Structure-activity relationship studies for the peptide portion of the bladder epithelial cell antiproliferative factor from interstitial cystitis patients

Article abstract of DOI:10.1021/jm8002763

We performed comprehensive structure-activity relationship (SAR) studies on the peptide portion of antiproliferative factor (APF), a sialylated frizzled-8 related glycopeptide that inhibits normal bladder epithelial and urothelial carcinoma cell proliferation. Glycopeptide derivatives were synthesized by solid-phase methods using standard Fmoc chemistry and purified by RP-HPLC; all intermediate and final products were verified by HPLC-MS and NMR analyses. Antiproliferative activity of each derivative was determined by inhibition of ³H-thymidine incorporation in primary normal human bladder epithelial cells. Structural components of the peptide segment of APF that proved to be important for biological activity included the presence of at least eight of the nine N-terminal amino acids, a negative charge in the C-terminal amino acid, a free amino group at the N-terminus, maintenance of a specific amino acid sequence in the C-terminal tail, and trans conformation for the peptide bonds. These data provide critical guidelines for optimization of structure in design of APF analogues as potential therapeutic agents.

Full text of DOI:10.1021/jm8002763

5974
J. Med. Chem. 2008, 51, 5974–5983
Structure-Activity Relationship Studies for the Peptide Portion of the Bladder Epithelial Cell
Antiproliferative Factor from Interstitial Cystitis Patients
Piotr Kaczmarek,† Susan K. Keay,*,‡,§ Gillian M. Tocci,† Kristopher R. Koch,‡ Chen-Ou Zhang,‡ Joseph J. Barchi, Jr.,|
David Grkovic,‡ Li Guo,‡ and Christopher J. Michejda†
Molecular Aspects of Drug Design Section, Structural Biophysics Laboratory, Center for Cancer Research, National Cancer Institute at
Frederick, Frederick, Maryland 21702, DiVision of Infectious Diseases, Department of Medicine, UniVersity of Maryland School of Medicine,
Baltimore, Maryland 21201, Research SerVice, Veterans Administration Maryland Health Care System, Baltimore, Maryland 21201, and
Laboratory of Medicinal Chemistry, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, Maryland 21702
ReceiVed March 13, 2008
We performed comprehensive structure-activity relationship (SAR) studies on the peptide portion of
antiproliferative factor (APF), a sialylated frizzled-8 related glycopeptide that inhibits normal bladder epithelial
and urothelial carcinoma cell proliferation. Glycopeptide derivatives were synthesized by solid-phase methods
using standard Fmoc chemistry and purified by RP-HPLC; all intermediate and final products were verified
by HPLC-MS and NMR analyses. Antiproliferative activity of each derivative was determined by inhibition
of 3H-thymidine incorporation in primary normal human bladder epithelial cells. Structural components of
the peptide segment of APF that proved to be important for biological activity included the presence of at
least eight of the nine N-terminal amino acids, a negative charge in the C-terminal amino acid, a free amino
group at the N-terminus, maintenance of a specific amino acid sequence in the C-terminal tail, and trans
conformation for the peptide bonds. These data provide critical guidelines for optimization of structure in
design of APF analogues as potential therapeutic agents.
Introduction
The peptide sequence of APF is identical to a segment of the
sixth transmembrane domain of the frizzled-8 protein, a Wnt
ligand receptor.7,8
Interstitial cystitis (ICa) is a devastating disease of the urinary
bladder that is characterized by thinning or even focal oblitera-
tion of the bladder epithelium. It affects approximately 1 million
Americans, and evidence suggests it occurs eight to nine times
more frequently in women than in men.1,2 The cause of this
disorder remains unknown. Several possible causes of IC have
been proposed including changes in neuronal function, autoim-
mune reactivity, infection, and toxin production.3-6 Urine from
IC patients has been shown to contain an antiproliferative factor
(APF) that decreases 3H-thymidine incorporation by human
bladder epithelial cells.6 A variety of techniques including total
synthesis were previously used to identify APF as a nonapeptide
(TVPAAVVVA) containing a 2,3-sialylated core 1 R-O-linked
disaccharide (Galꢀ1-3GalNAc, the Thomsen-Friedenreich an-
tigen, or “TFag”) linked to the N-terminal threonine residue (i.e.,
Neu5AcR2-3Galꢀ1-3GalNAcR-O-TVPAAVVVA, Figure 1).7
Early studies indicated that purified native APF increased
E-cadherin expression and decreased proliferation of bladder
epithelial cells in vitro,9 and both native and synthetic APF were
shown to inhibit the proliferation of normal bladder epithelial
as well as cells derived from urothelial carcinomas at picomolar
to low nanomolar concentrations.7,10 Therefore, APF and
perhaps more efficacious synthetic derivatives potentially
represent an innovative group of antitumor agents with a novel
mode of action. In addition, pull-down experiments utilizing a
biotinylated derivative of APF identified its bladder epithelial
cell receptor as the cytoskeleton associated protein 4, CKAP4.11
Preliminary SAR information obtained during the original
complete characterization and synthesis of APF indicated that
the terminal sialic acid residue is not necessary for activity but
that the R-linked TF-disaccharide of the peptide is required (i.e.,
Galꢀ1-3GalNAcꢀ-O-TVPAAVVVA and the nonglycosylated
nonapeptide were completely inactive).7
We have now performed additional extensive SAR studies
on the peptide portion of the APF molecule to better understand
the structural elements that are required for antiproliferative
activity. The synthesis of congeners containing structural
modifications to the peptide portion of APF (TVPAAVVVA)
and the effects of these modifications on the biological activity
of APF are presented.
* To whom correspondence should be addressed. Address: Research
Division, Room 3B-184, Baltimore Veterans Affairs Medical Center, 10
North Greene Street, Baltimore, MD 21201. Phone: 410-605-7000, extension
6450. Fax: 410-605-7837. E-mail: skeay@medicine.umaryland.edu.
†
Structural Biophysics Laboratory, Center for Cancer Research, National
Cancer Institute at Frederick.
‡
University of Maryland School of Medicine.
Veterans Administration Maryland Health Care System.
Laboratory of Medicinal Chemistry, Center for Cancer Research,
§
|
National Cancer Institute at Frederick.
a Abbreviations: IC, interstitial cystitis; 12-Ado, 12-aminododecanoic
acid; APF, antiproliferative factor; 2ClTrt resin, 2-chlorotrityl resin;
Ac2O, acetic anhydride; AcOH, acetic acid; Aib, aminoisobutyric acid;
Aze, azetidine 2-carboxylic acid; BEP, 2-bromo-1-ethylpyridinium
tetrafluoroborate; DCM, dichloromethane; DIPEA, N,N-diisopropylethy-
lamine; HATU, O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium
hexafluorophosphate; HOAt, 1-hydroxy-7-azabenzotriazole; Hyp, trans-
4-hydroxyproline; NMP, 1-methyl-2-pyrrolidinone; Hyp(tBu), O-tert-
butyryl-trans-4-hydroxyproline; PyAOP, (7-azabenzotriazol-1-yloxy)-
tripyrrolidinophosphonium hexafluorophosphate; Pip, pipecolic acid;
TFag, Thomsen-Friedenreich antigen; TFA, trifluoroacetic acid; TFE,
2,2,2-trifluoroethanol.
Results
We determined whether changes to certain structural aspects
of the peptide segment influenced the biological activity of APF
by systematically replacing or modifying amino acids residues
from the N-to-C termini of the sequence. Derivatives are
grouped on the basis of amino acid substitutions or modifications
made in three separate segments of APF: the N-terminal Thr-
Val segment (Table 1), the Pro-Ala segment (Table 2), and the
10.1021/jm8002763 CCC: $40.75
2008 American Chemical Society
Published on Web 09/13/2008

SAR Studies of the Bladder Epithelial Cell APF
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19 5975
Figure 1. Structures of APF and as-APF.
C-terminal tail (AVVVA) segment (Tables 3-5). L-Amino acids
were used for the synthesis of all derivatives unless otherwise
indicated.
We previously determined that the endobiotic factor (Neu5-
AcR2-3Galꢀ1-3GalNAcR-O-TVPAAVVVA, Figure 1), the
nonsialylated analogue (Galꢀ1-3GalNAcR-O-TVPAAVVVA,
1), the sialylated compound with a lactosamine unit in place of
the TFag disaccharide R-O-linked to threonine (Neu5AcR2-
3Galꢀ1-4GlcNAcR-O-TVPAAVVVA), the sialylated compound
with a lactosamine unit and substitution of the first two amino
acids Thr-Val with Ser-Leu (Neu5AcR2-3Galꢀ1-4GlcNAcR-
O-SLPAAVVVA), and the same compound in nonsialylated
form (Galꢀ1-4GlcNAcR-O-SLPAAVVVA) are all essentially
equipotent in biological antiproliferation assays. On the basis
of these results, activity for each analogue described in this
report was compared simultaneously to activity of the most
synthetically accessible of these analogues, the nonsialylated
form of the endobiotic (Galꢀ1-3GalNAcR-O-TVPAAVVVA,
1), hereafter referred to as asialo-APF, or “as-APF”.
Figure 2. Antiproliferative activity of specific APF derivatives (modifica-
tions to the N terminus). Inhibition of tritiated thymidine incorporation by
primary normal bladder epithelial cells was determined for each derivative
at the concentrations indicated. Experiments were run in triplicate on two
separate occasions. Data are expressed as the mean percent change in
thymidine incorporation relative to a cell control treated with diluent
(acetonitrile/H2O at 1:1) alone; bars indicate standard error of the mean
for all six data points: ([) Galꢀ1-3GalNAc-O-TVPAAVVVA; (0) Galꢀ1-
3GalNAcR-O-TLPAAVVVA; (4) Galꢀ1-3GalNAcR-O-SLPAAVVVA;
(1) Galꢀ1-3GalNAcR-O-SVPAAVVVA.
Modifications to the N-Terminus (Thr1-Val2). We began
by first replacing threonine-valine in 1 with serine-leucine,
an “isosteric” substitution that maintained identical atomic mass
while essentially “transferring” a methylene unit from the
N-terminal threonine to Val2 (2, Table 1), resulting in a
derivative similar to the Galꢀ1-4GlcNAcR-O-SLPAAVVVA
derivative described in the previous paper.7 This modification
resulted in 2 orders of magnitude loss of potency (Table 1)
(Figure 2). In comparison, simple removal of the threonine
methyl group in this location (i.e., the lone substitution of Thr1
with Ser, 3) resulted in even greater (4 orders of magnitude)
loss of activity compared to the parent as-APF molecule 1, and
the lone substitution of Val2 with Leu (4) resulted in inactivation
(Table 1, Figure 2). These results indicate that the number and
positioning of methyl groups in this location are important for
as-APF activity.
Certain other minor modifications to the N-terminal two
amino acids also affected as-APF activity (Table 1). For
example, acetylation of the N-terminal threonine (5) and
extension of the peptide sequence with Tyr (a preceding amino
acid in the sequence of frizzled-8 protein,8 6) both resulted in
approximately 2 orders of magnitude loss of potency, providing
evidence for the importance of the threonine amino group.
Interestingly, replacement of Val2 with Tyr (7) resulted in
complete inactivation of as-APF, indicating that the side chain
of the amino acid in this location is critical for activity.
Modifications to Pro3-Ala4. Except for the N-terminal
glycosylated threonine, the amino acid residues of as-APF are
made up of only three different amino acids, one of these being
proline. The cyclic nature of the proline side chain is often a
source of conformational adjustment in a peptide/protein
sequence, being involved in turns and changes in directionality
of the sequence following this residue. The proline in APF
appears to be important for its activity, as substitution of proline
with all but one of the modified amino acids tested was
detrimental to biological function (Table 2). For example,
substitution of L-proline with D-proline (8), pseudoproline
(Ser(ΨMe,Me pro), 9) (Figure 3), or azetidine 2-carboxylic acid
(Aze) (10) (Figure 3) completely abolished activity (Table 2).
While certain other substitutions did not completely destroy
activity, substitution of Pro3 with Ala (11), trans-4-hydrox-
yproline (12) (Figure 3), or O-tert-butyryl-trans-4-hydroxypro-
line (13) resulted in 2-3 orders of magnitude decrease, and
substitution of Pro3 with N-methylalanine (14) resulted in 4
orders of magnitude decrease in biological activity. Only
substitution of Pro3 with pipecolic acid, the six-membered ring
analogue of proline (15), resulted in complete retention of as-
APF’s biological activity (Figure 3).
Substitution of AAVVVA with 12-Aminododecanoic
Acid. Because APF is a highly hydrophobic peptide with only
the N-terminal glycosylated threonine offering any measure of

5976 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19
Table 1. Substitutions or Modifications of the N-Terminus (Thr1-Val2)
Kaczmarek et al.
a As a result of the variability of cell response in the biological assay, the activity of each congener was normalized to the activity of 1 run simultaneously
on the same plate according to the following equation: % ) [IC50 (as-APF)/IC50 (derivative)] × 100; the average IC50 value of 1 was ∼1 nM. Percent of
activity is expressed relative to 1, which served as a standard control on each plate. Derivatives with no significant activity at <25 µM concentration (the
cutoff limit for the biological assay) were considered to be inactive. b NS ) not significant at p > 0.05.
hydrophilicity, we next considered the possibility that the three
N-terminal amino acids (TVP) may be important for specific
interaction with the receptor while the following hydrophobic
C-terminal amino acids may interact nonspecifically with (e.g.,
intercalate into) the lipid-containing cell membrane. We there-
fore determined whether complete replacement of AAVVVA
with the amino-substituted fatty acid 12-aminododecanoic acid
(12-Ado) (16) affected biological activity. This derivative proved
to be completely inactive, however (Table 3), indicating a
requirement for one or more additional specific structural
characteristics of the carboxy-terminal peptide segment.
Modifications of Carboxy-Terminal Amino Acids 5-9
(AVVVA). To determine the length of the C-terminal tail that
is necessary for activity, we first tested as-APF containing only
five of the nine amino acids (i.e., truncated by four amino acids
at the carboxy terminal end (17)) and determined that this
derivative was completely inactive. We therefore next began
truncating as-APF beginning at the carboxy terminal end. as-
APF containing all but the carboxy-terminal alanine (18) had
full activity, but as-APF truncated by only one additional amino
acid (19) proved to be completely inactive (Table 3). Taken
together, these findings indicate that a minimum of the eight
N-terminal amino acids is necessary to maintain a required
structural element of as-APF.
We noted that the 5 amino acid C-terminal “tail” of APF
contains the AXXXA sequence, a common R-helical motif in
proteins.12 We therefore hypothesized that the amino acid
sequence of this segment of as-APF may also be important for
interaction with its receptor because similar motifs have been
shown to function in protein-protein dimerization.13-15 To test
this hypothesis, we reversed Val8 and Ala9 (20) or replaced
either Ala9 or Ala5 with more branched but similarly charged
amino acids (such as Leu9, 21; or Val5, 22), all of which changes
resulted in loss of most or all biological activity (Table 4). While
Ala9 is not required for as-APF activity, these findings provide
evidence that Ala5 and Ala9 may both be important for optimal
activity of APF containing nine amino acids.
The phenomenon of protein-protein dimerization can also
occur for GXXXG or SXXXS motifs, so we next tested as-
APF derivatives containing GXXXG or SXXXS in place of

SAR Studies of the Bladder Epithelial Cell APF
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19 5977
Table 2. Substitutions or Modifications of the Pro3-Ala4 Segment
a As a result of the variability of cell response in the biological assay, the activity of each congener was normalized to the activity of 1 run simultaneously
on the same plate according to the following equation: % ) [IC50 (as-APF)/IC50 (derivative)] × 100; the average IC50 value of 1 was ∼ 1 nM. Percent of
activity is expressed relative to 1, which served as a standard control on each plate. Derivatives with no significant activity at <25 µM concentration (the
cutoff limit for the biological assay) were considered to be inactive. b NS ) not significant at p > 0.05.
AXXXA. Additional evidence for the importance of alanine in
the fifth and ninth positions was provided by the partial
inactivation resulting from replacement of both Ala5 and Ala9
with serine (Ser5,9, “SXXXS”, 23) and complete inactivation
resulting from replacement with glycine (Gly5,9, “GXXXG”,
24) (Table 4). However, substitution of Val6-8 with alanine
residues with retention of Ala5 (25) also resulted in decreased
as-APF activity, while substitution of Val6-8 with glycine
residues (26), substitution of Val7 with D-valine (27), or
substitution of Val6 and Val8 with isoleucines (28) resulted in
complete inactivation. Taken together, all of the above findings
suggest that both the presence of alanine in the fifth position
and the presence of valine in the sixth through eighth positions
are important for optimal activity of as-APF.
activity. Interestingly, the addition of cysteine to the carboxy
terminus (30) resulted in a decrease in APF activity of 2 orders
of magnitude while the addition of lysine with a much larger
Nꢀ-attached dansyl group in the 10th position (31) resulted in
no loss of activity (Figure 5). Whether this latter finding
indicates additional interaction between the dansyl group and
the APF receptor remains to be determined. However, the
addition of either Glu (32) or Lys (33) (Figure 5) to the carboxy
terminus in the 10th position (for possible subsequent cycliza-
tion, see below) resulted in the complete loss of activity, some
of which was restored by neutralizing the charge on the Glu or
Lys side chains while maintaining the C-terminal carboxylate
[(34) and (35) (Figure 5)] (Table 5). These findings suggest that
the presence of either a positively or negatively charged side
chain in the 10th position is detrimental to APF activity.
Finally, we synthesized an as-APF derivative in which the
entire peptide portion was cyclized from the amino group on
Carboxyamidation of Ala9 in as-APF (29) also resulted in
decreased activity (Table 5), suggesting the possibility that a
negative charge in the ninth position may be important for APF

5978 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19
Kaczmarek et al.
Table 3. Substitution of AAVVVA with 12-Aminododecanoic Acid and Truncated Glycopeptides
a As a result of the variability of cell response in the biological assay, the activity of each congener was normalized to the activity of 1 run simultaneously
on the same plate according to the following equation: % ) [IC50 (as-APF)/IC50 (derivative)] × 100; the average IC50 value of 1 was ∼1 nM. Percent of
activity is expressed relative to 1, which served as a standard control on each plate. Derivatives with no significant activity at <25 µM concentration (the
cutoff limit for the biological assay) were considered to be inactive. b NS ) not significant at p > 0.05.
the N-terminus to the carboxyl group on the C-terminus (36).
Although the complete inactivity of head-to-tail cyclized APF
peptide could be evidence for the importance of both C- and
N-terminal charges, it is also possible that this derivative’s
inactivity resulted from conformational changes occurring as a
result of cyclization.
with various other modified amino acids that can affect
conformation also resulted in complete, or substantial, loss of
activity. Ring size, functionality, and polarity can affect the
conformation and potencies of proline-substituted APF deriva-
tives, as shown by the decreased activity following substitution
with D-proline, pseudoproline, azetidine 2-carboxylic acid, trans-
4-hydroxyproline, O-tert-butyryl-trans-4-hydroxyproline, ala-
nine, and N-methylalanine. In the D-proline derivative, the
internal backbone torsion angle ꢁ effectively changes sign,
which in turn changes the orientation of the peptide segments
on either side of the Pro residue relative to native APF, resulting
in complete inactivation. Inactivation of as-APF following
substitution of proline with pseudoproline (the latter of which
often results in a high percentage of cis amide bonds preceding
this residue17) may indicate a requirement for a trans conforma-
tion, a finding compatible with NMR data showing that APF
does not contain any cis peptide bonds (unpublished observa-
tions). In comparison, substitution of L-proline with pipecolic
acid had no apparent effect on activity, suggesting this derivative
maintains a similar conformation to as-APF.
The decreased activity of as-APF following replacement of
the proline with alanine or N-methylalanine might be explained
by the fact that these derivatives, while likely to be more flexible
than the parent APF congener, can have a small number of the
angles in that location topochemically identical with bioactive
APF, allowing some activity. In addition, the relatively greater
activities of 11-14 or the parent congener as compared to 10
is most likely explained by the unfavorable restriction of
conformation for the azetidine derivative.
Discussion
We previously showed that glycosylation of APF is necessary
for biological activity7 and have now determined that several
structural aspects of the 9 amino acid peptide segment are also
important for structural integrity of the active compound.
Our data clearly show that the biological activity of APF is
very sensitive to changes in the N-terminus of the glycopeptide.
The N-terminal two amino acids (Thr-Val) can be substituted
with Ser-Leu with 2 orders of magnitude loss of activity, but
sole substitution of Thr with Ser (3) resulted in even greater
loss of potency, indicating that the number and positioning of
the methyl groups in this location are important for as-APF
activity. A recent report showed that the Ψ angle preferences
are very different in simple GalNAc-containing glycoaminoacids
depending on whether the amino acid is serine or threonine.16
Whether this is operational in the N-terminal-substituted APF
derivatives is at present unknown, but NMR studies are in
progress to address this issue. However, the lone substitution
of Val2 with Leu resulted in complete inactivation, indicating
that an additional methyl group in this location might prevent
optimal interaction between as-APF and its receptor. In addition,
the 100-fold decrease in activity caused by either extension of
the N-terminus with tyrosine (the amino acid preceding threo-
nine in the frizzled 8 protein sequence8) or acetylation of the
N-terminal amino group provides evidence for the functional
importance of the very specific positioning of a positively
charged N-terminal amino group relative to the sugar moieties
for maintenance of as-APF activity.
Our data also indicate that Ala5 and Val6-8 are very important
for optimal biological activity and that a minimum of eight
amino acids is required for biological activity. The decrease in
activity resulting from replacement of Ala8 or Ala9 with amino
acids containing larger side chains is compatible with the
hypothesis that both amino acids may form a flat surface on an
R-helix that may be important for interaction with the APF
receptor.12 However, the equal activity of 1 and 18 indicates
Conformation of as-APF in the area of the proline residue
also appears to be very important, as substitution of L-proline

SAR Studies of the Bladder Epithelial Cell APF
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19 5979
Table 4. Substitutions of C-Terminal Amino Acids 5-9 (AVVVA)
a As a result of the variability of cell response in the biological assay, the activity of each congener was normalized to the activity of 1 run simultaneously
on the same plate according to the following equation: % ) [IC50 (as-APF)/IC50 (derivative)] × 100; the average IC50 value of 1 was ∼1 nM. Percent of
activity is expressed relative to 1, which served as a standard control on each plate. Derivatives with no significant activity at <25 µM concentration (the
cutoff limit for the biological assay) were considered to be inactive. b NS ) not significant at p > 0.05.
that this interaction, if it occurs, may only be required for Ala5.
CD measurements have not revealed an ordered structure of
the parent congener 1 in water solution (see Supporting
Information), and comprehensive NMR studies of as-APF in
water solution confirm the lack of ordered structure in water
(data not shown). However, there is some evidence for an
ordered structure in higher concentrations (45%) of trifluoro-
ethanol, making it reasonable to hypothesize that the carboxy
terminal tail of as-APF may be able to adopt an R-helical-like
conformation in a cellular milieu and/or upon interaction with
its receptor. Extensive molecular dynamics studies based on the
limited NMR restraints available clearly show that the C-
terminal stretch of amino acids (AVVVA) in as-APF can adopt
folded structures with concomitant adjustments in the rotamer
distribution about the anomeric bond of the disaccharide after
1 ns (data to be published elsewhere). NMR studies of as-APF
and several less potent analogues in the presence of its receptor
in both aqueous and lipid environments are in progress to
determine the precise structural features associated with maxi-
mum activity of these glycopeptides.
In addition, a negatively charged species at the carboxy-
terminal end appears to be very important for as-APF activity.
Moreover, when a negative charge state is maintained in that
location, additional steric bulk can be tolerated at this end of

5980 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19
Table 5. Modifications of the C-Terminus
Kaczmarek et al.
a As a result of the variability of cell response in the biological assay, the activity of each congener was normalized to the activity of 1 run simultaneously
on the same plate according to the following equation: % ) [IC50 (as-APF)/IC50 (derivative)] × 100; the average IC50 value of 1 was ∼1 nM. Percent of
activity is expressed relative to 1, which served as a standard control on each plate. Derivatives with no significant activity at <25 µM concentration (the
cutoff limit for the biological assay) were considered to be inactive. b NS ) not significant at p > 0.05.
the peptide, allowing for the synthesis of active derivatives
containing fluorescent labels on the C-terminus to follow
temporal and spatial aspects of the APF-cellular receptor
interaction.
Inhibition of bladder epithelial cell proliferation by several
of the APF derivatives appeared to be fairly abrupt for each
dilution series, showing greater than 90% inhibition at the first
active concentration. There are several possible explanations
for this observation. Because the primary cells and derivatives
used for these studies were very precious and small changes in
structure generally resulted in dramatic changes in activity (to
<1% of the original synthetic APF), the assay for these studies
used 10-fold dilutions of the APF derivatives. It is possible that
smaller increments in the dilution series for each derivative
might have uncovered additional midpoints of activity. However,
it is also possible that a critical concentration is required for
each derivative to reach a threshold for activity and that this
concentration changes as a result of a change in structure. Such
a dependence might occur if APF aggregation is needed for
binding to the receptor and/or if a critical APF/receptor ratio is

SAR Studies of the Bladder Epithelial Cell APF
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19 5981
Figure 5. Antiproliferative activity of specific APF derivatives
(modifications to carboxy-terminal alanine). Inhibition of tritiated
thymidine incorporation by primary normal bladder epithelial cells was
determined for each derivative at the concentrations indicated. Experi-
ments were run in triplicate on two separate occasions. Data are
expressed as the mean percent change in thymidine incorporation
relative to a cell control treated with diluent (acetonitrile/H2O at 1:1)
alone; bars indicate standard error of the mean for all six data points:
([) Galꢀ1-3GalNAc-O-TVPAAVVVA; (0) Galꢀ1-3GalNAcR-O-TV-
PAAVVVAK(Ac); (4) Galꢀ1-3GalNAcR-O-TVPAAVVVAK(dansyl)
(1) Galꢀ1-3GalNAcR-O-TVPAAVVVAK.
Figure 3. Antiproliferative activity of specific APF derivatives
(modifications to Pro3-Ala4). Inhibition of tritiated thymidine incorpora-
tion by primary normal bladder epithelial cells was determined for each
derivative at the concentrations indicated. Experiments were run in
triplicate on two separate occasions. Data are expressed as the mean
percent change in thymidine incorporation relative to a cell control
treated with diluent (acetonitrile/H2O at 1:1) alone; bars indicate
standard error of the mean for all six data points: (O) Galꢀ1-3GalNAc-
O-TVPAAVVVA; (0) Galꢀ1-3GalNAcR-O-TLS(ΨMe,Mepro)-AAV-
VVA; (4) Galꢀ1-3GalNAcR-O-TV-Hyp-AAVVVA; (1) Galꢀ1-
3GalNAcR-O-TV-Aze-AAVVVA; ([) Galꢀ1-3GalNAcR-O-TV-Pip-
AAVVVA.
to be present.7 This report demonstrates that the peptide portion
of as-APF must also have at least eight N-terminal amino acids.
A summary of these requirements is illustrated in Figure 6A.
Our data also suggest the possible additional requirement for a
trans conformation for the Pro-Ala peptide bond. In addition,
for optimal activity the peptide portion of as-APF must contain
(1) a specific amino acid sequence with alanine in position 5
and valines in positions 6-8, (2) the conformation allowed by
proline or pipecolic acid in position 3, (3) a very specific
arrangement of methyl groups on the two N-terminal amino
acids, (4) an amino acid no bulkier than alanine in the ninth
position, and (5) a free N-terminal amino group and a free
C-terminal carboxy group. These features are highlighted in
Figure 6B.
Figure 4. Antiproliferative activity of specific APF derivatives
(changes in amino acids 6-8 (VVV)). Inhibition of tritiated thymidine
incorporation by primary normal bladder epithelial cells was determined
for each derivative at the concentrations indicated. Experiments were
run in triplicate on two separate occasions. Data are expressed as the
mean percent change in thymidine incorporation relative to a cell control
treated with diluent (acetonitrile/H2O at 1:1) alone; bars indicate
standard error of the mean for all six data points: (1) Galꢀ1-3GalNAc-
O-TVPAAVVVA; (0) Galꢀ1-3GalNAcR-O-TVPAAGGGA; (4) Galꢀ1-
3GalNAcR-O-TVPAAAAAA.
Materials and Methods
General. Amino acids and resins were purchased from AnaSpec,
Inc. (San Jose, CA) or EMD Chemicals (San Diego, CA), PyAOP,
AcOH, and Ac2O from Sigma Aldrich (St. Louis, MO), HOAt and
HATU from AK Scientific, Inc. (Mountain View, CA), and solvents
from American Bioanalytical (Natick, MA). Peptide synthesis was
performed on a Nautilus 2400 Parallel synthesizer (Argonaut,
Technologies, Foster City, CA). Preparative HPLC was performed
on a Waters 600 instrument with UV detection (Waters 2487) on
reverse phase C18 or C8 silica (mobile phase: solvent A, H2O/0.1%
TFA, solvent B, CH3CN in 0.1% TFA). NMR analyses were
performed on a Varian INOVA instrument operating at 500 MHz
for proton from 15 to 40 °C in either D2O or H2O/D2O, 9:1. Water
suppression was accomplished by standard WATERGATE or WET
pulse sequences for observation of amide protons. CD measure-
ments were performed on an AVIV 202 spectrometer in water (50
µM, pH 6.0) and neat TFE (50 µM).
Patients. Normal controls who were asymptomatic for urinary
tract disease and undergoing cystoscopy following abdominal or
pelvic surgery as standard of care consented to provide biopsy for
the generation of normal bladder epithelial cell explants. These
participants were all at least 18 years old and enrolled in accordance
with guidelines of the Institutional Review Board of the University
of Maryland School of Medicine.
required for each derivative to mediate a cell signaling response.
We have performed concentration-dependent 1H NMR experi-
ments on as-APF that showed no change in chemical shift over
a 1000-fold concentration range, indicating that the endobiotic
form does not aggregate (data not shown). However, additional
studies to determine whether aggregation might be important
for the activity of other APF derivatives, as well as to determine
the mode of APF/receptor interaction(s), are in progress.
Conclusions
On the basis of these data, we were able to arrive at some
definitive, and some tentative, conclusions on the requirements
of the peptide portion of as-APF for its antiproliferative activity
in bladder epithelial cells. We previously determined that as-
APF must have an R-linked carbohydrate for biological activity
Synthesis of APF Derivatives. The synthesis of the peptide
segments of the glycopeptides were carried out in 0.1 mM scale
by solid-phase methods by using standard Fmoc chemistry on

5982 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19
Kaczmarek et al.
Figure 6. Schematic diagram indicating (A) essential and (B) important structural requirements for as-APF activity.
2ClTrt resin. Protected amino acids (0.5 mmol) were coupled using
HATU (0.5 mmol) and HOAt (0.5 mmol) reagents in the presence
of DIPEA (1.0 mmol). The Fmoc group was removed with 20%
piperidine in NMP, and a mixture of BEP/HOAt/DIPEA (0.05
mmol/0.05 mmol/0.15 mmol) in NMP was used for coupling of
Fmoc-Thr(Ac4Galꢀ1-3Ac2GalNAcR-O-)-OH or Fmoc-Ser(Ac4Galꢀ1-
3Ac2GalNAcR-O-)-OH (0.05mmol) to the peptide chain. Acetyl
groups were removed on the solid support using 10% hydrazine
monohydrate in MeOH,18 and each glycopeptide was cleaved from
the resin with TFA/DCM/H2O (50/49/1) or TFA/H2O (95/5). All
intermediates and the final products were verified by HPLC-MS;
purity of >95% was confirmed for all compounds by HPLC trace
analysis at 227 nm (see Supporting Information). Glycopeptides
were purified by RP-HPLC on either a C8 or C18 column with
gradient elution with H2O (0.1% TFA) and MeCN (0.1% TFA).
Synthesis of Glycopeptide 5. The glycopeptide was synthesized
using the general procedure described above. After the attachment
of Fmoc-Thr(Ac4Galꢀ1-3Ac2GalNAcR-O-)-OH, the Fmoc group
was removed with 20% piperidine in NMP and the deprotected
amino group was acetylated using Ac2O/DIPEA (2:5) in DCM.
Synthesis of Glycopeptide 9. The synthesis of glycopeptide 9
was performed using the same general procedure described above
with the exception that the Val-Ser(ΨMe,Mepro) segment was
coupled as a dipeptide unit. To protect the pseudoproline unit, the
glycopeptide was cleaved from the resin using a TFE/DCM (2/8)
mixture.
performed on a Rink amide resin. Prior to the first coupling step,
the Fmoc group was removed with 20% piperidine in NMP.
Synthesis of Glycopeptide 35. After the acylation of the 2ClTrt
resin with Dde-Lys(Fmoc)-OH, the Fmoc group was removed and
the deprotected amino group was acetylated using a Ac2O (2 mmol)/
DIPEA (5 mmol) mixture in dry DCM. The Dde group was then
removed with 2% hydrazine in DMF, and synthesis of the remaining
glycopeptide was performed using the general method described
above.
Synthesis of Cyclic Glycopeptide 36. Ac4Galꢀ1-3Ac2GalNAcR-
O-TVPAAVVVA was synthesized using the general procedure
described above on 2ClTrt resin. The Fmoc group was removed
with 20% piperidine in NMP, and the glycopeptide was cleaved
using TFA/DCM/H2O (50/49/1). After HPLC purification of the
crude glycopeptide, 30 mg (0.02 mmol) was dissolved in 2:1 DCM/
DMF (45 mL) and stirred for 24 h in the presence of PyAOP/
HOAt/DIPEA (0.1 mmol/0.1 mmol/0.1 mmol). The formation of
33 was then confirmed by HPLC-MS. Following evaporation of
the solvent, the glycopeptide was dissolved in H2O/MeCN and
lyophilized. After additional HPLC purification the glycopeptide
was dissolved in 10% hydrazine monohydrate in MeOH. All acetyl
groups were then removed, and the solution was neutralized with
AcOH and evaporated. The dry glycopeptide was then dissolved
in AcOH/H2O/MeCN and lyophilized. Further HPLC purification
led to pure 36 (5.5 mg, 23% yield).
Cell Culture. Cystoscopy was performed under general anes-
thesia, and 4 mm2 pieces of transitional epithelium with submucosal
bladder tissue were obtained for the growth of primary bladder
epithelial cells, as previously described.6,7 Primary normal bladder
epithelial cells were propagated in DMEM-F12 (Media-Tech,
Herndon, VA) with 10% heat-inactivated fetal bovine serum (FBS),
1% antibiotic/antimycotic solution, 1% L-glutamine, 0.25 units/mL
insulin (all from Sigma, St. Louis, MO), and 5 ng/mL hEGF (R&D
Systems, Minneapolis, MN) at 37 °C in a 5% CO2 atmosphere and
characterized by binding of AE-1/AE-3 pancytokeratin antibodies
(Signet, Dedham, MA).
Synthesis of Glycopeptide 13. The general procedure described
above was used to synthesize 13, which was then cleaved from the
resin using a TFE/DCM (2:8) mixture to maintain the protective
groups on the Hyp moiety.
Synthesis of Glycopeptide 14. The glycopeptide was synthesized
as described above with the exception that coupling of the amino
acid that precedes the N-methylamino acid in the sequence was
repeated twice.
Synthesis of Glycopeptide 29. This compound was synthesized
using the general procedure described above except that it was

SAR Studies of the Bladder Epithelial Cell APF
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19 5983
3H-Thymidine Incorporation. Cell proliferation was measured
by 3H-thymidine incorporation into explanted normal human
bladder epithelial cells, plating 1.5 × 104 cells/well onto a 96-well
cell culture plate (VWR 29442-054), in 150 µL/well MEM
containing 10% heat inactivated FBS, 1% antibiotic/antimycotic
solution, and 1% L-glutamine (all from Sigma), resulting in a
doubling time of 48-72 h, as previously described.6,7 Each purified
lyophilized synthetic APF congener was resuspended in acetonitrile/
distilled water (1:1) and applied to the cells in serum-free MEM
(containing only L-glutamine and antibiotics/antimycotics); cell
controls received acetonitrile/distilled water diluted in serum-free
MEM alone. Cells were then incubated at 37 °C in a 5% CO2
atmosphere for 48 h. The cell contents were harvested and
methanol-fixed onto glass fiber filter paper, and the amount of
radioactivity incorporated was determined. Significant inhibition
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3
of H-thymidine incorporation was defined as a mean decrease in
counts per minute of greater than 2 standard deviations from the
mean of control cells for each plate. Inhibition of cell proliferation
was determined from a semilog plot of dose-response for each
APF derivative; IC50 was determined as the concentration of each
derivative that caused a mean 50% inhibition of thymidine
incorporation compared to the mean of untreated cell controls.
Statistical Analysis. The thymidine incorporation (APF biologi-
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separate runs, with 1 run simultaneously in triplicate on the same
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Acknowledgment. The authors thank Toby Chai (University
of Maryland School of Medicine) for providing bladder biopsy
specimens from which the explanted bladder epithelial cells were
propagated and R. Andrew Byrd (National Cancer Institute,
National Institutes of Health) for helpful discussions. This work
was supported by funding from the National Institutes of Health
(NIDDK Grant R01 DK52596), as well as in part by funding
from the Intramural Research Program of the National Cancer
Institute, National Institutes of Health. We also gratefully
acknowledge the assistance of the Biophysics Resource, Struc-
tural Biophysics Laboratory, Center for Cancer Research,
National Cancer Institute.
Supporting Information Available: HPLC traces, NMR spectra,
and CD data of selected derivatives. This material is available free
of charge via the Internet at http://pubs.acs.org.
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