Piptides: New, Easily Accessible Chemotypes For Interactions With Biomolecules

Article abstract of DOI:10.1002/anie.202015203

Small molecule probe development is pivotal in biomolecular science. Research described here was undertaken to develop a non-peptidic chemotype, piptides, that is amenable to convenient, iterative solid-phase syntheses, and useful in biomolecular probe discovery. Piptides can be made from readily accessible pip acid building blocks and have good proteolytic and pH stabilities. An illustrative application of piptides against a protein-protein interaction (PPI) target was explored. The Exploring Key Orientations (EKO) strategy was used to evaluate piptide candidates for this. A library of only 14 piptides contained five members that disrupted epidermal growth factor (EGF) and its receptor, EGFR, at low micromolar concentrations. These piptides also caused apoptotic cell death, and antagonized EGF-induced phosphorylation of intracellular tyrosine residues in EGFR.

Full text of DOI:10.1002/anie.202015203

A Journal of the Gesellschaft Deutscher Chemiker
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Accepted Article
Title: Piptides: New, Easily Accessible Chemotypes For
Interactions With Biomolecules
Authors: Maritess Arancillo, Jaru Taechalertpaisarn, Xiaowen Liang,
and Kevin Burgess
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202015203
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10.1002/anie.202015203
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RESEARCH ARTICLE
Piptides: New, Easily Accessible Chemotypes For Interactions
With Biomolecules
Maritess Arancillo,[a] Jaru Taechalertpaisarn,[a] Xiaowen Liang,[b] and Kevin Burgess*[a]
[a]
M. Arancillo, Dr. J. Taechalertpaisarn, Prof. K. Burgess
Department of Chemistry, Texas A & M University
Box 30012, College Station, TX 77842, USA
Email: burgess@tamu.edu
[b]
Dr. X. Liang
Center for Infectious and Inflammatory Diseases, Institute of Biosciences and Technology, Texas A&M Health Science Center
Houston, TX 77030, USA
Supporting information for this article is given via a link at the end of the document.
Abstract: Small molecule probe development is pivotal in
biomolecular science. Research described here was undertaken to
develop a non-peptidic chemotype, piptides, that is amenable to
convenient, iterative solid-phase syntheses, and useful in
biomolecular probe discovery. Piptides can be made from readily
accessible pip acid building blocks and have good proteolytic and pH
stabilities. An illustrative application of piptides against a protein-
protein interaction (PPI) target was explored. The Exploring Key
Orientations, EKO, strategy was used to evaluate piptide candidates
for this. A library of only 14 piptides contained five members that
disrupted epidermal growth factor (EGF) and its receptor, EGFR, at
low micromolar concentrations. These piptides also caused
apoptotic cell death, and antagonized EGF-induced phosphorylation
of intracellular tyrosine residues in EGFR.
-Peptides (Figure 1c) share some of the same
disadvantages as peptoids, and they tend to be more flexible
than peptides. An exception is systems comprised of ,-amino
acids (eg cyclic systems), but these cannot be readily
functionalized with the full complement of side chains
corresponding to genetically encoded amino acids.[13]
Introduction
Small molecule probes that perturb biomacromolecular
function are useful in medicinal science.[1] High throughput
screening can be used to obtain such probes, but these
strategies are not always fruitful, justifiable, or affordable.[2]
Screening becomes more efficient if it features chemotypes that
can be quickly, reliably, and inexpensively assembled into
diverse libraries using combinatorial methods.[3] Such strategies
are optimally useful if they can be performed by researchers
possessing only basic synthetic skills. Further streamlining may
be achieved using “privileged chemotypes” that have a bias to
positively interact with the target biomacromolecules.[4]
Peptides are privileged chemotypes because they interact
with biomolecules using native pharmacophores (Figure 1a).
Cyclization[5] and N-methylation[6] strategies can make them
even better probes by rendering them more rigid, proteolytically
stable, and cell permeable,[6a, 7] but these modifications require
more synthetic expertise.
Peptoids[8] (N-substituted oligoglycines, Figure 1b) with
diverse N-substituents are easy to prepare, via solid-phase
syntheses,[9] from commercially available amines. However,
they tend to be more flexible than peptides, which must lose
more entropy to interact with a target, hence rarely bind with
dissociation constants <1M.[10] Analogs with enhanced rigidity
are known (eg cyclic[11] and ones with α-chiral centers[12]) but
making them requires well-developed synthetic skills. Moreover,
population of both cis and trans conformers of tertiary amide
bonds, of which there are often many, complicate peptoid NMR
spectra.
Figure 1. Comparison of a peptides, b peptoids, c -peptides, and d piptides.
1
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Recognizing the limitations outlined above, we set out to
establish a new chemotype design with the following unique
combination of attributes:
•
•
•
ease of solid-phase syntheses;
primary structure diversity that parallels peptides;
more rigidity (less significant degrees of freedom) than corresponding
peptides, peptoids, or side chain functionalized -peptides;
monomer repeats that spatially resemble alternate amino acids of the
corresponding peptides (ie “side chain periodicities” of atomic spacings
between side chains);
•
•
•
•
•
incorporation of piperazine units, as in many pharmaceuticals;
pH characteristics that vary around the physiological range;
proteolytic and pH stability; and,
structures that sample unexplored patent space.
It is non-trivial to devise chemotypes that meet all these
criteria, but the one featured here does: piptides 1 (Figure 1d).
This contribution illustrates how researchers can design
initial hit piptides to perturb protein-protein interactions (PPIs),
with epidermal growth factor (EGF) and transforming growth
factor- (TGF) interacting their receptor EGFR as an illustrative
case. EGFR is a validated target for cancer therapeutics.[14]
Modalities for regulating signals from EGFR are used in the
clinic, but they all tend to feature biologicals (eg humanized
monoclonal antibodies), or kinase inhibitors.[15] Attempts to
block EGF•EGFR or TGF•EGFR by targeting the PPI interfaces
with small molecules have been largely unsuccessful, yet we
were able to identify several preliminary hits from a library of
only 14 piptide-based compounds.
Scheme 1. Generalized syntheses of pip acids. Lower case one-letter codes
are used to delineate amino acid side chains (R) and relate them to the closest
amino acid; primed letters indicate protected side chains (eg d’ for the –
t
CH2CO2 Bu of Asp and k’ for the –(CH2)4NHBoc of Lys).
Denosylation of Phe- and Thr(OtBu)-derived pip acids L-4f
and L-4t’, respectively, gave the piperazines 6 used to initiate
trial solution phase syntheses. Several denosylation methods
were investigated, and the conditions shown in Scheme 2 were
the best found.
Results and Discussion
Piptides can be constructed from fragments 2, pip acids.
Prior to our work there were only two synthetic strategies to a
small number of pip acids, and both of these rendered only
racemic material.[16]
(2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HBTU)[19] was used to activate the C-free
N-protected units 5 to give the C,N-diprotected dipiptides 7 then
tripiptides 8 (Scheme 2a). Scheme 2b illustrates how piptides
with repeating sequences can be made by dividing
intermediates like 8, selective deprotection of either terminus,
then recombination.
Solid-phase piptide syntheses may be guided by the
following concepts. The protection strategy is similar to common
peptide syntheses because side chain-protected amino acids
appropriate for FMOC-peptide syntheses[18a] are used to make
the functionalized N-protected pip acid derivatives 5. In that
case, use of resins functionalized with TFA-sensitive handles
allows successive couplings of side chain-protected pip acids
with retention of their masking groups. Finally, cleavage of the
target piptide with simultaneous side chain deprotection, could
be achieved via treatment with scavenger cocktails containing
TFA. Uncoupled, supported, piptide N-termini are better stained
using the chloranil test[20] than via ninhydrin,[21] just as for
peptides syntheses where Pro is the N-terminal residue. Thus,
visual chloranil tests indicate situations in which two or more
coupling cycles are necessary to drive the reactions to
completion. Denosylation of supported piptides (cf for supported
peptides[22]), and the coupling reactions used to form them, may
Tri(4-nitrobenzene sulfonate) {tri-nosyl or tri-4Ns} 3 had
been reacted with primary arylamines under microwave
conditions to give N-aryl,N-4Ns-piperazines.[17] Compounds 3
could be converted into a variety of N-4Ns-pip acid esters 4, then
selectively deprotected to their C-free N-protected forms 5 (and,
under other conditions, to C-protected N-free analog 6, vide
infra; Scheme 1). These N-protected pip acids tend to be solids.
They can be made from amino acid building blocks with
protected-functionalized side chains, ie exactly the ones
commonly used in the FMOC approach to peptides,[18] and
appropriate for solid-phase syntheses on trifluoroacetic acid
(TFA)-sensitive resins. This is important because incorporation
of functionalized side chains into peptidomimetic chemotypes
can be time-consuming and experimentally tedious.
2
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RESEARCH ARTICLE
optionally be accelerated using microwave reactors (Scheme 3).
Scheme 3 also illustrates how hybrids of piptides, and peptides
can be produced easily by coupling -amino acids to piptide N-
termini, as in the synthesis of compounds 10 shown.
decomposition after 24 h (Figure S1b). To test for pH-mediated
hydrolysis, the same piptide was maintained in aqueous media
under acidic, basic, and neutral conditions; it showed essentially
no decomposition at low pH but around 30 % decomposition
was observed at pH 10 (Figure S1c).
Short piptide-containing sequences such as 10 would not
be expected to fold into any preferred conformation in solution.
Indeed, circular dichroism spectra collected for LLL-10ysl in
methanol showed only very weak molar ellipticities, ie its CD
spectrum was essentially flat (Figure S2).
Scheme 2. a Linear, and b divergent-convergent, solution phase syntheses of
piptides.
Isolated yields are indicated in Scheme 3, but these
parameters are notoriously variable in solid-phase syntheses
due to the small amounts of material cleaved from the support,
losses in prep-HPLC purification, and because all yields based
on support loading are hard to measure accurately. Analytical
HPLC UV/ELSD analyses of crude materials from illustrative
solid-phase syntheses are given in the SI; these data show that
the purity of piptides cleaved from the resin is high. Moreover,
solid-phase syntheses of piptides are not restricted to two or
three repeat residues; Scheme S1 shows preparation of a 9-mer
piptide.
Scheme 3. Illustrative solid-phase synthesis of piptides on TentaGel S-RAM
resin (blue spheres).
Exploring Key Orientations on Secondary structures
(EKOS)[24] was used to evaluate biases of preferred
conformations of 10aaa. In EKOS, conformations of the mimic
are simulated, preferred ones (within 3 kcal/mol of the lowest
energy conformation identified) are systematically overlaid on
ideal secondary structures according to their three Cα – Cβ
vectors, and the fit of the superimpositions are evaluated in
terms of the root mean square deviations (RMSDs) of the six Cα
and Cβ coordinates involved. We have shown,[24-25] superior
secondary structure mimics overlay with RMSD values <0.5 Å.
Figure S3 shows 10aaa is an excellent mimic of strand-turn-
strand, and parallel and antiparallel -sheets. A Ramachandran
plot (Figure S4) shows preferred conformers of 10aaa are
Two experiments were performed to assess relative
stabilities of piptides and peptides towards hydrolysis. To test
enzymatic proteolysis, piptide LLL-10fff and the closely related
peptide LLL-11fff (Figure S1a) were incubated at 37 °C in pH 7.4
PBS with Pronase® (a protease mixture used in proteomic
studies).[23]
conditions (t1/2 ~ 2 h), whereas the piptide showed no significant
The peptide degraded rapidly under these
3
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RESEARCH ARTICLE
concentrated in a narrow range of -bond angles, indicative of
conformational rigidity.
Epidermal growth factor receptor (EGFR) is overexpressed
at varying levels in most types of cancer cells.[14] High surface
densities of EGFR favor dimerization, making cells
overexpressing EGFR abnormally sensitive to stimulation by
their complementary endogenous protein growth hormones
(EGF and TGF), hence promoting unconstrained cell growth.[26]
For instance, secretion of TGF is associated with various
cancer types including breast, lung,[15a] kidney, melanoma,
liver,[27] and glioblastomas.[26] In general, there seems to be a
causal link between increased EGF or TGF expression and
tumor development.[28]
Conversely, blockade of EGFR
suppresses tumor cell growth in vitro and in vivo.
EGFR antagonists used for cancer chemotherapy can be
divided into “biologicals” (typically antibodies, eg cetuximab,
panitumumab, and trastuzumab) that perturb ligand•EGFR
interactions,[15a-f] and kinase inhibitors (eg gefitinib, erlotinib,
lapatinib) that suppress signaling from the receptors.[15e-h]
However, small molecule inhibitors of the EGF kinase domain
are not ideal probes for EGFR-mediated signaling because they
tend to inhibit other kinases. Furthermore, in the clinic, EGFR
kinase inhibitors (and even humanized mAbs for this target) are
vulnerable to intrinsic and acquired resistance.[15b]
The non-ligated, “inactive” form of EGFR rests in an
autoinhibited conformation. On introduction of TGF or EGF,
two of the same protein ligands bind two identical domains along
the EGFR periphery causing conformational changes, but the
EGFR•EGFR interface is maintained.[29] Mutagenesis of EGF
and of TGF have revealed key residues for interactions with
EGFR. For instance, affinity between TGF and EGFR seems
to disproportionately rely on 42Arg and 48Leu of the ligand.[30]
A
molecular dynamics study[31] and X-ray crystallography (1MOX)
has also revealed several residues of TGF that are involved in
hydrogen bonding and salt bridge formation with EGFR,
including 44Glu and 46Ala.
In the current study, the EKO strategy[32] was used to
evaluate piptide-based chemotypes for their potential to disrupt
Figure 3. ELISA assays to a detect inhibition of binding 10 M EGF-biotin to
EGFR anchored to a microplate, and to b determine IC50 values.
Here, EKO analyses indicated preferred conformers of
chemotype 10 overlaid on EGF or TGF at the TGF•EGFR
interface (Figure 2).
Figures 3b – g illustrates different
stereoisomers of 10aaa overlaid well on 39Val, 44Glu, and 46Ala,
in both possible orientations (ie N-to-C, and C-to-N). Thus,
Figure 2b shows the C-terminus of DLD-10aaa superimposed on
39Val, whereas DDD-10aaa best matched its N-terminus on 39Val
in Figure 2e. Observations such as these, where “pseudo-
symmetrical” chemotypes, like 10, overlay with either N-to-C
polarity, are unsurprising because EKO considers only the side
chain orientations and not the scaffold core.
protein ligand•EGFR interactions.
Briefly, EKO compares
favored conformations of small molecules that present three
amino acid sidechains, with PPI interface regions, based on
degree of fit of side chain C and C coordinates. Validation for
the strategy has been reported in the context of the HIV-1
protease dimer,[32] the anti-thrombin dimer,[33] uPA•uPAR,[34] and
PCSK9•LDLR.[35]
Figure 2. EKO analyses of piptide-based chemotypes on TGF•EGFR. a TGF•EGFR dimer interface (1MOX). b-g EKO implicated stereoisomers of 10aaa
overlaid on TGF 39Val, 44Glu, and 46Ala. Note structure 1MOX indicates 44Glu and 46Ala are involved in H-bonding with EGFR. Figures in purple represent the
root mean squared deviation of mimic and protein-ligand at the six C and C coordinates involved.
4
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ELISA primary assays revealed five of the 14 EKO-
implicated chemotypes perturbed EGF•EGFR at micromolar
concentrations with satisfactory dose-response profiles. Figure
3 calibrates their inhibition of EGF•EGFR relative to a blank with
no protein ligand (equivalent to 100% “inhibition” because there
is no EGF binding to EGFR) and to 10 M EGF-biotin (under
these conditions EGF-biotin saturates the available EGFR hence
“inhibition” is set to 0%). That graphic also includes data for
LLL-10faa, which is a “partial control” insofar as it has the same
chemotype core, but some of the side chains and
stereochemistries are not ones predicted to be appropriate from
the EKO analyses. Compound LLL-10faa also has hydrophobic
sidechains, which could promote non-specific binding, and,
indeed, in the event, that partial control showed only relatively
low inhibition with no dose-response (Figure 3a).
Concentrations of the three best inhibitors from the primary
ELISA were varied at closer intervals to obtain IC50 values (M
throughout; Figure 3b): LDL-10eav, 30.5  1.2; LLL-10aev, 33.3
 1.7; DDL-10vae, 35.8  2.2.
tested using HEK293 (EGFR-negative) cells and showed little to
no cytotoxicity (Figure S6).
Flow cytometry experiments indicated gefitinib and LDL-
10eav caused cytotoxicity via early apoptosis (annexin V
staining), more than necrosis (propidium iodide; Figure 5b and
S7). Western blot assays were performed to determine if an
illustrative piptide-based probe impedes EGF-induced pTyr at
EGFR. Figure 5c illustrates that phosphorylation could be
suppressed almost completely by LDL-10eav in competition with
EGF.
Figure 4. Direct binding of compounds 10 to EGFR immobilized on Biacore
sensor chip surface (SPR) shown as a sensorgram.
In SPR experiments, binding of LDL-10eav and DDL-10vae
to EGFR was shown to be reversible as indicated by complete
dissociation to baseline level. However, LLL-10aev showed
more significant secondary binding and aggregation on the
receptor surface, as indicated by incomplete dissociation after
extended washing. The Kd values calculated from SPR were
LDL-10eav, 41.1  13.2; LLL-10aev, 50.6  13.8; DDL-10vae,
30.7  10.2 M (Figure 4). Competition experiments were
performed to complement these direct binding assays. In these,
the compounds were tested in competition with 30 nM TGF or
27 nM EGF on immobilized EGFR. Ratios of Kd values of EGF
or TGF for EGFR with and without the featured compounds
were obtained; values greater than 1 indicate diminished binding
of the natural protein ligands to EGFR. In the event, the
compound predicted to have the lowest Kd for EGFR in the direct
binding SPR studies, DDL-10vae, had the most negative impact
on the binding of TGF to EGFR in the competitive assay, as
expected (Figure S5c).
additive
50 ng/mL EGF +
none
LDL-10eav
EGF
Cytotoxicity assays were performed on the five hit
compounds featured in Figure 5a with the expectation that
reduced viabilities would result if these bind EGFR. A549
human lung cancer cells were used in this study because they
overexpress EGFR.[36] Figure 5a shows all the five of these
featured compounds reduced viability more than the partial
negative control LLL-10faa, but less than gefitinib (a FDA-
approved EGFR kinase inhibitor).[37] Compounds were also
Figure 5. Cellular data for piptide-based chemotypes. a A549 cell viabilities
as monitored using alamarBlue. b Flow cytometry data for A549 cells treated
with either 15 M gefitinib or 50 M LDL-10eav. c Inhibition of EGFR pTyr for
LDL-10eav at concentrations (M) decreasing from 112.5, 75, 50, 25, 12.5 in
competition with EGF (uniformly used at 50 ng/mL).
5
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Piptides can be prepared expeditiously by researchers with
only moderate synthesis skills; and they would become even
more accessible if pip acids became commercially available.
Our data on EGFR indicate piptide-based chemotypes can be
starting point probes for PPI targets. Five viable hits emerged
from a library of only 14 molecules selected by evaluation with
EKO; their Kd values (30 – 90 M) are modest, but detection of
any measurable binding is a notable success in studying small
molecules to perturb PPIs, particularly because solubilized
EGFR is probably not in
a native conformation when
immobilized on a gold surface for SPR. Moreover, the best
binders from these five hits were active in cellular assays
(induce cytotoxicity via apoptosis and inhibit EGF-mediated
phosphorylation of intracellular Tyr residues in EGFR).
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This study does not feature intracellular targets, but
researchers might be interested in applying the strategy to such
PPIs. An attractive feature of piperazines is that they can be
reversibly protonated, and this can facilitate passive diffusion
through membranes. Piptides with appropriate side chains
might conceivably be somewhat cell permeable, and Table S1
shows some calculated data to explore this point.
The closest parallel to piptides in the literature is
oligooxopiperazines.[38] Oligooxopiperazines have been used for
disrupting PPIs,[39] but are harder to access, and the range of
side chains so far incorporated is less. Further, the two
chemotypes are simply different, thus probably suitable for
complementary applications.
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Acknowledgements
We thank the following grant agencies for funding: DoD BCRP
Breakthrough Award (BC141561), CPRIT (RP170144 and
RP180875), and The Robert A. Welch Foundation (A-1121),
Texas A&M University (RP180875), NIH (R01EY029695) and
NSF (CHE1608009). The NMR instrumentation at Texas A&M
University was supported by a grant from the National Science
Foundation (DBI-9970232) and the Texas A&M University
System. The SPR studies were conducted in the protein
interaction core at the Institute of Biosciences and Technology,
TAMU, Houston, TX.
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Keywords: cancer • EGF • peptidomimetics • protein–protein
interactions • solid-phase synthesis
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Entry for the Table of Contents
Piperazines + peptides = piptides! These new chemotypes are accessible from natural amino acids in either configuration, hence
can conveniently display all the side chain pharmacophores found in peptides and proteins. They can be made in solution or on
supports and are proteolytically-/pH-robust. Piptide-based disruption of EGF•EGFR was chosen as an initial challenge and Exploring
Key Orientations (EKO) was used to evaluate candidates.
8
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