N-tert-Prenylation of the indole ring improves the cytotoxicity of a short antagonist G analogue against small cell lung cancer.

Natural prenylated indoles have been proposed as potential anticancer agents. To exploit this discovery for developing new peptide therapeutics, we report the first studies whereby incorporation of prenylated indoles into primary sequences has been achieved. We developed a route to synthesise Nα-Fmoc-protected tryptophan derivatives in which the prenyl group is linked to the N-indole core, using Pd(ii)-mediated C-H functionalisation of 2-methyl-2-butene. Based on the Substance P antagonist G (SPG), a well-known Small Cell Lung Cancer (SCLC) anticancer agent, we designed a new penta-peptide sequence to include a prenyl moiety on one of the tryptophan residues. The N-tert-prenylated tryptophan analogue was assembled into the pentameric peptide using standard solid phase peptide synthesis or liquid phase synthesis by fragment coupling. In vitro screening showed that the N-tert-prenylation of the indole ring on the tryptophan residue located near the C-terminal of the penta-peptide enhanced the cytotoxicity against H69 (IC50 = 2.84 ± 0.14 µM) and DMS79 (IC50 = 4.37 ± 0.44 µM) SCLC cell lines when compared with the unmodified penta-peptide (H69, IC50 = 30.74 ± 0.30 µM and DMS79, IC50 = 23.00 ± 2.07 µM) or the parent SPG sequence (IC50 > 30 µM, both cell lines). SCLC almost invariably relapses with therapy-resistant disease. The DMS79 cell line was established from a patient following treatment with a number of chemotherapeutics (cytoxan, vincristine and methotrexate) and radiation therapy. Treating DMS79 tumour-bearing nude mice provided a human xenograft model of drug resistance to test the efficacy of the prenylated peptide. A low dose (1.5 mg kg-1) of the prenylated peptide was found to reduce tumour growth by ∼30% (P < 0.05) at day 7, relative to the control group receiving vehicle only. We conclude that the availability of the Fmoc-Trp(N-tert-prenyl)-OH amino acid facilitates the synthesis of prenylated-tryptophan-containing peptides to explore their therapeutic potential.

Improved Tat-mediated plasmid DNA transfer by fusion to LK15 peptide.

The use of cell penetrating peptides (CPPs), such as Tat-derived peptide, to deliver DNA into cells is limited as evidenced by the low transfection efficiency of their DNA complexes. Here, we demonstrate that covalent attachment of membrane active peptide LK15 to Tat peptide improves gene transfer. Our results demonstrate that Tat peptide was able to form complexes with DNA, but their transfection efficiency was insufficient as assessed by luciferase assay. The attachment of LK15 to Tat significantly improved the physiochemical properties of the DNA complexes, rendered the complexes membrane active and enhanced the gene expression in HT29 and in HT1080 cultured cells. The enhanced transfection ability of Tat-LK15 compared to Tat is likely to be due mainly to the higher uptake of DNA. Finally, we evaluated the penetration and transfection ability of Tat and Tat-LK15 in multicellular tumour spheroids (MCTS) to mimic in vivo delivery to tumours. The results showed that the penetration and transfection ability of Tat and Tat-LK15/DNA complexes were limited to the rim of HT29 spheroids. Taken together, our data shows improvement in the transfection efficiency of Tat peptide by covalent attachment to LK15. Further advancements are needed before any potential applications in tissues as the penetration into the core of MCTS remains severely restricted.

Cloaking cytolytic peptides for liposome-based detection and potential drug delivery.

Potent cytolytic peptides with specific tethering and cloaking sites have been synthesised and used to release payload from liposomes in a quantitative manner. A functionally located cloaking site has been modified specifically by simple conjugation without adversely affecting the cytolytic properties of the peptide. The cytolytic activity of modified peptides was then efficiently (>98%) cloaked and uncloaked by ligand-protein or hapten-antibody interactions. The principle of a dual response peptide has been demonstrated using an avidin-cloaked pH-sensitive peptide. Biospecific cloaking/uncloaking provided a new sensitive (approximately 12 pmol) homogeneous diagnostic and also appears potentially suited to bioresponsively targeted release of antimicrobial, anticancer and other drugs now delivered using liposomes.