γ-AApeptide-based small-molecule ligands that inhibit Aβ aggregation

Article abstract of DOI:10.1039/c3cc46685j

We report the design, synthesis, characterization and evaluation of a novel class of γ-AApeptide one-bead-one-compound (OBOC) library, from which a small γ-AApeptide was identified to effectively prevent and disassemble Aβ aggregation. the Partner Organis

Full text of DOI:10.1039/c3cc46685j

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c-AApeptide-based small-molecule ligands that
inhibit Ab aggregation†
Cite this: Chem. Commun., 2014,
50, 5206
Haifan Wu,a Yaqiong Li,a Ge Bai,a Youhong Niu,a Qiao Qiao,a Jeremiah D. Tipton,b
Chuanhai Cao*c and Jianfeng Cai*a
Received 1st September 2013,
Accepted 10th October 2013
DOI: 10.1039/c3cc46685j
www.rsc.org/chemcomm
We report the design, synthesis, characterization and evaluation of a
novel class of c-AApeptide one-bead-one-compound (OBOC) library,
from which a small c-AApeptide was identified to effectively prevent
and disassemble Ab aggregation.
One of the most important goals in modern chemical biology and
biomedical sciences is to identify molecular ligands that recognize
peptides or proteins of interest with high specificity and affinity.1
Combinatorial chemistry is a powerful approach for ligand screening
as it creates a diverse library of compounds, which provides unbiased
opportunity for ligand identification when the structural information
of targets is not available or not helpful in the rational design.1 In fact,
most bioactive molecules are identified through screening efforts.2,3
As peptides have favourable protein binding capabilities and a well-
established synthetic protocol, early efforts were dedicated to the
identification of selective peptide ligands against a variety of targets.4–7
Unnatural peptidomimetic ligand libraries have been of significant
interest recently, as these ligands contain unnatural backbones
and therefore possess enormous structural diversity and
enhanced stability against proteolysis. The examples of peptido-
mimetic ligands include peptoids,1,8–13 b-peptides,14 N-acylated
polyamine,15,16 etc. However, except for peptoids, the applica-
tions of peptidomimetic ligand libraries are rare.
Fig. 1 Synthesis of the g-AApeptides. (a) An a-peptide and a g-AApeptide.
(b) The synthesis of g-AApeptides. (c) N-Alloc protected g-AApeptide
building blocks and acylating agents (carboxylic acids and acyl chlorides)
used in the preparation of a g-AApeptide combinatorial ligand library.
g-AApeptides are a new class of peptidomimetics developed in
our lab very recently in order to advance the application of peptido-
mimetics in chemical biology and biomedical sciences (Fig. 1a).17–24
As they contain N-acylated-N-aminoethyl amino acid units (Fig. 1a)
derived from g-PNAs,25 they are termed ‘‘g-AApeptides’’.20 Each unit
(building block) of the g-AApeptide is comparable to a dipeptide
residue in a canonical peptide. As such, g-AApeptides essentially
project an identical number of functional groups as conventional
peptides of the same length. Half of the side chains of g-AApeptides
are chiral, which may impose conformational bias to the molecular
ligands similar to conventional peptides, and presumably lead to the
identification of ligands with improved specificity and affinity.1 Since
the other half of the side chains are introduced through acylation of
the nitrogen on the backbone by carboxylic acids or acyl chlorides,
there is virtually limitless potential of generating g-AApeptide libraries
a Department of Chemistry, University of South Florida, 4202 E. Fowler Ave, Tampa,
FL 33620, USA. E-mail: jianfengcai@usf.edu; Fax: +1 831-874-3203;
Tel: +1 813-974-9506
b Proteomics and Mass Spectrometry, USF CDDI, 3720 Spectrum Blvd., Tampa,
FL 33612, USA
c College of Pharmacy, University of South Florida, 4202 E. Fowler Ave, Tampa,
FL 33620, USA. E-mail: ccao@health.usf.edu
† Electronic supplementary information (ESI) available: Development of a g-
AApeptide library, ligand screening and identification, and experimental details.
See DOI: 10.1039/c3cc46685j
5206 | Chem. Commun., 2014, 50, 5206--5208
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with chemically diverse functional groups. Moreover, g-AApeptides
are highly resistant to proteolytic degradation,20,26 making them
ideal candidates as molecular probes or therapeutic agents.
To further expand the biological potential of g-AApeptides,
herein we report the development of a g-AApeptide OBOC library
via the split-and-pool method. N-Alloc protected g-AApeptide
building blocks17,19,20,22,27 were used to prepare g-AApeptides
of diverse functional groups (Fig. 1b and c).27
To find out if MS/MS could be potentially used to solve the
unknown g-AApeptide structure, we analyzed the fragmentation
pattern of one known g-AApeptide to prove that MS/MS is
capable of determining the sequences of g-AApeptides. As shown
in Fig. S1 (ESI†), this g-AApeptide produces fragments in clear
patterns and the sequence can be deduced unambiguously.
We then proceeded to the next step by establishing a one-
bead-one-compound (OBOC) g-AApeptide library. The library
was prepared by the split-and-pool method which produces beads
with only one compound displayed on one bead (Scheme S2, ESI†).4 A
methionine residue was first attached to the TentaGel beads (160 002,
150 mm, 520 000 beads per g), which facilitates the cleavage from
beads by CNBr treatment.28 Then 4 N-alloc-protected g-AApeptide
building blocks, and 5 acylating agents (including carboxylic acids or
acid chlorides) (Fig. 1c) were used to generate the combinatorial
library of 4-building-block g-AApeptides (comparable to 8-mer
peptides in length). In theory, this library would contain
4 Â 5 Â 4 Â 5 Â 4 Â 5 Â 4 Â 6 = 192 000 compounds.
Fig. 2 Screening of the g-AApeptide library. (a) Schematic representation
of the on-bead screening of the g-AApeptide library against the Ab40
peptide. The last picture was taken under a fluorescent microscope installed
with a triple filter pass. Excitation is 550 nm and emission is 605 nm. (b) The
identified g-AApeptide from the on-bead screening (HW-155-1) and the
control peptide KLVFF.
To explore the potential of this g-AApeptide library as an
excellent source of protein/peptide ligands, the library was
screened against the Ab40 peptide, which is one of the major
etiological factors for Alzheimer’s disease (AD) and plays a central
role in the pathogenesis of AD.29,30 Although it is of high signifi-
cance to quickly identify novel anti-Ab aggregation inhibitors, there
is no effective approach to achieve this.31
We hypothesized that the g-AApeptide library can be used to
identify ligands that bind to Ab, and their anti-aggregation
efficiency can be determined by functional assay afterwards. As
such, we incubated beads with the Ab40 peptide, followed by the
treatment with anti-Ab antibody 6E10 (Fig. 2a). Then an anti-mouse
IgG-dylight 549 conjugate was added. Dylight 549 produces strong
orange fluorescence, in which region TentaGel beads have low
background fluorescence.28 Out of B192 000 beads, two putative
hits (approximately 0.001% hit rate) were identified and collected.
The low rate may suggest the high selectivity of the library. One of
the structures was identified by MS/MS unambiguously (Fig. S2,
ESI†). This lead, designated as HW-155-1, was re-synthesized on
Rink-amide resin (Fig. 2b). The hydrophobic core residues 16–20 of
the Ab peptide, KLVFF, which inhibits Ab aggregation both in vitro
and in vivo,32–35 was also synthesized and used for comparison.
These two sequences were investigated for their capability to
inhibit the aggregation of Ab40 by ThT assay. Consistent to
previous reports, KLVFF is a weak disruptor of Ab aggregates.
100 mM of KLVFF can only inhibit less than 50% of Ab aggrega-
tion (Fig. 3a). Surprisingly, 1 mM of HW-155-1 already inhibits
B50% of Ab aggregation (Fig. 3b), indicating that it is at least
Fig. 3 ThT assay of compounds against Ab40. (a) The change of fluorescence
in the first 2 h of incubation of KLVFF with Ab40; (b) the change of fluorescence
in the first 2 h of incubation of HW-155-1 with Ab40; (c) the ratio of aggregation
after 24 h. Aggregation control (100%) is set as the change of fluorescence of
2.5 mM Ab40 in Tris (pH 7.5) buffer. The concentration of ThT is 5 mM. Excitation:
440 nm; emission: 482 nm.
100-fold more potent than KLVFF. A similar inhibitory effect is one of the most potent small molecules that disrupt the
was observed even after 24 h (Fig. 3c). Therefore, HW-155-1 aggregation of Ab40.
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as well as the optimization of the lead g-AApeptide HW-155-1
for the intervention of AD.
Notes and references
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5208 | Chem. Commun., 2014, 50, 5206--5208
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