Angewandte
Communications
Chemie
Antitumor Agents
Re-engineering the Immune Response to Metastatic Cancer:
Antibody-Recruiting Small Molecules Targeting the Urokinase
Receptor
Anthony F. Rullo, Kelly J. Fitzgerald, Viswanathan Muthusamy, Min Liu, Cai Yuan,
Mingdong Huang, Minsup Kim, Art E. Cho, and David A. Spiegel*
Abstract: Developing selective strategies to treat metastatic
cancers remains a significant challenge. Herein, we report the
first antibody-recruiting small molecule (ARM) that is capable
of recognizing the urokinase-type plasminogen activator
receptor (uPAR), a uniquely overexpressed cancer cell-surface
marker, and facilitating the immune-mediated destruction of
cancer cells. A co-crystal structure of the ARM-U2/uPAR
complex was obtained, representing the first crystal structure of
uPAR complexed with a non-peptide ligand. Finally, we
demonstrated that ARM-U2 substantially suppresses tumor
growth in vivo with no evidence of weight loss, unlike the
standard-of-care agent doxorubicin. This work underscores the
promise of antibody-recruiting molecules as immunotherapeu-
tics for treating cancer.
Antibody-recruiting molecules (ARMs) are bifunctional
molecules capable of delivering endogenous antibodies to
disease-causing entities, leading to their destruction and/or
clearance by the immune system. Our group and others have
previously developed ARMs that target various cancers and
infectious agents. These novel immunotherapies have the
potential both to complement protein-based agents while
overcoming their challenges, such as poor oral bioavailability,
high molecular weights, and immunogenicity.[5]
Our group previously reported an ARM capable of
targeting uPAR-expressing cancer cells for immune-mediated
cell death.[5] Although effective in vitro, the reported con-
struct (termed ARM-U1) contained the uPA protein at its
target binding terminus (TBT), imparting many of the
limitations of biologics. We therefore hypothesized that the
therapeutic potential of this agent could be significantly
improved upon by replacing the uPA protein with a high-
affinity uPAR-binding small molecule (Figure 1A).
Herein, we report the design, synthesis, and in vitro and
in vivo evaluation of a second-generation, low-molecular-
weight (< 1000 amu) ARM derivative termed ARM-U2. In
place of uPA, ARM-U2 incorporates a restructured analogue
of IPR-803, a uPAR inhibitor identified previously using
a virtual screen, into its TBT.[6] ARM-U2 targets the uPA
binding site on uPAR with low nanomolar affinity, induces
immune-mediated phagocytosis and cytotoxicity of uPAR-
expressing cells in culture, and also inhibits tumor progression
in vivo, possessing comparable efficacy to the standard-of-
care agent doxorubicin but without the substantial weight loss
associated with doxorubicin treatment. We also report a co-
crystal structure of the ARM-U2/uPAR complex, which to
our knowledge represents the first uPAR/small-molecule
inhibitor complex to appear in the literature. This work
underscores the promise of small-molecule immunothera-
peutics for treating patients with uPAR-expressing metastatic
cancers.
T
umor metastasis involves the invasion of cancer cells into
surrounding tissues, a process often accelerated by cell-
surface proteases. One such protease, the urokinase-type
plasminogen activator (uPA), breaks down extracellular
matrix proteins and activates migration-inducing signal
cascades upon binding to the urokinase-type plasminogen
activator receptor (uPAR).[1] Both uPA and uPAR expression
are substantially upregulated in invasive cancer cells com-
pared to healthy cells or benign tumors.[2] In clinical settings,
uPAR levels have been shown to correlate with metastatic
potential and poor clinical outcomes.[1a] As such, uPAR has
gained recognition as a promising target for treating meta-
static cancers from diverse tissues of origin, including breast,
colon, stomach, and bladder.[3,4]
[*] Dr. A. F. Rullo, Dr. V. Muthusamy, Prof. Dr. D. A. Spiegel
Department of Chemistry, Yale University
225 Prospect Street, New Haven, CT 06511 (USA)
E-mail: david.spiegel@yale.edu
K. J. Fitzgerald, Prof. Dr. D. A. Spiegel
Department of Pharmacology, Yale School of Medicine
333 Cedar Street, New Haven, CT 06520 (USA)
In designing ARM-U2, we first sought to identify a site in
the IPR-803 scaffold at which to affix a linker connecting the
TBT to a 2,4-dinitrophenyl (DNP) antibody-binding terminus
(ABT). To this end, we performed computationally assisted
docking experiments,[7] which suggested that functionaliza-
tion of the IPR-803 core at position 2 of ring C with
triethylene glycol derived PEG-3 linker 9 would be sufficient
to drive solvent exposure of the ABT (1; Figure 1B). Also,
computational studies performed previously and in our
laboratory suggested that electrostatic interactions between
R53 and the carboxylate of IPR-803 are critical for driving
M. Liu, Dr. C. Yuan, Prof. Dr. M. Huang
State Key Laboratory of Structural Chemistry
Fujian Institute of Research on the Structure of Matter
Chinese Academy of Sciences
155 Yang Qiao West Road, Fuzhou, Fujian 350002 (China)
M. Kim, Prof. Dr. A. E. Cho
Department of Bioinformatics, Korea University
2511 Sejong-ro, Sejong, 339-700 (Korea)
Supporting information for this article is available on the WWW
3642
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2016, 55, 3642 –3646