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

Article abstract of DOI:10.1002/cbic.201600257

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.

Full text of DOI:10.1002/cbic.201600257

Accepted Article
Title: Naturally Inspired Peptide Leads: Alanine Scanning Reveals an Actin-
Targeting Thiazole Analogue of Bisebromoamide
Authors: Heather J Johnston; Sarah K Boys; Ashraff Makda; Neil O Carragher; Ali-
son Nicola Hulme
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soon as possible and may be different to this Accepted Article as a result of editing.
Readers should obtain the VoR from the journal website shown below when it is pu-
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To be cited as: ChemBioChem 10.1002/cbic.201600257
Link to VoR: http://dx.doi.org/10.1002/cbic.201600257
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ChemBioChem
10.1002/cbic.201600257
FULL PAPER
Naturally Inspired Peptide Leads: Alanine Scanning Reveals an
Actin-Targeting Thiazole Analogue of Bisebromoamide
Heather J. Johnston,[a] Sarah K. Boys,[a] Ashraff Makda,[b] Neil O. Carragher[b] and Alison N. Hulme*[a]
Abstract: Systematic alanine-scanning of the linear peptide
bisebromoamide (BBA), isolated from a marine cyanobacterium, is
enabled by targeting the solid phase peptide synthesis of thiazole
analogues. The synthetic Tz-BBA analogues have comparable
cytotoxicity (nM) to bisebromoamide and cellular morphology assays
indicate that they target the actin cytoskeleton. Pathway inhibition in
human colon tumour (HCT-116) cells has been explored using
reverse phase protein array (RPPA) analysis, which shows a dose-
dependent response of 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.
syntheses have been reported[10] both of which rely on stepwise
construction of tri-peptide fragments in solution, followed by late
stage introduction, or construction, of the sensitive thiazoline
(Tzl) ring [Scheme 1 (b)]. These prior studies have generated a
very limited amount of SAR data, and indicate that either
enantiomeric orientation of the methyl group on the thiazoline
ring is equally active.[10] Crucially, subsequent isolation of the
nor-methyl analogue at this position (nor-BBA), has shown that it
also retains activity.[9] These data allowed us to design a more
pH stable thiazole analogue[11] of bisebromoamide [Tz-BBA
(Bis1), Scheme 1 (c)] with confidence, and to propose an SPPS
route to its construction which would facilitate systematic
investigation of the SAR of this intriguing peptide.
Introduction
Non ribosomal peptide synthase (NRPS)-derived natural
products provide a wealth of therapeutic leads due to the
chemical and structural diversity which they encompass.[1] Since
NRPS peptides often contain heavily modified analogues of the
proteinogenic amino acids which make up their ribosomal
counterparts,[2] they tend to be more resistant to proteolysis and
thus ripe for development as drugs. One of the greatest
challenges to studying the bioactivity of NRPS peptide leads is
their synthesis, and the modification of this synthetic process to
allow systematic structure-activity relationship (SAR) studies to
be conducted.[3,4] Reducing this challenge to two simpler
problems of: (i) efficient building block synthesis; and (ii) building
block coupling under standard solid phase peptide synthesis
(SPPS) conditions, provides an attractive approach and
facilitates SAR studies (e.g. peptide scanning)[5] which are
commonly employed in the proteinogenic domain.
aa4 aa3 aa2 aa1
aa7 aa6 aa5
The linear peptide bisebromoamide [BBA (1), Scheme 1
(a)] was isolated from a marine cyanobacterium Lyngbya sp. by
the Suenaga group in 2009.[6] BBA was shown to have an IC50 of
0.04 µg/mL against HeLa S3 cells and an average GI50 of 40 nM
when tested against a panel of 39 human cancer cell lines.[6]
BBA has been shown to inhibit the phosphorylation of ERK in
NRK cells,[6] and both ERK and Akt in two renal cell carcinoma
lines.[7] BBA has also been shown to be the first linear peptide to
act as an actin filament stabilizer.[8] While the biosynthesis of
BBA 1 has not yet been elucidated, the presence of D-amino
acids and heavily modified proteinogenic amino acids led
Suenaga et al. to suggest a NRPS origin.[2,9] To date, two total
aa7
aa6
aa4,5
aa3 aa2 aa1
[a]
[b]
H. J. Johnston, S. K. Boys, Dr. A. N. Hulme
EaStCHEM School of Chemistry, The University of Edinburgh,
David Brewster Road, Edinburgh, EH9 3FJ, UK
E-mail: Alison.Hulme@ed.ac.uk
Scheme 1. (a) Marine cyanobacterium-derived linear amide, bisebromoamide
(BBA, 1); (b) Previous solution phase synthetic strategies reliant on the late-
stage introduction, or construction, of the sensitive 4-MePro-Tzl motif (the
conjugate of aa4 & aa5); (c) & (d) This work, stepwise construction of thiazole-
bisebromoamide (Tz-BBA, Bis1) by SPPS, incorporating a central, stable 4-
MePro-Tz building block (aa4,5 highlighted in red).
A. Makda, Prof. N. Carragher
Edinburgh Cancer Research Centre, Institute of Genetics and
Molecular Medicine, University of Edinburgh, Crewe Road South,
Edinburgh EH4 2XR, UK
Supporting information for this article is given via a link at the end of
the document. Primary data files can be found at
http://dx.doi.org/10.7488/ds/1417

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Results and Discussion
conditions,[19] allowing the desired product Tz-BBA Bis1 to be
1
isolated, purified by RP-HPLC[23] and analyzed by 2D NMR. H
NMR data for the 4-MePro ring shows marked differences
between cis- and trans-substituted isomers;[14] detailed analysis
of NMR data for peptides Bis1-6 supported the conclusion that
negligible epimerization of the 4-MePro-Tz motif had occurred
during SPPS.
Amide-bond disconnection across Tz-BBA Bis1 led to the
identification of seven key fragments: aa1 2-(1-oxo-
propyl)pyrrolidine (Opp); aa2 N-methyl phenylalanine (NMe-
Phe); aa3 D-leucine (D-Leu); aa4,5 the cyclised and oxidised
conjugate of 4-methylproline and cysteine (4-MePro-Tz); aa6 N-
methyl-3-bromotyrosine (NMe-D-BrTyr); aa7 alanine (Ala); and
pivalic acid (Piv) which caps the N-terminus. Targeting an SPPS
based strategy for synthesis required each of the six amino acid-
based fragments in nitrogen protected forms. Practical issues
associated with the amino acid sequence[12] and linker group
compatibility compelled the usage of Boc-protecting groups. The
three non-commercial fragments were synthesised as shown in
Scheme 2. Grignard addition to commercial Weinreb amide 2[13]
gave the Opp fragment 3 in excellent yield (97%). Synthetic 4-
methyl-proline tert-butyl ester 4[14] was converted to Boc
carbamate 5 under standard conditions, and DIBAL-H reduction
of the ester to give prolinal derivative 6 proceeded smoothly.
The resultant aldehyde was coupled to L-cysteine methyl ester
following the precedent of Shiori et al.[15] to give intermediate
thiazolidine 7 as a mixture of diastereomers (~1:1.5 by NMR).
Reproducible oxidation to the corresponding thiazole 8 (MnO2)
was shown to be dependent on pre-activation of the oxidant by
heating.[15] Basic hydrolysis of the methyl ester gave the 4-
MePro-Tz fragment 9 (55% overall yield from 4) in excellent
optical purity. Finally, the phenol in bromo-tyrosine derivative 10
(readily accessed using selective mono ortho-bromination by
NBS)[16] was protected as its tert-butyl ether 11 using a
modification of the procedure reported by Sambri et al..[17] N-
methylation to give 12 and subsequent ester cleavage gave the
desired NMe-D-BrTyr fragment 13 (66% overall yield from 10).
The use of a hydrazine-based linker[18] was required to
facilitate the direct attachment of the C-terminal Opp moiety in
the SPPS synthesis of Tz-BBA Bis1. The hydrazide adducts
which result are stable to basic and non-aqueous acidic
conditions, show little evidence of racemization and provide
products in high levels of purity. We were attracted to the
semicarbazide linker described by Albericio et al.,[19] which has
been used by several groups for the synthesis of peptidyl
aldehydes and ketones.[20] Modified resin 14 was readily
prepared from aminomethyl-polystyrene by treatment with CDI
followed by tert-butyl carbazate (Scheme 3);[19] successful TFA-
mediated deprotection of intermediate species 15 was confirmed
using the TNBS test.[19] The Boc-protected Opp moiety 3 was
attached as its hydrazide in the presence of acetic acid.[20a]
Subsequent amino acid coupling cycles were achieved using
anhydrous conditions for Boc deprotection, followed by
Oxyma/DIC-mediated coupling with the appropriate amino
acid.[21] In all cases, only three equivalents of the coupling amino
acid were used, but where the N-terminus was comprised of a
secondary amino acid a double coupling cycle was employed.
For the final coupling and capping steps [after the addition of
Scheme 2. Opp, 4-MePro-Tz and NMe-D-BrTyr fragment synthesis. Reagents
and Conditions: a) EtMgBr (1 M in THF), THF, 0 ºC – rt, 4 h, 97%; b) Boc2O,
DIPEA, CH2Cl2, rt, 16 h, 95%; c) DIBAL-H, CH2Cl2, 78 ºC, 2 h, 97%; d) Et3N,
Toluene, 0 ºC – rt, 16 h, 98%; e) MnO2, MeCN, 60 ºC, 24 h, 77%; f) NaOH (1
M aq), MeOH, THF, 0 ºC – rt, 16 h, 80%; g) Boc2O, Sc(OTf)2, CH2Cl2, rt, 24 h,
77%; h) NaH, THF, MeI, DMF, 0 ºC – rt, 16 h, 95%; i) NaOH (1 M aq), MeOH,
THF, 0 °C  rt, 24 h, 90%.
Boc-BrTyr(OtBu)],
a
two-step deprotection process was
employed to allow selective cleavage of the Boc carbamate in
the presence of the tert-butyl ether.[22] Cleavage from the resin
and deprotection were achieved under aqueous acidic

ChemBioChem
10.1002/cbic.201600257
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yield).[25] Switching the terminal pivalic acid capping moiety to
acetic acid gave the final analogue (Bis6, Table 1) for use in
SAR studies.
The effects of Bis1-6 on the growth of the human colon
cancer cell line, HCT116, were probed using an IncuCyte Zoom
platform. HCT116 cells express an activating Ras mutation
representing an aggressive and common form of human colon
cancer.[26] The dual Src-Abl tyrosine kinase inhibitor dasatinib
and the DMSO vehicle (0.1% v/v) were used in control assays.
Confluence assays monitoring HCT116 cell growth clearly
indicated both a dose-dependent and time-dependent response
for compounds Bis1-4, with Bis1 appearing to be the most
active (Figure S1A). Apoptosis was measured dynamically using
the caspase 3 biosensor NucView[27] and indicated that Bis1-4
exerted their cytotoxic activity through an apoptotic mechanism
(Figure S1A). Compounds Bis5 and Bis6 showed little or no
activity either in repressing cell growth, or inducing apoptosis.
The relative activity of all the analogues was further evaluated
by testing each Tz-BBA analogue as an 8-point half-log dose
response in a 3 day alamarBlue® cell viability assay. Following
72 h incubation with each Tz-BBA analogue, cell viability was
measured by fluorescent conversion of the alamarBlue® reagent
and dose response curves were fitted to each concentration
series to calculate the relative EC50 upon cell viability;
compounds Bis1-4 showed nM EC50 activities whilst Bis5 and
Bis6 showed no significant activity (Figure 1). These initial
results indicated that substitution of either the N-terminal
pivalate capping group, or aa6 (the NMe-D-BrTyr residue)
significantly reduces the activity of Tz-BBA analogues.
Scheme 3. Semicarbazide resin synthesis and SPPS coupling. Reagents and
Conditions: a) (i) CDI, DMF, rt,
3 h; (ii) BocNHNH2, DMF, rt, 3 h; b)
i
TFA:CH2Cl2 (1:1), rt, 1 h, followed by 2 × 10 min wash with 10% Pr2NEt 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 ºC – rt , 2
h; (ii) TBAF (1 M in THF), THF, rt, 20 min; f) Boc-protected aa2, aa3, aa4,5, aa6,
aa7 or pivalic acid, Oxyma, DIC, DMF, rt, 1 h; g) TFA:H2O (4:1), rt, 1 h.
Table 1. Sequences of the Tz-BBA alanine scan analogues used to probe the
structure activity relationships of BBA.
N-
aa7
aa6
aa4,5
aa3
aa2
aa1
NMe-
D-BrTyr
NMe-
D-BrTyr
NMe-
D-BrTyr
NMe-
4-Me-
Pro-Tz
4-Me-
Pro-Tz
4-Me-
Pro-Tz
4-Me-
Pro-Tz
4-Me-
Pro-Tz
4-Me-
Pro-Tz
Bis1
Bis2
Bis3
Bis4
Bis5
Bis6
Piv
Ala
D-Leu
NMe-Phe
Opp
Piv
Piv
Piv
Piv
Ac
Ala
Ala
Ala
Ala
Ala
D-Leu
D-Leu
Ala
NMe-Phe
Ala
Ala-NH2
Opp
NMe-Phe
NMe-Phe
NMe-Phe
Opp
D-BrTyr
Ala
D-Leu
D-Leu
Opp
NMe-
D-BrTyr
Opp
Peptide scanning, like SPPS, is a technique used more
commonly in the development of ribosomal peptides, however it
can also be used for non-ribosomal species and it provides a
wealth of information.[5] The limited studies on BBA (1) to date
have not allowed a comprehensive assessment of the activity
which results from each individual amino acid residue,[9,10] most
probably because of the relative complexity of previous synthetic
routes, combined with the comparative instability of amino acids
adjacent to a thiazoline moiety. By systematically replacing
amino acid residues aa2, aa3, and aa6 in the Tz-BBA target Bis1
with alanine,[24] three analogues (Bis3-5, Table 1) were
successfully synthesized by SPPS using the semicarbazide
resin (2-8% overall yield). Replacement of the C-terminal Opp
moiety with alanine (Bis2, Table 1) was achieved by use of the
Rink amide resin to give the C-terminal amide (16% overall
Analogue
Bis1
Bis2
Bis3
Bis4
EC50 (nM)
45
71
483
178
Figure 1. Cell viability for the human colon cancer cell line HCT-116. Dose
response curves and calculated EC50 values determined by alamarBlue® cell
viability assay after incubation of HCT116 cells with compounds Bis1-6 for
72 h (dose range: 0.003-10 μM). Conversion of the alamarBlue® reagent to
the activated fluorescent resorufin cell viability indicator was calculated across
DMSO (vehicle) and compound treated samples. Tz-BBA analogue molar
concentration expressed on a log scale (X-axis). Dose response of compound-
treated cells expressed as a percentage of control (DMSO) values (Y-axis).
Cell viability dose-response plots and EC50 values represent the mean from 3
independent experiments. Error bars: +/- Standard Deviation from n=3.

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Figure 2. Cytoskeletal morphology assays on the human colon cell line HCT-116. Cells were treated for 48 h with 1 M Tz-BBA analogues Bis1-6. Cells where
subsequently permeabilized and fixed in paraformaldehyde prior to staining nuclei with the DNA binding dye, DAPI (blue) and Filamentous-actin fibers (F-actin)
with phalloidin conjugated to Alexa Fluor 548 (green). Images were acquired using an automated ImageXpress microXL high-content imaging platform (Molecular
Devices) using a 20× objective and fields of view where selected to represent HCT-116 cell morphology for DMSO (control) and each treatment group. Images
are for: a) DMSO; b) Cytochalasin D; c) Bis1; d) Bis2; e) Bis3 (representative of data for Bis3 and Bis4); f) Bis 6 (representative of data for Bis5 and Bis6).
To further investigate the mechanism of action of the
active analogues (Bis1-3) Reverse Phase Protein Array (RPPA)
profiling[28] was conducted against a panel of 62 targets on a
Zeptosens planar waveguide RPPA platform.[29] The Zeptosens
platform provides precise quantification of changes in the
abundance of multiple protein and phosphorylated protein
species in biological samples following exposure to compound
dose- and time-series, thereby providing an unbiased evaluation
of compound mechanism-of-action at the post-translational
pathway level.[30] The 62 pathway markers were selected to
cover a broad range of key cancer driver pathways, canonical
signaling nodes and the ERK and Akt signaling pathways
previously demonstrated to be regulated by BBA in renal
carcinoma cells (see Table S1 for antibodies and pathways
studied). The only clearly discernable inhibition of pathway
signaling was upon protein kinase C (PKC) and discernable
reduction in total protein expression levels was of the insulin
receptor substrate protein IRS-1 (see Figure S2). Both of which
appeared to be more affected by analogues Bis1 and Bis2 than
the other active analogue Bis3. In contrast to the results of
previous studies performed on HeLa[6] and renal carcinoma cells
lines,[7] for the HCT116 human colon cancer cell line RPPA
analysis showed no inhibition of phosphorylation of MEK or Akt
by any of the analogues tested (see Figure S2). IRS-1 is an
intracellular signaling adaptor protein which can act as a docking
site for SH2-containing proteins including PI3K and Grb2, thus
linking it to the PI3K/Akt/mTOR and MAPK (ERK) pathways.[31]
However, IRS proteins can be phosphorylated on serine
residues through negative feedback loops, which inhibits their
function.[32] Thus the expression of IRS-1, which itself varies
from cell type to cell type, may not reflect the functional status of
this adaptor protein.[31] The relatively “clean” profile obtained by
RPPA demonstrates that the Tz-BBA analogues do not result in
global changes in stress pathways and canonical signaling
events, and are suggestive of a selective mechanism-of-action.
Perhaps most striking in terms of defining a mechanism for
the cytotoxicity were the morphological images which were
collected using an ImageXpress high content microscope
(Figure 2). All four active Tz-BBA analogues showed distinct
morphological changes relative to the DMSO control (Figure 2a),
but N-terminal analogues Bis5 and Bis6 gave rise to no
significant changes (Figure 2f). The lead compound Bis1 and its
C-terminal aa1 modified alanine analogue Bis2 show
morphological changes including clear disruption and
aggregation of F-actin filaments and reduced cell-substrate
adhesion (Figure 2c,d) which are similar to those shown by
compounds such as cytochalasin
D
(Figure 2b) and
jasplakinolide which impair actin dynamics.[33] The other
analogues Bis3 and Bis4 show different morphological changes
(Figure 2e), including more elongated cells with prominent F-
actin filaments and promotion of cell-substrate adhesion; these
changes either result from a different mechanism, or perhaps
from the lower overall activity of these analogues. The
morphological changes induced by Bis1 and Bis2 are in good
accord with those previously reported for the parent compound
BBA in HeLa cells,[8] and indicate that the switch from thiazoline

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DIPEA in DMF (3 mL) was added to the resin and the resin was agitated
for 10 min at rt. This was repeated with fresh solution for a further 10 min,
then the resin was washed with DMF (3 × 5 mL), CH2Cl2 (3 × 5 mL),
MeOH (3 × 5 mL) and CH2Cl2 (3 × 5 mL). The TNBS test was performed
and the deprotection was repeated if any colourless beads were
observed. Semicarbazide resin 14 was used immediately.
to thiazole to facilitate the generation of these Tz-BBA
analogues by SPPS has not impaired their activity.
Conclusions
Attachment of the Ketone-containing Opp residue: A solution of Boc-
protected ketone 3 (2.4 mmol, 3.0 eq.) in anhydrous CH2Cl2 (5 mL) and
2% glacial acetic acid was added to the resin and the resin was agitated
for 18 h at rt.
Amino Acid coupling: The Boc-protected amino acid residue (2.4 mmol,
3.0 eq.) and Oxyma (0.34 g, 2.4 mmol, 3.0 eq.) were dissolved in DMF (3
mL) and DIC (0.37 mL, 2.4 mmol, 3.0 eq.) was added. The reaction
mixture was agitated for 5 min before addition to the resin along with
iPr2NEt (0.21 mL, 1.2 mmol, 1.5 eq.), and the resin was then agitated at rt
for 1 h. The reaction mixture was removed and the resin washed with
DMF (3 × 5 mL), CH2Cl2 (3 × 5 mL), MeOH (3 × 5 mL) and CH2Cl2 (3 × 5
mL).
Boc Deprotection: A 1:1 mixture of TFA and CH2Cl2 (5 mL) was added
to the resin and the resin was agitated for 5 min. This was repeated with
fresh solution for a further 20 min, then the resin was washed with
CH2Cl2 (3 × 5 mL), MeOH (3 × 5 mL) and CH2Cl2 (3 × 5 mL). The TNBS
test was performed and the deprotection was repeated if any colourless
beads were observed.
Selective Boc deprotection: The resin was solvated in dry CH2Cl2 (8
mL) and chilled (dry ice) before addition of 2,6-lutidine (1.38 mL, 12.0
mmol, 15 eq.) and TMSOTf (1.74 mL, 9.60 mmol, 12 eq.). The resin was
agitated at 78 ºC for 15 min then at rt for a further 90 min. The reaction
mixture was removed and the resin washed with CH2Cl2 (3 × 5 mL),
MeOH (3 × 5 mL) and CH2Cl2 (3 × 5mL). TBAF (4 mL, 1 M in THF) was
then added and the resin was agitated for 10 min. This was repeated with
fresh solution, then the resin was washed with CH2Cl2 (3 × 5 mL), MeOH
(3 × 5 mL) and CH2Cl2 (3 × 5 mL).
Since its initial isolation in 2009, bisebromoamide (BBA)
has been identified as targeting the ERK and Akt pathways in
renal cell carcinoma cell lines,[7] and has been shown to be the
first linear peptide actin-targeting molecule.[8] Through the
generation of thiazoline analogues (Tz-BBA) we have been able
to pursue an SPPS-based approach towards the investigation of
its SAR for the first time, generating four analogues (Bis1-4)
which showed nM cytotoxic activity against a human colon
tumour cell line, HCT-116. Systematic alanine scanning has
revealed that while the C-terminus may be altered without
significantly affecting the activity of the resultant analogues;
alteration of N-terminal residues (Bis5), or replacement of the N-
terminal pivalate cap (Bis6) removes all activity. RPPA analysis
suggests a very specific mode of action for the most active of
these Tz-BBA analogues (Bis1 and Bis2) in HCT-116 cells,
which results in reduced activity of PKC and reduced expression
of IRS-1. This is particularly exciting since the oncogenic protein
IRS-1 is over-expressed in a wide range of cancers.[31]
Two analogues (Bis1 and Bis2) have been shown to
induce similar morphological changes in HCT-116 cells to those
generated by the parent linear peptide in HeLa cells at
comparable dosage levels; these changes are consistent with F-
actin disruption and aggregation. Of these analogues, Ala-NH2
terminated Tz-BBA derivative Bis2 is particularly attractive as a
lead compound, since its SPPS synthesis can be conducted
using high-yielding Rink amide resin chemistry, rather than the
more-challenging semicarbazide-based resin synthesis which
was employed in the construction of the other analogues. Thus
our systematic SPPS-based approach towards the SAR analysis
of this NRPS-derived linear peptide has allowed the
identification of a new and promising lead for use in the design
of anti-cancer therapeutics, or as a tool to study the role of IRS-1
expression and F-actin aggregation.
Cleavage of Peptide from Semicarbazide Resin:
A mixture of
TFA:H2O (5 mL, 4:1) was added to the resin and the resin was agitated
at rt for 1 h. The reaction solution was filtered and collected, then
evaporated to dryness under nitrogen. The resulting residue was then
purified by column chromatography (CH2Cl2:MeOH) to obtain the desired
product.
Experimental procedures for the Rink amide coupling, together with
purification procedures and spectroscopic data for peptides Bis1-6 are
presented in the Supporting Information.
High-Content Imaging Methods
Kinetic cell growth and Apoptosis assays were performed using the
IncuCyte-ZOOM® Live cell kinetic imaging system (Essen Bioscience).
Cytoskeletal morphology assays were performed on the ImageXpress-
microTM High content imaging system (Molecular Devices).
Experimental Section
Experimental procedures and spectroscopic data for the preparation of
fragments Opp (3), 4-MeProTz (9) and NMe-D-BrTyr (13) are presented
in the Supporting Information.
Cell Viability Assay: Cells were seeded in a 96-well plate format at the
appropriate cell concentration (5,000 cells/well) and incubated for 48 h
before treatment. The media in each well was then replaced with fresh
media containing each Tz-BBA analogue Bis1-6 as an 8-point half-log
dose response and incubated for 3 days. alamarBlue® cell viability
reagent (10% v/v) was added to each well and the plate was incubated
for 1 h. Fluorescence emission was detected using a PerkinElmer
EnVision® multilabel reader (excitation filter at 540 nm and emissions
filter at 590 nm). All conditions were normalized to the DMSO treated
cells (100%) and curves fitted using GraphPad Prism using a sigmoidal
variable slope curve. The data presented in Figure 1 represent the mean
and standard deviation from 3 independent experiments.
SPPS Conditions for Semicarbazide Resin 14
Preparation of Resin 14: Aminomethyl polystyrene (300-500 mesh, 4.0
mmol/g) (0.20 g, 0.80 mmol, 1.00 eq.) was swollen in CH2Cl2 (5 mL) for
30 min, then washed with DMF (3 × 5 mL). CDI (0.65 g, 4.0 mmol, 5 eq.)
in DMF (5 mL) was added to the resin and the resin was agitated for 3 h
at rt. The resin was washed with DMF (3 × 5 mL), then tert-butyl
carbazate (0.53 g, 4.00 mmol, 5 eq.) in DMF (5 mL) was added to the
resin and the resin was agitated for a further 3 h at rt to give intermediate
15. The resin was washed with DMF (3 × 5 mL) and CH2Cl2 (3 × 5 mL),
then a 1:1 mixture of TFA and CH2Cl2 (5 mL) was added to the resin and
the resin was agitated for 1 h. The resin was washed with CH2Cl2 (3 × 5
mL), MeOH (3 × 5 mL) and CH2Cl2 (3 × 5 mL), then a solution of 10%
Apoptosis Assay: Cells were seeded in a 96-well plate format at the
appropriate cell concentration (5,000 cells/well) and incubated for 24 h
before treatment. The media in each well was then replaced with fresh

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10.1002/cbic.201600257
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media containing each Tz-BBA analogue Bis1-6 as an 8-point half-log
dose response and incubated for 5 days. Untreated cells were incubated
with DMSO (0.1% v/v). Apoptosis was detected using the cell-permeable
caspase biosensor NucViewTM (Biotium) which was added to each well at
1 M at the same time as compound treatment. The IncuCyte® imaging
using the ZeptoREADERTM instrument (Zeptosens-Bayer). For each-sub-
array, five separate images were acquired using different exposure times
ranging from 0.5-10 s. Microarray images representing the longest
exposure without saturation of fluorescent signal detection were
automatically selected for analysis using the ZeptoViewTM 3.1 software. A
weighted linear fit through the 4-fold concentration series was used to
calculate relative fluorescence intensity (RFI) value for each sample
replicate. Local normalization of sample signal to the reference BSA grid
was used to compensate for any intra- or inter-array/chip variation.
The panel of 62 protein analytes (see Table S1) were normalized by the
following global normalization procedure using the entire antibody panel:
(a) Determine median for each antibody across the sample set. (b) Divide
each raw linear value by the median within each antibody to obtain the
median-centred ratio. (c) Calculate the median from median-centred ratio
for each sample across the entire panel of antibodies. This median
functions as a correction factor for protein loading adjustment. (d) Divide
raw RFI data by the correction factor to obtain the normalized values.
Global normalized data for each Tz-BBA analogue is subsequently
normalized to DMSO control for each time point and plotted as bar
graphs (see Figure S2).
software automatically quantifies cell confluence and NucViewTM
/
Caspase positive objects at sequential time points following compound
addition. This provided a kinetic read out of cell growth and apoptosis
induction in cells following addition of each Tz-BBA analogue.
Representative data and images from
a least three independent
experiments performed across separate weeks are presented in Figure
S1.
Cytoskeletal Morphology Assays: Cells were seeded in a 96-well plate
format at the appropriate cell concentration (5,000 cells/well) and
incubated for 24 h before treatment. The media in each well was then
replaced with fresh media containing each Tz-BBA analogue Bis1-6 as
an 8-point half-log dose response and incubated for 48 h. Untreated cells
were incubated with DMSO (0.1% v/v). All images were acquired on the
automated ImageXpress microXL high-content imaging platform
(Molecular Devices) using a ×20 PanFluor ELWD Ph1 DM objective and
a 16-bit camera (binning resolution of 1). Four separate fields of view per
well were acquired using laser-based autofocus parameters optimized for
cell plates and cell type. All immunostaining procedures were performed
at room temperature in 96-well plates. All volumes are 100 μL unless
otherwise stated. Cells were fixed following direct addition of 8%
paraformaldehyde in PBS to cells (final concentration 4%) and were
incubated for 20 min. Cells were washed three times with PBS then
incubated with a mixture of Phalloidin conjugated to Alexa Fluor 548
(Molecular probes; diluted 1:500) and 4’,6-diamidino-2-phenylindole
(DAPI; Sigma D8417) for 45 min in the absence of light. Cells were
washed three times with PBS again before imaging. Representative
images are presented in Figure 2.
Acknowledgements
We thank Cancer Research UK (Grant Ref C21383/A6950),
EPSRC and BBSRC (studentship to SKB, BBS/S/H/2005/13535)
for funding. We also thank Research Councils UK for an
academic fellowship award to NOC.
Keywords: non-ribosomal peptide • structure activity
relationship • solid phase synthesis • alanine scan • reverse
phase protein array• cell morphology • IRS-1 • F-actin
Reverse Phase Protein Array Methods
The abundance of total protein and phosphorylated protein epitopes was
quantified using the Zeptosens reverse phase protein microarray platform
as previously described.[30] Briefly, HCT116 cells were seeded at 4 × 105
cells/well in 6-well plates. Cells were pre-incubated in treatment-free
media for 48 h prior to addition of each Tz-BBA analogue (Bis1-3) at 6-
point half log dose response ranging from 3 – 0.03 M for 30 min, 3 h
and 24 h. Control samples were treated with media containing 0.1% (v/v)
DMSO. Following exposure, the drug-containing medium was removed,
and the cells were lysed with CLB1 buffer (Zeptosens, Bayer) for 30 min
according to the manufacturer’s instructions. Cell lysates were
normalized to a uniform protein concentration with spotting buffer CSBL1
(Zeptosens-Bayer) prior to preparing a final 4-fold concentration series
of; 0.2; 0.15; 0.1 and 0.75mg/ml. The diluted concentration series of each
sample was printed onto Zeptosens protein microarray chips
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Entry for the Table of Contents
FULL PAPER
Heather J. Johnston, Sarah K. Boys,
Ashraff Makda, Prof. Neil O. Carragher
and Dr. Alison N. Hulme*
Page No. – Page No.
Naturally Inspired Peptide Leads:
Alanine Scanning Reveals an Actin-
Targeting Thiazole Analogue of
Bisebromoamide
Targeting actin by design: Systematic alanine scanning of the marine-derived,
linear NRPS peptide bisebromoamide reveals a thiazole analogue with nM
cytotoxicity which is readily synthesised by solid phase peptide synthesis. High
content imaging suggests a mode of action similar to jasplakinolide.