Small molecule ligands for active targeting of TrkC-expressing tumor cells

Article abstract of DOI:10.1021/ml300227d

A small molecule motif was used in active targeting to deliver cytotoxic substances into tumor cells that express the TrkC receptor. Underlying this study was the hypothesis that internalization of targeted conjugates into cells would be facile if mediated by receptor binding and receptor-ligand internalization. Initial experiments using 6-mercaptopurine gave encouraging data but demonstrated the importance of maintaining solubility and high cytotoxicity. Conjugates of the targeting agent with a cytotoxic rosamine (similar to a rhodamine) were more successful. Targeting of TrkC was observed, validated in a series of competition experiments featuring other TrkC ligands, and accumulation into lysosomes was observed, as expected for receptor-mediated internalization.

Full text of DOI:10.1021/ml300227d

Letter
pubs.acs.org/acsmedchemlett
Small Molecule Ligands for Active Targeting of TrkC-Expressing
Tumor Cells
Eunhwa Ko, Anyanee Kamkaew, and Kevin Burgess*
Department of Chemistry, Texas A & M University, Box 30012, College Station, Texas 77842, United States
S
* Supporting Information
ABSTRACT: A small molecule motif was used in “active targeting”
to deliver cytotoxic substances into tumor cells that express the TrkC
receptor. Underlying this study was the hypothesis that internal-
ization of targeted conjugates into cells would be facile if mediated by
receptor binding and receptor−ligand internalization. Initial experi-
ments using 6-mercaptopurine gave encouraging data but demon-
strated the importance of maintaining solubility and high cytotoxicity.
Conjugates of the targeting agent with a cytotoxic rosamine (similar
to a rhodamine) were more successful. Targeting of TrkC was
observed, validated in a series of competition experiments featuring
other TrkC ligands, and accumulation into lysosomes was observed,
as expected for receptor-mediated internalization.
KEYWORDS: TrkC, neurotrophin, targeting, cancer, small molecule ligand
urrent U.S.-approved small molecule pharmaceuticals for
Cthe treatment of cancer tend to have low therapeutic
indices, that is, similarly toxic to cancerous and healthy tissue.
Therapeutic indices of anticancer compounds can be increased
by selective delivery; this is a form of “targeting”. Unfortunately,
the word “targeting” is ambiguous in biomedical research. For
instance, compounds that inhibit enzymes overexpressed in
tumor cells are sometimes described as “targeted”, but they are
not guided to the target via an extracellular chemical interaction.1
Conversely, some physical or physiochemical properties (e.g., en-
hanced permeability and retention and pH effects) favor accu-
mulation of nanoparticles or acid-sensitive compounds in tumors;
for the purposes of this work, we refer to this as passive targeting.
Passive targeting can be useful, but the particles can still accu-
mulate in healthy tissue surrounded by either relatively imper-
meable vasculature or abnormally acidic interstitial fluid. Active
targeting occurs when proteins or small molecule ligands form
chemical interactions with macromolecules selectively expressed
on the surface of the targeted cells.2
At least two possible factors can favor active targeting: (i)
affinity of the ligand for that cell surface receptor and (ii) inter-
nalization of the ligand into the cells by the receptor. Actively
targeted cytotoxicity occurs if one or both of these effects are in
play. One cell surface receptor that could be targeted is TrkC.
We hypothesized that this receptor is a good target because
several tumor types overexpress TrkC, including neuroblastoma,3
medulloblastoma,4 and breast cancer.5 Moreover, this receptor
internalizes the natural (neurotrophin) ligands that bind to it,
fulfilling criterion (ii).6
Figure 1. Targeting ligands used in this study and the nontargeting
control compound 2mor-6MP.
The research that is described in this letter was undertaken to
demonstrate active targeting of TrkC-expressing tumor cells that
could be affected using novel small molecule targeting ligands.
Received: August 3, 2012
Accepted: September 30, 2012
Published: October 9, 2012
© 2012 American Chemical Society
1008
dx.doi.org/10.1021/ml300227d | ACS Med. Chem. Lett. 2012, 3, 1008−1012

ACS Medicinal Chemistry Letters
Letter
Figure 2. Antiproliferative assays. (a) IY-IY-6MP selectively targets
TrkC expressing cells (red) and not the parent line (WT, purple; data
are shown with controls that demonstrate both of the IY-IY amino
acid side-chains and the 6MP fragment were essential for cytotoxicity).
(b) IY-IY-Ros shows more cytotoxicity for the TrkC-expressing cells,
TrkC (red) and 4T1 (purple), than non-TrkC-expressing cells, WT
(blue). Error bars were based on three runs.
Figure 3. Dose-dependent reduction of IY-IY-Ros cytotoxicity (red)
in competition with the TrkC ligands NT-3, 3.5 nM (blue), or IY-IY-
TEG, 20 μM (green), which occurs for (a) TrkC cells but not for (b)
WT cells. The concentrations of NT-3 and IY-IY-TEG were kept
constant throughout the experiments.
Specifically, a triazine-bridged compound IY-IY-TEG was used as
a basis for this study (Figure 1). That compound was previously
shown to bind TrkC-expressing cells and elicit agonistic effects
but only in synergy with the parent TrkC ligand, a protein
growth factor called NT3.7 Side-chain pharmacophores (shown
in red) in IY-IY-TEG and related molecules were shown to be
pivotal to binding.
The central hypothesis here is that the partial agonist activity
of the IY-IY motif could be overwhelmed by incorporating a
highly cytotoxic fragment in the same molecule, leading to selec-
tive binding and internalization for TrkC-expressing cells. 6-
Mercaptopurine (6MP)8 and a rosamine (Ros, a highly cytotoxic
fluorescent probe)9 were used for their cell-killing effects. We
theorized that permeation of IY-IY-6MP and IY-IY-Ros into cells
would be disfavored because of size and polarity effects, unless
active targeting was involved. To evaluate our hypothesis, cytoto-
xicities of IY-IY-6MP toward a cell line that does not express
TrkC (wild-type NIH3T3 abbreviated to WT)10,11 were com-
pared with the same cell line stably transfected with TrkC
(NIH3T3 + TrkC abbreviated to TrkC; expression levels 10−
20 × 103 receptors cell−1).
controls that, in the event, support this conclusion. Thus, the
nontargeted analogue 2mor-6MP (where the IY-IY motif is
substituted by morpholine; Figure 1) is not significantly cytotoxic
to the TrkC or WT cells and neither is the targeted but non-
cytotoxic compound IY-IY-TEG.
It was impossible to determine an IC50 for IY-IY-6MP because
the concentrations required exceeded the solubility of the com-
pound. A relatively high amount was needed in solution because
the cytotoxicities of N9-alkylated derivatives of 6MP are less than
the parent system (ED50 = 50 μM for 6MP, 400 μM for N9-butyl,
and 600 μM for N9-ethyl in Chinese hamster ovary cells).12 For
this reason, the focus of the study shifted to IY-IY-Ros; the
rosamine fragment of this molecule is significantly more cytotoxic
(IC50 0.39 0.22 μM9 for Ros vs 2.79 0.69 μM8 for 6MP; both
for MCF-7, human breast cancer cell line).
Figure 2b compares cytotoxicity data for IY-IY-Ros on the
TrkC cells (TrkC; IC50 = 15.80 0.18 μM) and on a murine
breast cancer line that also expresses TrkC (4T1; IC50 = 19.88
2.53 μM) with that for the non-TrkC expressing NIH3T3 cells
(WT; IC50 = 27.58 1.38 μM). More cell death occurred for the
TrkC-expressing cells than for the non-TrkC-expressing ones
when ligand concentrations above about 18 μM were used. Cyto-
toxicity data for an analogue with the inverted amino acid sequence
YI-YI-Ros was used as a negative control (the YI-YI motif does
not bind TrkC-expressing cells).7 The cytotoxicity of YI-YI-Ros
Figure 2a shows cytotoxicity data for IY-IY-6MP; the red line
is for the TrkC-expressing cells (TrkC), and the purple one is
for WT NIH3T3 cells. The conjugate is more cytotoxic toward
cells that express TrkC. All the other data in Figure 2a are for
1009
dx.doi.org/10.1021/ml300227d | ACS Med. Chem. Lett. 2012, 3, 1008−1012

ACS Medicinal Chemistry Letters
Letter
Figure 4. (a) 2mor-Ros and MitoTracker colocalize in TrkC cells, whereas (b) IY-IY-Ros colocalizes with LysoTracker in the same cells and (c) in
murine 4T1 breast cancer cells that also express TrkC.
with respect to the TrkC and WT cells was almost the same. More-
over, these data were also almost identical to that for IY-IY-Ros on
the WT cells (IC50 = 25.80 2.24 and 25.79 1.79 μM); these
data further support the targeting effect of the IY-IY motif.
Further evidence for the targeting effect of the IY-IY motif
was obtained from competition studies (Figure 3). The cyto-
toxicity of IY-IY-Ros was reduced in a dose-dependent way by
natural protein (NT3) and by synthetic (IY-IY-TEG) ligands
but only for the TrkC (Figure 3a) and not for the WT (Figure 3b)
cells. Moreover, no competition was observed for the control with
the inverted sequence YI-YI-Ros (see Figure S1 in the Supporting
Information).
Complexes of NT3 with TrkC are internalized by cells and
localize in the lysosome.6 Conversely, the rosamine dye corre-
sponding to Ros permeates into cells and accumulates in the
mitochondria.9 Experiments were performed in the current study
1010
dx.doi.org/10.1021/ml300227d | ACS Med. Chem. Lett. 2012, 3, 1008−1012

ACS Medicinal Chemistry Letters
Letter
to probe if the rosamine fragment with the bridging triazine
(2mor-Ros) was imported in the same way as the dye without
that fragment (Ros). Figure 4a shows the distribution of the
MitoTracker label in TrkC cells, distribution of 2mor-Ros in the
same cell, and the degree of overlay. These data indicate that
2mor-Ros localizes in the mitochondria, just as the parent dye does.
When the Trk cells were exposed to IY-IY-Ros, this targeted
(cytotoxic) dye was internalized within 30 min. Fluorescence
microscopy indicated the dye colocalized with LysoTracker
(Figure 4b) and not with MitoTracker; that is, it accumulated in
the same intracellular compartment as the TrkC-NT3 complex and
not in the same place as the rosamine dye. When another TrkC-
expressing line, murine 4T1 cells, was treated with IY-IY-Ros,
then this targeted (cytotoxic) dye also localized in the lysosome
(Figure 4c). These data support the hypothesis that the IY-IY
motif binds the TrkC receptor and is internalized.
The featured targeted compounds IY-IY-6MP and IY-IY-
Ros are not well suited for in vivo studies, for different rea-
sons. The water solubility and cytotoxicity of IY-IY-6MP are
inadequate for in vivo work. For IY-IY-Ros, the reasons are
different; while these studies were in progress, our col-
laborator Dr. Hong Boon Lee tested the parent rosamine and
found it caused severe weight loss in animals with inade-
quate concurrent reduction in tumor mass (personal com-
munication). Consequently, studies to use the same targeting
entities in conjunction with other cytotoxic compounds are
underway.
Only a few small molecules are known for their active target-
ing effects; the important ones are as follows. Folic acid targets
the folate receptor13 expressed on many human cancers.14 Mimics
of the RGD peptide15 target integrins,16,17 and riboflavin-based
compounds have been used to target its receptor expressed on
some tumor types.18,19 Bile acids target the vitamin D receptor
in colon cancer, and this has been exploited.20 Lectin-based
molecules21 have been employed to direct therapeutic agents to
the asialoglycoprotein receptor in the liver,22 and cholestenoic
acid, a ligand for the liver X receptor,23 also has value. γ-Amino-
n-butyric acid (GABA) or the GABA derivative baclofen tar-
geting the GABAB receptor have been used in a novel approach
for pancreatic cancer.24 Overall, there is a great deal more interest
in antibody−drug conjugates than in targeting small molecule−
drug conjugates.
Given the exquisite affinities of antibodies (mAbs) for antigens, it
is easy to understand why mAbs have attracted so much interest in
targeting approaches. However, we postulated above that affinity
and cell permeability are both important, and large proteins such as
mAbs tend not to permeate into cells, whereas small molecules can.
Selective cytotoxicity studies of the kind performed here show the
net effect of affinity and cell permeability.
AUTHOR INFORMATION
■
Corresponding Author
*Corresponding Author FAX: +1 979 845 8839. E-mail:
burgess@tamu.edu.
Funding
We thank The National Institutes of Health (GM087981) and
The Robert A. Welch Foundation (A-1121) for financial support.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Some of the IY monomers used in the study were made by Dr.
David Chen, and some of the rosamines were made by Dr.
Liangxing Wu, (both previously at TAMU); we thank them for
these. We also thank Dr. Rola Barhoumi and Dr. Robert
C. Burghardt for help with cell imaging.
REFERENCES
■
(1) Brannon-Peppas, L.; Blanchette, J. O. Nanoparticle and targeted
systems for cancer therapy. Adv. Drug Delivery Rev. 2004, 56, 1649−
1659.
(2) Peer, D.; Karp, J. M.; Hong, S.; Farokhzad, O. C.; Margalit, R.;
Langer, R. Nanocarriers as an emerging platform for cancer therapy.
Nat. Nanotechnol. 2007, 2, 751−760.
(3) Yamashiro, D. J.; Nakagawara, A.; Ikegaki, N.; Liu, X. G.;
Brodeur, G. M. Expression of TrkC in Favorable Human Neuro-
blastomas. Oncogene 1996, 12, 37−41.
(4) Segal, R. A.; Goumnerova, L. C.; Kwon, Y. K.; Stiles, C. D.;
Pomeroy, S. L. Expression of the Neurotrophin Receptor TrkC is
Linked to a Favorable Outcome in Medulloblastoma. Proc. Natl. Acad.
Sci. U.S.A. 1994, 91, 12867−12871.
(5) Jin, W.; Kim, G.-M.; Kim, M.-S.; Lim, M.-H.; Yun, C.-H.; Jeong,
J.; Nam, J.-S.; Kim, S.-J. TrkC plays an essential role in breast tumor
growth and metastasis. Carcinogenesis 2010, 31, 1939−1947.
(6) Butowt, R.; von Bartheld, C. S. Sorting of internalized
neurotrophins into an endocytic transcytosis pathway via the Golgi
system: Ultrastructural analysis in retinal ganglion cells. J. Neurosci.
2001, 21, 8915−8930.
(7) Chen, D.; Brahimi, F.; Angell, Y.; Li, Y.-C.; Moscowicz, J.;
Saragovi, H. U.; Burgess, K. Bivalent Peptidomimetic Ligands of TrkC
are Biased Agonists, Selectively Induce Neuritogenesis, or Potentiate
Neurotrophin-3 Trophic Signals. ACS Chem. Biol. 2009, 4, 769−781.
(8) Saczewski, F.; Maruszak, M.; Bednarski, P. J. Synthesis and
cytotoxic activity of imidazo[1,2-a]-1,3,5-triazine analogues of 6-
mercaptopurine. Arch. Pharm. Chem. Life Sci. 2008, 341, 121−125.
(9) Lim, S. H.; Wu, L.; Burgess, K.; Lee, H. B. New cytotoxic
rosamine derivatives selectively accumulate in the mitochondria of
cancer cells. Anti-Cancer Drugs 2009, 20, 461−468.
(10) Eibl, J. K.; Chapelsky, S. A.; Ross, G. M. Multipotent
neurotrophin antagonist targets brain-derived neurotrophic factor
and nerve growth factor. J. Pharmacol. Exp. Ther. 2010, 332, 446−454.
(11) Rabin, S. J.; Bachis, A.; Mocchetti, I. Gangliosides Activate Trk
Receptors by Inducing the Release of Neurotrophins. J. Biol. Chem.
2002, 277, 49466−49472.
Medicinal chemistry has been heavily biased toward the dis-
covery and syntheses of cytotoxic compounds, whereas develop-
ment of small molecule ligands for active targeting has received
much less attention. We suggest design and testing of small
molecule ligands for active targeting of cancer cells; indeed, for
specific cell types in general, it is both important and, so far,
somewhat neglected.
(12) Nelson, J. A.; Vidale, E. Formation of 6-thioguanine and 6-
mercaptopurine from their 9-alkyl derivatives in mice. Cancer Res.
1986, 46, 137−140.
(13) Low, P. S.; Henne, W. A.; Doorneweerd, D. D. Discovery and
Development of Folic-Acid-Based Receptor Targeting for Imaging and
Therapy of Cancer and Inflammatory Diseases. Acc. Chem. Res. 2008,
41, 120−xs129.
ASSOCIATED CONTENT
■
S
* Supporting Information
(14) Antony, A. C. Folate receptors. Annu. Rev. Nutr. 1996, 16, 501−
521.
(15) Plow, E. F.; Haas, T. A.; Zhang, L.; Loftus, J.; Smith, J. W.
Synthetic procedures for IY-IY-6MP, 2mor-6MP, IY-IY-Ros,
YI-YI-Ros, and 2mor-Ros. This material is available free of
charge via the Internet at http://pubs.acs.org.
Ligand binding to integrins. J. Biol. Chem. 2000, 275, 21785−21788.
1011
dx.doi.org/10.1021/ml300227d | ACS Med. Chem. Lett. 2012, 3, 1008−1012

ACS Medicinal Chemistry Letters
Letter
(16) Hynes, R. O. Integrins: A family of cell surface receptors. Cell
1987, 48, 549−554.
(17) Desgrosellier, J. S.; Cheresh, D. A. Integrins in cancer: Biological
implications and therapeutic opportunities. Nat. Rev. Cancer 2010, 10,
9−22.
(18) Plantinga, A.; Witte, A.; Li, M.-H.; Harmon, A.; Choi, S. K.;
Banaszak Holl, M. M.; Orr, B. G.; Baker, J. R.; Sinniah, K. Bioanalytical
Screening of Riboflavin Antagonists for Targeted Drug Delivery: A
Thermodynamic and Kinetic Study. ACS Med. Chem. Lett. 2011, 2,
363−367.
(19) White, H. B., III; Merrill, A. H., Jr. Riboflavin-binding proteins.
Annu. Rev. Nutr. 1988, 8, 279−299.
(20) Makishima, M.; Lu, T. T.; Xie, W.; Whitfield, G. K.; Domoto,
H.; Evans, R. M.; Haussler, M. R.; Mangelsdorf, D. J. Vitamin D
receptor as an intestinal bile acid sensor. Science 2002, 296, 1313−
1316.
(21) Andre, S.; Frisch, B.; Kaltner, H.; Desouza, D. L.; Schuber, F.;
Gabius, H.-J. Lectin-mediated drug targeting: Selection of valency,
sugar type (Gal/Lac), and spacer length for cluster glycosides as
parameters to distinguish ligand binding to C-type asialoglycoprotein
receptors and galectins. Pharm. Res. 2000, 17, 985−990.
(22) Ashwell, G.; Harford, J. Carbohydrate-specific receptors of the
liver. Annu. Rev. Biochem. 1982, 51, 531−554.
(23) Song, C.; Liao, S. Cholestenoic acid is a naturally occurring
ligand for liver X receptor α. Endocrinology 2000, 141, 4180−4184.
(24) Schuller, H. M.; Al-Wadei, H. A. N.; Majidi, M. GABAB
receptor is a novel drug target for pancreatic cancer. Cancer 2008, 112,
767−778.
1012
dx.doi.org/10.1021/ml300227d | ACS Med. Chem. Lett. 2012, 3, 1008−1012