Haitham Abusara Osama, Freeman Sally, Aojula Harmesh Singh
Division of Pharmacy and Optometry, School of Health Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester M13 9PT, UK.
Division of Pharmacy and Optometry, School of Health Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester M13 9PT, UK.
Eur J Med Chem. 2017 Sep 8;137:221-232. doi: 10.1016/j.ejmech.2017.05.053. Epub 2017 May 27.
The pentapeptide, tert-Prenyl-NH (DMePhe-DTrp-Phe-DTrp(N-tert-prenyl)-Leu-NH), has recently been reported by our group to exhibit properties of substance P (SP) antagonist G against small cell lung cancer (SCLC). In this study, we undertook a systematic structure activity investigation to optimise this lead compound to improve its in vitro anti-tumour activity and biocompatibility. A series of d-tryptophan (D-Trp) derivatives were synthesised, with a range of aliphatic N-alkyl chains (methyl to pentyl) on the indole nitrogen (N). These were incorporated into the pentapeptide sequence by substitution of the N-tert-prenylated D-Trp 4th residue with the N-alkylated D-Trp derivatives. These pentapeptides were significantly more potent than tert-Prenyl-NH, with the N-butyl modification generating the most cytotoxic peptides. Compared to tert-Prenyl-NH, a single butyl modification on the 4th D-Trp residue (Butyl-NH) showed a ∼3 fold enhancement in cytotoxicity in either the chemo-naive H69 or the DMS79 (originating from a patient treated with chemotherapeutics and radiation therapy) SCLC cell lines. In addition, the di-butylated sequence on the 2nd and 4th D-Trp residues (Butyl-NH) gave ∼4.5 times higher cytotoxicity against the H69 cell line and a ∼2 fold increase against the DMS79 cell line, compared to tert-Prenyl-NH. The favoured position for butyl modification was the 4th D-Trp residue, as the Butyl-NH peptide gave lower cytotoxicity on both cell lines. Butylated peptide sequences, when exposed to neat mouse plasma for 24 h at 37 °C, were found to resist degradation with >80% remaining intact compared to ∼58% for tert-Prenyl-NH. The degradation pathway in plasma occurs via de-amidation of the C-terminus, confirmed by mass spectrometry and RP-HPLC analysis. The butyl modification also conferred resistance to metabolism when tested using S9 liver fraction from mouse. The optimum analogue responsive against the DMS79 cell line was the Butyl-NH pentapeptide, which revealed a concentration dependent increase in apoptosis: the level of late apoptotic cells rose from ∼36% at 2 μM to ∼96% at 6 μM, as determined by flow cytometry, compared to the unmodified peptide that showed no such effect. Concluding, the butyl substitutions offered the best perspective for high cytotoxicity, induction of apoptosis and metabolic compatibility thereby comprising an improved broad spectrum SP antagonist candidate for treatment of SCLC.
我们团队最近报道了五肽tert-Prenyl-NH（DMePhe-DTrp-Phe-DTrp(N-叔戊烯基)-Leu-NH）对小细胞肺癌（SCLC）具有P物质（SP）拮抗剂G的特性。在本研究中，我们进行了系统的构效关系研究，以优化该先导化合物，提高其体外抗肿瘤活性和生物相容性。合成了一系列d-色氨酸（D-Trp）衍生物，在吲哚氮（N）上带有一系列脂肪族N-烷基链（从甲基到戊基）。通过用N-烷基化的D-Trp衍生物取代N-叔戊烯基化的D-Trp第4位残基，将这些衍生物引入五肽序列中。这些五肽比tert-Prenyl-NH的活性显著更高，其中N-丁基修饰产生的细胞毒性肽最强。与tert-Prenyl-NH相比，在第4位D-Trp残基上进行单一丁基修饰（Butyl-NH）在未接受过化疗的H69或DMS79（源自一名接受过化疗和放疗的患者）SCLC细胞系中，细胞毒性增强了约3倍。此外，与tert-Prenyl-NH相比，在第2位和第4位D-Trp残基上进行二丁基化的序列（Butyl-NH）对H69细胞系的细胞毒性高约4.5倍，对DMS79细胞系的细胞毒性增加了约2倍。丁基修饰的最佳位置是第4位D-Trp残基，因为Butyl-NH肽在两种细胞系上的细胞毒性较低。当丁基化的肽序列在37℃下于纯小鼠血浆中暴露24小时时，发现其能抵抗降解，剩余完整的比例>80%，而tert-Prenyl-NH约为58%。血浆中的降解途径是通过C末端的脱酰胺作用，这已通过质谱和RP-HPLC分析得到证实。当使用小鼠肝脏S9组分进行测试时，丁基修饰也赋予了抗代谢能力。对DMS79细胞系反应最佳的类似物是Butyl-NH五肽，其显示出凋亡的浓度依赖性增加：通过流式细胞术测定，晚期凋亡细胞的水平从2μM时的约36%上升至6μM时的约96%，而未修饰的肽则没有这种作用。总之，丁基取代在高细胞毒性、诱导凋亡和代谢相容性方面提供了最佳前景，从而构成了一种用于治疗SCLC的改进型广谱SP拮抗剂候选物。
