Naturally Inspired Peptide Leads: Alanine Scanning Reveals an Actin-Targeting Thiazole Analogue of Bisebromoamide

Abstract

Systematic alanine scanning of the linear peptide bisebromoamide (BBA), isolated from a marine cyanobacterium, was enabled by solid-phase peptide synthesis of thiazole analogues. The analogues have comparable cytotoxicity (nanomolar) to that of BBA, and cellular morphology assays indicated that they target the actin cytoskeleton. Pathway inhibition in human colon tumour (HCT116) cells was explored by reverse phase protein array (RPPA) analysis, which showed a dose-dependent response in IRS-1 expression. Alanine scanning reveals a structural dependence to the cytotoxicity, actin targeting and pathway inhibition, and allows a new readily synthesised lead to be proposed.

Keywords: alanine scan; cell morphology; nonribosomal peptides; reverse phase protein array; solid-phase synthesis; structure-activity relationships.

Scheme 1

A) Marine cyanobacterium‐derived linear amide, bisebromoamide (BBA, 1); B) Previous solution‐phase synthetic strategies rely on the late‐stage introduction (or construction) of the sensitive 4‐MePro‐Tzl motif (the conjugate of aa⁴ and aa⁵); C), D) In this work, stepwise construction of thiazole‐bisebromoamide (Tz‐BBA, Bis1) by SPPS incorporates a central, stable 4‐MePro‐Tz building block (aa^4,5 red).

Scheme 2

Synthesis of Opp, 4‐MePro‐Tz and N‐Me‐d‐BrTyr fragments. a) EtMgBr (1 m in THF), THF, 0 °C–RT, 4 h, 97 %; b) Boc₂O, DIPEA, CH₂Cl₂, RT, 16 h, 95 %; c) DIBAL‐H, CH₂Cl₂, −78 °C, 2 h, 97 %; d) Et₃N, toluene, 0 °C–RT, 16 h, 98 %; e) MnO₂, MeCN, 60 °C, 24 h, 77 %; f) NaOH (1 m aq), MeOH, THF, 0 °C–RT, 16 h, 80 %; g) Boc₂O, Sc(OTf)₂, CH₂Cl₂, RT, 24 h, 77 %; h) NaH, THF, MeI, DMF, 0 °C–RT, 16 h, 95 %; i) NaOH (1 m aq), MeOH, THF, 0 °C–RT, 24 h, 90 %.

Scheme 3

Semicarbazide resin synthesis and SPPS coupling. a) i: CDI, DMF, RT, 3 h; ii: Boc‐NHNH₂, DMF, RT, 3 h; b) TFA/CH₂Cl₂ (1:1), RT, 1 h, followed by 2×10 min wash with 10 % iPr₂NEt in DMF; c) 3, AcOH/CH₂Cl₂ (2:98), RT, 16 h; d) i: TFA/CH₂Cl₂ (1:1), RT, 25 min; ii: iPr₂NEt wash prior to coupling; e) i: 2,6‐lutidine, TMSOTf, CH₂Cl₂, 0 °C–RT, 2 h; ii: TBAF (1 m in THF), THF, RT, 20 min; f) Boc‐protected aa², aa³, aa^{4 ,5}, aa⁶, aa⁷ or pivalic acid, Oxyma, DIC, DMF, RT, 1 h; g) TFA/H₂O (4:1), RT, 1 h.

Figure 1

HCT116 cell viability. Dose–response curves determined by an alamarBlue cell viability assay after incubation with Bis1–6 (0.003–10 μm) for 72 h. Conversion of the alamarBlue reagent to the activated fluorescent resorufin cell viability indicator was calculated for DMSO (vehicle) and compound‐treated samples. Viability is expressed relative to control (DMSO). Dose–response plots are mean±SD (n=3). EC₅₀ values of 45 (Bis1), 71 (Bis2), 483 (Bis3) and 178 nm (Bis4) were also obtained.

Figure 2

HCT116 cytoskeletal morphology after treatment with 1 μm Bis1–6 for 48 h. Cells were permeabilised and fixed in paraformaldehyde prior to staining nuclei (DAPI, blue) and filamentous‐actin fibres (F‐actin; phalloidin conjugated to Alexa Fluor 548, green). Images were acquired in an automated ImageXpress microXL high‐content imaging platform (Molecular Devices) with a 20× objective; images show HCT116 cell morphology for DMSO (control) and each treatment group: A) DMSO; B) cytochalasin D; C) Bis1; D) Bis2; E) Bis3 (representative also for Bis4); F) Bis 6 (representative also for Bis5).