Communications
DOI: 10.1002/anie.201103273
Live Cell Imaging
Bioorthogonal Probes for Polo-like Kinase 1 Imaging and
Quantification**
Ghyslain Budin, Katherine S. Yang, Thomas Reiner, and Ralph Weissleder*
Polo-like kinase 1 (PLK1) is a critical mediator of the cell
cycle, regulating mitotic progression. Specific functions of
PLK1 during mitosis include chromosome segregation, cen-
trosome maturation, bipolar spindle formation, regulation of
the anaphase-promoting complex/cyclosome (APC/C), coor-
dination of cytokinesis, and regulation of the DNA damage
checkpoint.[1–3] This serine/threonine kinase also plays an
essential role in mitotic entry by promoting Cdk1-cyclin B
activation and nuclear translocation through phosphorylation
of Cdc25C,[4,5] Wee1,[6] Myt1,[7] and cyclin B.[8,9] PLK1 consists
of two domains, an N-terminal catalytic serine/threonine
kinase domain and a C-terminal polo-box domain (PBD).[3]
The PBD recognizes specific phosphorylated targeting
sequences that are essential for interaction of PLK1 with its
substrates and for PLK1 localization to centrosomes, spindles,
kinetochores, and the midzone/midbody during mitosis.[10–13]
Given the role of PLK1 in mitotic progression it is perhaps
not surprising that overexpression has been shown to lead to
oncogenic transformation.[14] Increased PLK1 levels have
been found in a wide variety of cancers, including breast, lung,
colorectal, ovarian, pancreatic, prostate and head and neck
cancers.[15] Since PLK1 expression correlates with cell pro-
liferation, it has been suggested as an early marker for cancer
detection, as well as a prognostic marker.[16] However, to date
it has been difficult to visualize and quantify PLK1 expression
directly in patient samples or in live cells due to the lack of
imaging agents available for intracellular/nuclear targets such
as PLK1. Having the ability to monitor PLK1 directly in live
cells or by whole body imaging (where cells cannot be fixed
and antibodies do not reach nuclear targets) would have far
reaching applications in the development of future PLK1
inhibitors and for a better understanding of its biology.
Among the available small-molecule inhibitors targeting
PLK1, BI 2536 is currently the most intensively studied and
potent inhibitor in clinical trials (Figure 1A).[17,18] BI 2536
binds to and inhibits PLK1, leading to mitotic arrest,
Figure 1. A) Chemical structure of BI 2536; B) Crystal structure of
PLK1 in complex with BI 2536 (PDB ID: 2RKU).[20]
disruption of cytokinesis, and apoptosis.[18] BI 2536 exhibits
over 10000-fold selectivity towards PLK1 as compared to 63
other kinases and only minimal activity against the closely
related kinases PLK2 and PLK3.[17,18] BI 2536 has been tested
on numerous human cancer cell lines in vitro, and confirmed
in xenograft models showing prominent anti-cancer activ-
ity.[18]
Based on the implications of PLK1 in human tumors, the
clinical development of PLK1 inhibitors and the inability to
visualize the target readily in vivo, we sought to develop
small-molecule PLK1 imaging agents based on BI 2536
scaffold, using a modular bioorthogonal approach. Specifi-
cally, we modified a BI 2536 precursor with trans-cyclooctene
(TCO) which can then react bioorthogonally with tetrazine
(Tz) moieties to rapidly test and develop lead candidates into
optical or isotope based imaging agents.[19] Here we report the
first use of BI 2536-TCO as an imaging agent for PLK1 in live
cells and as a tool to quantify protein expression levels.
The crystal structure of PLK1 in complex with BI 2536
(PDB ID 2RKU)[20] shows that the N-methylpiperazine
residue does not participate in ligand–protein interaction
and is oriented towards the solvent (Figure 1B). Based on the
crystal structure of PLK1 and BI 2536, we reasoned that a
modification on the aromatic carboxylic acid of BI 2536
should not majorly affect the affinity of the drug for PLK1.
BI 2536-TCO was synthesized in eight steps with an
overall yield of 4% (Scheme 1).[21] Briefly, esterification of
the commercially available d-2-aminobutyric acid followed
by a reductive amination with cyclopentanone afforded
compound 2 in 97% yield. The secondary amine was reacted
with 2,4-dichloro-5-nitropyrimidin to form 3. The reduction of
[*] Dr. G. Budin,[+] Dr. K. S. Yang,[+] Dr. T. Reiner, Prof. R. Weissleder
Center for Systems Biology, Massachusetts General Hospital
185 Cambridge Street, Boston, MA 02114 (USA)
E-mail: rweissleder@mgh.harvard.edu
Prof. R. Weissleder
Harvard Medical School
200 Longwood Avenue, Boston, MA 02115 (USA)
[+] These authors contributed equally to this work.
[**] This work was supported by the National Institutes of Health (NIH)
grant number RO1EB010011, K.S. was supported by a NIH grant
T32-CA79443 and T.R. was supported by a grant from the German
Academy of Sciences Leopoldina (LPDS 2009-24). We thank Joshua
Dunham and Alex Zaltsman for image processing, and Dr. Robert
Yang and Prof. Peter Sorger for assistance with cellWoRx and Image
Rail.
Supporting information for this article is available on the WWW
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ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2011, 50, 9378 –9381