Design of high-affinity peptide conjugates with optimized fluorescence quantum yield as markers for small peptide transporter PEPT1 (SLC15A1)

Article abstract of DOI:10.1016/j.bmcl.2008.03.044

We employed a computational approach to design and synthesize a series of fluorescently labeled hPEPT1 substrates. Five Alexa Fluor-350-labeled peptides were assessed for their in vitro inhibitory activity in hPEPT1-transfected CHO cells. At least four labeled peptides show potent inhibitory activity toward hPEPT1-mediated uptake of [³H]-GlySar and three compounds displayed a significant cellular uptake specifically mediated by hPEPT1.

Full text of DOI:10.1016/j.bmcl.2008.03.044

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 2555–2557
Design of high-affinity peptide conjugates with optimized
fluorescence quantum yield as markers for small
peptide transporter PEPT1 (SLC15A1)
Praveen M. Bahadduri, Abhijit Ray, Akash Khandelwal and Peter W. Swaan*
Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MD 21201, USA
Received 10 January 2008; revised 12 March 2008; accepted 17 March 2008
Available online 20 March 2008
Abstract—We employed a computational approach to design and synthesize a series of fluorescently labeled hPEPT1 substrates.
Five Alexa Fluor-350TM-labeled peptides were assessed for their in vitro inhibitory activity in hPEPT1-transfected CHO cells. At least
four labeled peptides show potent inhibitory activity toward hPEPT1-mediated uptake of [3H]-GlySar and three compounds dis-
played a significant cellular uptake specifically mediated by hPEPT1.
Ó 2008 Elsevier Ltd. All rights reserved.
The human small peptide transporter, hPEPT1, is a clin-
ically important intestinal solute carrier protein critically
involved in the absorption of its natural substrates, food
protein-derived di- and tripeptides. Additionally, it
mediates the intestinal uptake of numerous drug classes,
such as b-lactam antibiotics, ACE-inhibitors, several
antivirals,1 as well as various prodrugs. For example,
valyl prodrugs of the nucleoside antiviral drugs acyclo-
vir and ganciclovir display 3- to 5-fold and 10-fold high-
er bioavailability upon oral administration in humans
compared to their parent compounds, respectively.2,3
The increased intestinal permeability can be attributed
solely to their affinity for intestinal hPEPT1. This trans-
porter’s relaxed substrate specificity, relatively high
capacity and low affinity (Km ꢀ1.1 mM for the proto-
typical PEPT1 substrate glycylsarcosine (GlySar))
makes it an ideal target for the design of peptidomimetic
drugs with increased intestinal permeability and
bioavailability.
out using stably expressed cell lines in a high-throughput
screening (HTS) setup. The success of this approach will
be determined by assay sensitivity for prototypical sub-
strates or new chemical entities (NCEs) when assessing
inhibition or transport via hPEPT1. The most com-
monly applied assay techniques involve laborious liquid
chromatography–mass spectrometry or costly radiola-
beled tracer methodologies. However, fluorescence of-
fers a convenient alternative for HTS applications due
to its sensitivity and applicability to a wide variety of
cell-based systems.
Several other groups have reported fluorescently labeled
peptides directed to develop hPEPT1 HTS assays, albeit
with limited success. Abe and colleagues4 synthesized
FITC and coumarin-3-carboxylic acid conjugated pep-
tide analogs that showed affinity for hPEPT1 expressed
in Caco2 cells, but were not transported. Other studies
reported AMCA (7-amino-4-methylcoumarin-3-acetic
acid) peptide conjugates5 and peptide analog-FITC es-
ters6 with hPEPT1 affinity. However, the fluorescent
dyes used in these studies have limited quantum yields
and may not be particularly suitable for HTS implemen-
tation.7 In fact, fluorescein dyes are known to display
self-quenching properties at high concentrations, there-
by limiting potential assay sensitivity. The present study
was designed to develop a metabolically stable fluoro-
genic substrate for hPEPT1 with superior fluorescence
quantum yield at a range of intracellular pH values.
Based on our previous knowledge of the hPEPT1
pharmacophore and structure-activity relationship,8 we
In the absence of a crystal structure for hPEPT1, the
rate at which new substrates and inhibitors can be iden-
tified or rationally designed is expectedly slow. Alterna-
tive experimental strategies to rapidly screen large
compound database for hPEPT1 affinity can be carried
Keywords: Alexa Fluor 350TM; hPEPT1; Peptide conjugation; Fluores-
cence; High-throughput screening; CoMFA; Transporter.
*
Corresponding author. Tel.: +1 410 706 0103; fax: +1 410 706
5017; e-mail: pswaan@rx.umaryland.edu
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.03.044

2556
P. M. Bahadduri et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2555–2557
Table 1. Predicted activities of Alexa Fluor-350TM derivatives from
CoMFA analysis
designed five novel Alexa Fluor 350TM (AF)-labeled pep-
tide analogs using computational design, synthesis, and
in vitro analysis of AF-labeled substrates designed spe-
cifically to target PEPT1.
Compounds
ÀlogKi
Ki (mM)
AF–LysGly
AF–LysVal
AF–LysSer
AF–LysAla
AF–LysGlyGly
4.550
4.459
4.590
4.611
4.700
0.028
0.035
0.026
0.024
0.019
We built a three-dimensional quantitative structure–
activity relationship (3D-QSAR) using Comparative
Molecular Field Analysis (CoMFA) from the extensive
dataset by Gebauer and colleagues.9 Molecules were
built and subsequently energy minimized in SYBYL
(v.7.1) using the Tripos force field.10 The most active
compound in the dataset, o-(methylphenyl)-D-tyrosyl-
L-alanine9 was used as a template and all the other mol-
ecules were superimposed to it using atom-based align-
ment. The resulting CoMFA steric and electrostatic
contour plots are shown in Figure 1. The model devel-
oped has good descriptive ability as evidenced by a r2
value of 0.989 with six components.
desired product. All in vitro studies were conducted with
stably transfected CHO cell lines expressing hPEPT1 as
described previously.8 Competitive inhibition studies
were performed at pH 6.0 for 30 min using [3H]-glycyl-
sarcosine (GlySar) (1 lM, 1 Ci/mmol) (Moravek, Brea,
CA) as a tracer. Initially, parent peptides were assessed
for their inhibitory potencies of [3H]-GlySar uptake in
hPEPT1-CHO cells. Subsequently, the inhibition of
100 lM and 1000 lM AF-labeled peptides (in DMSO,
not exceeding 1% final DMSO concentration) on [3H]-
GlySar (1 lM) accumulation in hPEPT1-CHO cells
was determined. To determine the cellular uptake of fluo-
rescently labeled peptides and to assess their mutual
competitive inhibition of GlySar, the effect of 10 mM
GlySar (Sigma, St. Louis, MO) on the uptake of
100 lM AF-labeled dipeptide was assayed in hPEPT1-
CHO cells. Cells were washed three times and lysed with
1 N NaOH for 1 h and neutralized with 10% HCl solu-
tion. Total cellular fluorescence was determined using a
Spectramax Gemini XS (Molecular Devices, Sunnyvale,
CA). Data were calibrated using fluorescence standard
curves and normalized to protein content.
The overall steric contribution (53%) to binding is high-
er than the electrostatic contribution (47%). The AF
analogs (Table 1) using pKi (ÀlogKi) (M) as dependent
variable and CoMFA descriptors as independent vari-
ables in the partial least square (PLS) analysis (data
not shown). Out of a set of 50 permutations, the five
highest scoring compounds were selected for synthesis
and in vitro testing.
The synthesis of dipeptide-conjugated AF (Molecular
Probes, Eugene, OR) was carried out by dissolving
BocLys(Z)OSu in a mixture of N,N-dimethyl formamide
(DMF), 1,4-dioxane, diisopropylamine (DIEA), and an
appropriate amino acid or dipeptide (Scheme 1). The
progress of the reaction was monitored by mass spectros-
copy for the disappearance of a peak at m/z 478.5. The
dipeptide is then deprotected by catalytic hydrogenation
using 10% Pd/C to remove the carbonyloxybenzyl (Z)
group. The deprotected compound was dissolved in
DMF, dioxane, and diisopropylethylamine followed by
the addition of AF carboxylic acid succinimidyl ester
to give the fluorescently labeled dipeptide. This was final-
ly deprotected using TFA in dichloromethane to give the
AF-labeled peptides have strong affinity for hPEPT1 as
demonstrated by their potent inhibition of [3H]-GlySar
uptake in hPEPT1-transfected CHO cells (Fig. 2).
Reduction in hPEPT1 affinity imparted by AF-conjuga-
tion is minimal as compared to the activity of the parent
peptide (Fig. 2). Of the compounds tested, the LysVal,
LysSer, and LysAla analogs showed the strongest rela-
tive inhibition of [3H]-GlySar uptake. Interestingly, the
cellular uptake of AF-labeled LysVal was significantly
greater (2- to 3-fold) compared to the other peptides
(Fig. 3). This would indicate that inhibition data do
not correlate with hPEPT1-mediated substrate translo-
cation. Inhibition of AF-labeled peptide uptake by a
100-fold GlySar excess indicates mutual inhibition be-
tween GlySar and AF-labeled analogs, indicative of a
shared translocation pathway, that is, hPEPT1
(Fig. 3). Interestingly, the LysVal conjugate of AF dis-
plays relatively high cellular uptake while only modestly
inhibiting [3H]-GlySar uptake (ꢀ40% at 1 mM), whereas
LysAla and LysSer conjugates exerted stronger [3H]-
GlySar uptake inhibition (<ꢀ22%) with only modest
cellular permeation. This would suggest that AF–LysVal
is a good substrate for PEPT1 compared to the other
dipeptide conjugates, but further studies are warranted
to fully characterize this phenomenon.
Figure 1. CoMFA contour map. The green and yellow regions indicate
that an increase in steric bulk favors or disfavors binding to hPEPT1,
respectively. The increase of negative charge near red region or positive
charge near blue region favors binding. The template compound is
shown in capped stick model.
Overall, AF offers significant advantages over other re-
ported fluorescently labeled peptides such as a large
Stokes shift (100 nm for AF vs 30 nm for FITC) and a
relatively high fluorescence quantum yield (0.89 AF–

P. M. Bahadduri et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2555–2557
2557
Scheme 1. Reagents and conditions: (i) DMF, 1,4-Dioxane, DIEA; (ii) 10% Pd/C, EtoAc, MeOH; (iii) DMF, 1,4-Dioxane, Alexa Fluor 350
carboxylic acid succinimidyl ester; (iv) CH2Cl2, TFA.
LysVal vs AMCA reference in water), which may enable
the use of these novel conjugates in a HTS setting. In
summary, this report describes the design and synthesis
of five novel AF-conjugated peptides with sub-millimo-
lar affinity levels for hPEPT1. Future studies should
determine whether cellular uptake can be monitored
without cellular disruption and in real-time to facilitate
HTS screening.
Acknowledgment
This research was funded by a grant from the National
Institutes of Health R03 DK075157 (to P.W.S.) in re-
sponse to the Molecular Libraries and Imaging Road-
map Initiative.
Figure 2. Relative inhibition of 1 lM [3H]-GlySar uptake (% control)
in hPEPT1-transfected CHO cells in the presence of fluorescently
labeled peptides (n = 3 SD).
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Figure 3. AF-labeled peptide uptake in hPEPT1-CHO cells. Bars
indicate cellular uptake of 100 lM labeled peptide in the absence (open
bars) and presence (closed bars) of 10 mM GlySar. (n = 3 SD).