Chemical control over immune recognition: A class of antibody-recruiting small molecules that target prostate cancer

Article abstract of DOI:10.1021/ja906844e

(Figure Presented) Prostate cancer is the second leading cause of cancer-related death among the American male population, and society is in dire need of new approaches to treat this disease. Here we report the design, synthesis, and biological evaluation

Full text of DOI:10.1021/ja906844e

Published on Web 11/04/2009
Chemical Control over Immune Recognition: A Class of Antibody-Recruiting
Small Molecules That Target Prostate Cancer
Ryan P. Murelli, Andrew X. Zhang, Julien Michel, William L. Jorgensen, and David A. Spiegel*
Department of Chemistry, Yale UniVersity, 225 Prospect Street, P.O. Box 208107, New HaVen, Connecticut 06520-8107
Received August 12, 2009; E-mail: david.spiegel@yale.edu
Prostate cancer is the second leading cause of cancer-related death
among the American male population, and it has been predicted
that one out of every six American men will develop prostate cancer
during their lifetime.1 Available treatment options, including
chemical/surgical castration, radiation therapy, and chemotherapy,
are often ineffective against advanced disease and are also often
associated with severe side effects.2 Thus, new approaches to treat
prostate cancer are highly desirable. To this end, monoclonal
antibody therapies have shown promise;2 however, no such agent
has yet successfully obtained FDA approval for the treatment of
prostate cancer. Furthermore, antibody drugs are limited by severe
side effects, lack of oral bioavailabiliy, and high cost.3 Here we
describe a novel technology for prostate cancer treatment that
we believe could address many of the limitations of currently
available therapies and that combines advantages of both small-
molecule-based and antibody-based strategies.
specific membrane antigen (PSMA). PSMA is a cell-surface protein
that is highly overexpressed on prostate cancer cells relative to
normal cells of the prostate, and its expression increases with clinical
stage.8 This protein has been exploited as a target in both prostate
cancer imaging9 and monoclonal antibody therapy for the disease.10
Figure 1. Schematic depiction of the reported approach to prostate cancer
targeting. An antibody-recruiting small molecule (ARM) binds the cell-
surface prostate cancer marker prostate-specific membrane antigen (PSMA),
thus recruiting antibodies to these cells for recognition and targeted killing
by the immune system. The bifunctional ARMs are composed of an
antibody-binding terminus (ABT), a linker region, and a cell-binding
terminus (CBT).
Figure 2. Structure-based design studies. (A) Modeled complex illustrating
the design of a CBT for use in ARM-Ps. (B) Structural model of the ternary
complex between the Fv region of an anti-DNP antibody, ARM-P, and the
PSMA dimer. (C) Known PSMA-binding small molecules and structures
of ARM-P derivatives utilized in this study. (A) and (B) were created with
the program VMD.11
Several small-molecule ligands have been developed that bind
PSMA selectively and with high affinity, including 2-PMPA (1)12
and the glutamate ureas (2) (Figure 2C).13 These compounds
competitively inhibit PSMA’s enzymatic activity and have been
successfully modified for imaging and targeted drug delivery
applications.14 At the outset of our studies, we were intrigued by
observations that 2 could accommodate a wide range of R groups
at C2, including various alkyl heterocycle substituents, with minimal
loss of inhibitory potency.13 We therefore reasoned that we might
be able to incorporate a linker to join the ABT and CBT at this
position.
Thus, starting from a crystal structure for the complex of PSMA
with 1,15 the corresponding complex with 2 (R ) H) was modeled
using the program BOMB (biochemical and organic model
builder).16 Stabilizing interactions with active-site zinc ions were
indicated, as well as hydrogen bonding and salt-bridge interactions
with Tyr700, Lys699, Arg534, Arg536, and Asn257 (Figure 2A).
This model was found subsequently to be consistent with the
recently published cocrystal structure of PSMA complexed with
urea-based ligands.17 Next, BOMB was used to construct complexes
of 2 with alternative DNP linking groups. Among plausible designs,
1-butyl-4-alkyl-1,2,3-triazole analogues (e.g., 3-6) were judged as
promising because of favorable electrostatic interactions with
The key component of our approach is what we call antibody-
recruiting small molecules targeting prostate cancer (ARM-Ps).
These are bifunctional materials capable of redirecting antibodies
already present in the human bloodstream to prostate cancer cell
surfaces and increasing the destruction of cancer cells by effector
cells of the immune system (Figure 1). As shown, ARMs are
composed of an antibody-binding terminus (ABT), a cell-binding
terminus (CBT), and a linker region. In this manuscript, it is
demonstrated that ternary complexes formed between ARM-Ps,
human prostate cancer cells (LNCaP cells), and antibodies recog-
nizing the 2,4-dinitrophenyl (DNP) group lead to targeted cell-
mediated cytotoxicity of LNCaP cells. The power of this approach
derives from the observation that anti-DNP antibodies are already
found in the human bloodstream in a high percentage of the human
population4 and are competent to mediate target-cell killing.5,6
Several approaches that utilize bifunctional materials to recruit
antibodies to human pathogens have appeared,7 but ARM-Ps are
the first class of antibody-recruiting small molecules that target
prostate cancer. The general strategy reported herein has the
potential to initiate novel directions in treating cancer and other
diseases.
Our first goal in constructing ARM-Ps was to design an
appropriate CBT, and to this end, we chose to target the prostate-
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C O M M U N I C A T I O N S
Arg463, π-stacking interactions with Tyr700, the orientation of the
linker toward the solvent, and ease of synthesis.
complex formation was observed under these conditions using
DU145 prostate cancer cells, which lack PSMA.23 Together, these
data confirm that small-molecule-mediated antibody recruitment is
dependent upon binding of ARM-P8 to both PSMA and anti-DNP
antibodies. Moreover, competition with 2-PMPA and bis-DNP
lysine was found to be concentration-dependent (Figure 4C). The
Ki value determined for 2-PMPA in these studies was found to be
5.0 nM, which is almost identical to that determined in enzymatic
assays (2.3 nM).23 This result implies that ARM-P binding to PSMA
does not benefit from increased affinity due to multivalent
presentation.7g,h Fluorescence microscopy experiments (Figure 4D)
further confirmed the flow cytometry data and demonstrated
localization of the ternary complex to the cell membrane. No
fluorescence was observed in the absence of ARM-P8.23 Endocy-
tosis of fluorescent features at 37 °C but not 4 °C is consistent
with the reported behavior of PSMA.24
To estimate viable linker lengths, ternary complexes (involving
PSMA, ARM-Ps, the Fv region of an anti-DNP antibody18) were
constructed using the program FIRST (Figure 2B).19 Constrained
geometric simulations20 were then performed to assemble the
complex with the ABT and CBT binding sites in close proximity.
The modeling suggested that at least six oxyethylene units would
be necessary to prevent steric clashes between the two proteins
and that longer linkers might be preferable to prevent excessive
dehydration of the protein-protein interface.
Figure 3. Representative PSMA inhibition curves for ARM-Ps. Ki values
werecalculatedfrommeasuredIC50 andKM valuesthroughtheCheng-Prusoff
equation21 and are reported as the average of three runs ( standard deviation.
Thus, ARM-Ps 3-6 were synthesized (Figure 2C; these are
called ARM-P4 through ARM-P12 for the number of oxyethylene
units in the linker) and tested for binding to PSMA through a
standard enzymatic inhibition assay.22 This assay measures the
ability of designed small molecules to inhibit PSMA-catalyzed
cleavage of the peptide substrate N-acetylaspartylglutamate (NAAG).
As depicted in Figure 3, compound 3 (ARM-P4) inhibited PSMA
with a Ki of 63 pM, a value similar to that of the most potent PSMA-
binding small molecule developed to date.17 Although we antici-
pated that the flexible PEG linker in 3 could lead to entropic
penalties in binding PSMA, perhaps stabilizing interactions with
the triazole, DNP, and/or PEG portions of ARM-P4 compensate.
Interestingly, compounds 4-6, which contain 6, 8, and 12 oxy-
ethylene units, exhibited decreased inhibitory potency relative to
3. The origin of the trend is currently under investigation.
To evaluate the capacity of ARM-P derivatives to template
ternary complex formation in a cellular environment, we performed
live-cell flow cytometry assays with PSMA-expressing LNCaP cells
and Alexa Fluor 488-conjugated anti-DNP antibodies. Since the
anti-DNP antibody represented the fluorescent component in these
studies, rightward shifts of flow cytometry histograms indicate
increased levels of ternary complex formation. These experiments
revealed an intriguing trend (Figure 4A): although ARM-P4 (3)
possessed the highest affinity in PSMA-binding assays, maximal
amounts of ternary complex were formed in the presence of ARM-
P8 (5). These results are consistent with the predictions of the
computational modeling studies (see Figure 2B, above), which
suggested that linker lengths n ) 4 and 6 could lead to unfavorable
steric interactions between the antibody and PSMA. The relative
decrease in ternary complex formation for ARM-P12 versus ARM-
P8 may simply result from the decreased affinity of ARM-P12 for
PSMA, consistent with results in Figure 3.
Figure 4. Evaluating ternary complex formation. (A, B) Representative
traces from flow cytometry experiments. (C) Dose dependence of competitor
concentration on ternary complex formation. (D) Epifluorescence (Fluor)
and bright-field (BF) microscopy experiments performed in the presence
of ARM-P8 at 37 and 4 °C.
Having established that ARM-P8 possessed optimal linker length
for forming ternary complexes, we tested its ability to induce cell-
mediated cytotoxicity of LNCaP prostate cancer cells. This process
is known to take place by way of interactions between Fc receptors
on cytotoxic effector cells contained in peripheral blood (such as
NK cells, macrophages, and dendritic cells) and the Fc (constant)
regions of antibodies.6 Thus, LNCaP cells were combined with
peripheral blood mononuclear cells (PBMCs), anti-DNP antibodies,
and ARM-P8, and cell death was measured using a commercially
available calcein-release assay (Figure 5).25 As expected (see
below), ARM-P8 at concentrations up to 30 nM led to enhanced
cell killing, while treatment with ARM-P4 led to no change in cell
viability. The antibody concentration employed in these experiments
was slightly below that found in human serum,4 and lower
concentrations were also found to be efficacious (Figure S5 in the
Supporting Information). ARM-P8-mediated cell killing was not
observed in DU145 cells or in the presence of 2-PMPA, which
suggests that the molecular target in these experiments is PSMA
(Figure S5). Also, no cytotoxicity was observed in either LNCaP
or DU145 cells after treatment with ARM-P8 in the absence of
effector PBMCs, indicating that this compound is not itself cytotoxic
(Figure S5).
ARM-P8 was therefore chosen for evaluation in subsequent
studies. Flow cytometry experiments performed with an excess of
the competing ligand 2-PMPA or bis-DNP lysine (Figure 4B),
revealed baseline levels of ternary complex. Furthermore, no ternary
Notably, the intriguing bell-shaped pattern observed in the cell-
killing measurements with ARM-P8 has been observed in various
situations in which bifunctional ligands template ternary linkages.26
Such behavior results from the binding dynamics of these systems:
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at large total concentrations of the bifunctional small molecule,
unbound material competes with the ternary complex, driving the
system toward formation of binary complexes. We view this self-
antagonistic trend in ARM-P8-mediated cytotoxicity experiments
as evidence that cell killing proceeds via reversible formation of a
ternary complex. Furthermore, from a clinical standpoint, such a
model reveals a unique advantage associated with this novel class
of bifunctional therapeutics: they are autoinhibitory and could serve
clinically as the antidote for their own overdose.
Supporting Information Available: Detailed experimental proce-
dures and compound characterization. This material is available free
of charge via the Internet at http://pubs.acs.org.
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Figure 5. Antibody-dependent cellular cytotoxicity assays. LNCaP (PSMA-
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In summary, we have reported the structure-based design of a
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(ARM-Ps) capable of binding to prostate-specific membrane antigen
with high affinity (pM to nM) and recruiting antibodies to PSMA-
expressing cells. We have also demonstrated that one member of
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treatment of human cancers. Its autoinhibitory pharmacology (see
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Acknowledgment. The authors thank John Hines, Thomas
Gniadek, and Jacob Appelbaum for helpful suggestions and
experimental assistance. This work was funded by the National
Institutes of Health through the NIH Director’s New Innovator
Award Program (DP22OD002913 to D.A.S.) and the National
Foundation for Cancer Research (W.L.J.). J.M. acknowledges
support from a Marie Curie International Fellowship from the
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