.5 µm) with a flow rate of 1.0 mL/min and at room temperature.Analytical feeds were monitored at 214 and 254 nm.The mobile phases used for all separations were 0.1% TFA (v/v) in acetonitrile (solvent A) and 0.1% TFA (v/v) in water (solvent B).A linear gradient of 95% to 5% B was used for 40 minutes at room temperature.

Reversed-Phase Preparative HPLC
Semi-preparative HPLC was run on a Pre-HPLC Agilent 1100 Series (United States) instrument using a semi-preparative column (CST Daiso Prep C18; 30 mm Â 250 mm, 10 μm particle size) The flow rate was 30 mL/min.Solution A was acetonitrile containing 0.1% TFA.Solution B was H 2 O containing 0.1% TFA.A linear gradient of 95% to 95% B was used for over 5 minutes, and a linear gradient of 95% to 15% B is then applied over 55 minutes at room temperature.All peptides had a purity of at least 95%.

Synthesis of TD-(R) and TD-(S)
The linear peptide was synthesized on 2-chlorotrityl chloride resin (100-200 mesh, loading amount 1.12 mmol/g, 0.89 g, 1 mmol) via the strategy of SPPS.Peptide couplings were performed using HOBt (0.41 g, 3 mmol, 3 equiv.),DIC (0.58 g, 4.5 mmol, 4.5 equiv.),DMF (25 mL), and shaken for 2 hours at 25°C.Following the completion of each stage of the reaction, the solvent was drained and the resin was washed with DCM and DMF.The Fmoc group was deprotected using 20% piperidine in DMF (20 mL) at 25°C for 15 minutes.After the linear peptide 12 was assembled, it was cleaved from the resin using a mixture of TFE and DCM (1:3, v/v, 30 mL) at 25°C for 4 hours to produce Boc-protected peptide 13.After intermediate 13 was purified by semi-preparative reverse-phase HPLC, it was dissolved in the solution of TFA and DCM (1:3, v/v, 30 mL) and stirred at 25°C for 4 hours to give 14.Then, the linear peptide 14 (0.2 g, 0.34 mmol, 1 equiv.)was dissolved in DCM (200 mL) with the concentration of 1 mg/mL, and then PyAOP (0.89 g, 1.7 mmol, 5 equiv.),HOAt (0.23 g, 1.7 mmol, 5 equiv.),and NMM (0.4 g, 3.4 mmol, 10 equiv.)were added and stirred at 25°C for 12 hours.The solvent was evaporated in vacuo to give the crude cyclic peptide TD-(R), which was purified by semi-preparative RP-HPLC.The purity and the structure of TD-(R) were confirmed by analytical HPLC and HRMS.TD-(R) was lyophilized into a white powder with a yield of 13% and a purity of 96.9%.HRMS m/z calcd.for

Fig. 2
Fig. 2 Structures of trichodestruxin D and its derivatives.

Fig. 3 (
Fig. 3 (A) Effects of 100 μmol/L TD-(R) and TD-(S), and TD-1, 2, 3, 4, 5, 6 on A549 cell viability.(B) Effects of 100 μmol/L TD-(R) and TD-(S), and TD-1, 2, 3, 4, 5, 6 on the migration of A549 cells.(C)The migration of A549 cells was observed under a microscope when the cells were treated with 100 μmol/L TD-(R), TD-3, TD-4, and TD-6.Data were presented as mean AE standard error of the mean of at least three repeats.Data from Fig.3Awere analyzed using one-way ANOVA with GraphPad Prism software, and p 0.05 was considered statistically significant.Data from Fig.3Bwas obtained by cell counting with Image J software and one-way ANOVA with GraphPad Prism software for Fig.3C.p 0.05 was considered statistically significant.NC: negative controlled group (cells were treated with DMEM basal medium containing 1% DMSO and placed in the upper chamber).PC: positive controlled group (untreated cells with DMEM complete culture medium into the upper chamber).ÃÃ p < 0.01, ÃÃÃ p < 0.001 compared with the DMSO group.DMEM, Dulbecco's Modified Eagle's medium.
.3549, found: 566.3547.TD-(S) was synthesized in the same procedure with a yield of 10% and a purity of 98.6%, as a white powder.HRMS m/z calcd.forC28H 48 N 5 O 7 þ [M þ H] þ 566.3549, found: 566.3584.A549 cells were seeded at 5 Â 10 3 per well in a clear 96-well plate and incubated at 37°C in 5% CO 2 overnight.The groups were divided into a control group (cells treated with DMEM basal medium containing 1% DMSO) and drug administration groups.The cells in drug administration groups were treated with each cyclic peptide (100 µmol/L) in triplicate for Pharmaceutical Fronts © 2024.The Author(s).Cyclodepsipeptide Trichodestruxin D Zou et al.