Light-mediated liberation of enzymatic activity: Small molecule caged protein equivalents

Article abstract of DOI:10.1021/ja803395d

Light-activatable ( caged ) proteins have been used to correlate, with exquisite temporal and spatial control, intracellular biochemical action with global cellular behavior. However, the chemical or genetic construction of caged proteins is nontrivial, with subsequent laborious introduction into living cells, potentially problematic competition with natural endogenous counterparts, and challenging intracellular incorporation at levels equivalent to the natural enzymes. We describe the design, synthesis, and characterization of small molecular equivalents of a caged Src kinase. These compounds are easy to prepare and function by inhibiting the action of the natural unmodified enzyme. Copyright

Full text of DOI:10.1021/ja803395d

Published on Web 07/19/2008
Light-Mediated Liberation of Enzymatic Activity: “Small Molecule” Caged
Protein Equivalents
Haishan Li,†,‡ Jung-Mi Hah,†,§ and David S. Lawrence*,†,‡
Department of Biochemistry, The Albert Einstein College of Medicine, 1300 Morris Park AVenue,
Bronx, New York 10461, and The Departments of Chemistry, Medicinal Chemistry & Natural Products, and
Pharmacology, Kenan Laboratories, The UniVersity of North Carolina, Chapel Hill, North Carolina 27599
Received May 7, 2008; E-mail: lawrencd@email.unc.edu
Protein kinases serve as key participants in signaling pathways by
catalyzing phosphoryl transfer from ATP to the serine, threonine, and/
or tyrosine residues of protein substrates. The eventual cellular response
to a stimulus is often dependent upon when and where a particular
kinase is activated within the cell. Consequently, the use of constitu-
tively active protein kinases, or various defective mutants thereof, can
only partly address the relationship between the action of specific
signaling enzymes and ensuing cellular behavior. The creation of
“caged” protein kinases, enzymes whose activities can be rapidly
unleashed upon exposure to light, has been reported.1 The photostimu-
lation of caged proteins inside living cells has also been described,
which provides exquisite control over where and when the enzyme is
activated.2 However, there are many challenges associated with the
application of caged proteins3 to cellular systems. First, chemically
modified proteins are primarily introduced into cells via microinjection,
a process that limits subsequent behavioral studies to single cell
analysis. Second, enzymes often suffer a series of post-translational
modifications during their intracellular lifetime. These modifications
can be difficult to replicate in large-scale bacterial enzyme preparations,
which are often necessary for the acquisition of sufficient quantities
for caging experiments. Third, the intracellular introduction of unnatural
enzymes, whether by microinjection or genetic expression, is unlikely
to recapitulate the normal endogenous levels of the wild-type enzyme.
Finally, the action of the caged protein might be difficult to interpret
in the presence of its natural intracellular counterpart. We describe a
conceptually different strategy for the acquisition of light-activated
enzymes, an approach that targets the endogenous enzyme and
therefore has the potential to circumvent the limitations outlined above.
We recently reported the design and construction of a potent
peptide-derived inhibitor 1 (Ki ) 26 ( 4 nM; IC50 ) 36 ( 2 nM)
for the Src tyrosine kinase.4 Src comprises three domains, which
play various roles in catalysis (SH1), regulation (SH2 and SH3),
or substrate recognition (SH1, SH2, and SH3; Figure 1).5 Com-
pound 1, a “bivalent” species, simultaneously associates with the
active site (SH1 domain) and the SH2 domain. A key feature of
Figure 1. A photocleavable caged protein equivalent.
for their respective binding sites on the target protein. The latter
property furnishes an inhibitor with a Src kinase affinity that
significantly exceeds (∼50-fold) the affinities of the individual
components alone (e.g., SH1 domain-targeted monovalent inhibitor
2; IC50 ) 1.9 ( 0.3 µM; Table 1).4 We reasoned that a bivalent
inhibitor, containing a photolabile site positioned at or near the
ꢀ-Ala tether, would retain its ability to inhibit the target enzyme
(Figure 1). However, photolysis should split the inhibitor and
thereby curtail its effectiveness. Such a photodeactivatable inhibitor,
which is functionally equivalent to a photoactivatable Src kinase,
can potentially address the challenges associated with caged proteins
outlined above.
We prepared a small collection of bivalent inhibitors (Table 1),
each of which contains one of three different photocleavable groups
(I-III). In order to ensure the release of enzymatic activity upon
photolysis, the bivalent analogue must be a potent inhibitor relative
to compound 2. The photocleavable group III furnished the most
powerful prephotolysis inhibitor (6) in the series 4-6. We also
Table 1. Stable (1 and 2) and Photolytically Sensitive (4-11) Src
Inhibitors
#
peptide
IC50 (nM)
1
2
4
5
6
7
8
9
Ba-EEEIFGEF-Dap(Hna)-ꢀA3-pYEEIEX
35 ( 2
1900
Ba-EEEIFGEF-Dap(Hna)X + AcpYEEIE-NH2 (3)
Ba-EEEIFGEF-Dap(Hna)-ꢀA-I-ꢀA-pYEEIEX
Ba-EEEIFGEF-Dap(Hna)-ꢀA-II-ꢀA-pYEEIEX
Ba-EEEIFGEF-Dap(Hna)-ꢀA-III-ꢀA-pYEEIEX
Ba-EEEIFGE-II-Dap(Hna)-ꢀA3-pYEEIEX
115 ( 9
38 ( 4
18 ( 5
220 ( 10
28000
7500
1600
56 ( 4
Ba-EEEI-III-GEF-Dap(Hna)-ꢀA3-pY-III-EIEX
Ba-EEEIFGE-III-Dap(Hna)-ꢀA3-pY-III-EIEX
Ba-EEEIFGEF-Dap(Hna)-ꢀA-III-ꢀA-pY-III-EIEX
Ba-EEEIFGEF-Dap(Hna)C-III-ꢀA-pYEEIE-NH2
10
11
the inhibitor is that the SH1- and SH2-directed components, which
are linked via a ꢀ-Ala tether, exhibit approximately equal affinities
† The Albert Einstein College of Medicine.
‡ The University of North Carolina.
§ Current address: Biomaterials Center Life Science Division, Korea Institute
of Science and Technology, P.O. Box 131, Cheongryang, Seoul, 130-650 Korea.
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10.1021/ja803395d CCC: $40.75
2008 American Chemical Society

C O M M U N I C A T I O N S
Figure 3. Presence of Src kinase on (A) nonirradiated and (B) irradiated
UltraLink beads modified with bivalent inhibitor 11 as assessed by exposure
to an Alexa Fluor488-labeled monoclonal Src antibody.
Figure 2. Enzymatic activity (∆F vs time) as a function of irradiation
time of compound 6 (1.9 µM). Assays were performed as previously
described.7 Experimental conditions: A (0 min hν), B (2 min hν), C (15
min hν), D (120 min hν), and E (0 min hν + 1.9 µM compound 2).
slowly diffusible via anchoring to relatively immobile substrates
(e.g., proteins, organelles, etc.) using targeting substituents (e.g.,
amino acid sequences, fatty acids, etc.). With this in mind, we
explored the following question: can an analogue of 6 be spatially
affixed to a high molecular object and still function in a light-
sensitive fashion?
synthesized several peptides that have two photocleavable sites
(8-10) using, as a guiding principle, the notion that double
photolytic cleavage should destroy all inhibitory activity. Unfor-
tunately, these compounds proved to be exceedingly poor inhibitors.
Presumably the additional photosensitive site disrupts SH1 binding
(8 and 9) and/or SH2 targeting (8-10). With these features in mind,
6 was selected for further study.
We first addressed whether 6 simultaneously associates with the
active site and SH2 regions as envisioned (Figure 1). Ac-
pTyrGluGluIle-amide is a validated SH2 domain ligand with a KD
of 1-5 µM.6 Inhibitory efficacy of 6 is progressively and
significantly compromised as a function of SH2 ligand concentration
(e.g., IC50 ) 325 ( 60 nM at 320 µM Ac-pTyrGluGluIle-amide),
the expected result for an SH2 domain-dependent, active-site-
targeted inhibitor (Supporting Information).
Src kinase activity is completely blocked in the presence of 1.9
µM 6 (Figure 2). Photolysis of compound 6 releases kinase activity
as measured by a previously described real time fluorescence assay.7
Furthermore, longer photolysis times generate higher rates of
substrate phosphorylation. This response is precisely analogous to
the direct photoactivation of a caged enzyme, in which a photo-
cleavable moiety is removed from a key residue required for
activity. In both instances, enhanced photolysis times create larger
quantities of active enzyme. Under optimized conditions, photolysis
restores up to 90% of the activity displayed by Src in the presence
of compound 2 (1.9 µM; i.e., the byproduct of photolysis) or up to
50% of Src activity in the absence of compound 2 (Supporting
Information). These results compare favorably with those obtained
for previously described caged protein kinases1 and phosphatases.8
An analogous series of experiments were performed using lysates
from COS-1 cell overexpressing wild-type Src kinase (Supporting
Information).
Light-mediated activation of caged inhibitors, activators, and
enzymes allows the investigator to control when the agent of interest
is switched on. In addition, light exposure time, intensity, or the
number of laser pulses provides a means to control the amount of
active species generated. By contrast, spatial control of activity (i.e.,
localized release) via spot illumination is generally reserved for
high molecular weight species with slow diffusion rates, such as
proteins.2 Small molecules are unlikely candidates for experimen-
tally meaningful localized release since they commonly exhibit high
diffusion rates. Nevertheless, small molecules can be rendered
Bivalent peptide 11, which contains a Cys-for-ꢀAla substitution
in the tether region, serves as an effective Src kinase inhibitor (Table
1). Compound 11 was covalently attached to Pierce UltraLink beads
and incubated with varying concentrations of Src kinase to generate
a standard binding curve (Supporting Information). Illumination
affords approximately 50% nonbound active enzyme as assessed
by two criteria. First, photolysis affords 45 ( 4% Src kinase activity
in the supernatant (i.e., following removal of beads), as assessed
using a real time fluorescence assay (Supporting Information).
Second, 50 ( 4% of Src kinase is physically retained by the beads
following photolysis (i.e., 50% is present in solution), as determined
by fluorescent imaging (Figure 3). These results are consistent with
solution studies described above and support the notion that protein
activity and location can be controlled with light using appropriately
designed inhibitory species. Application to cell-based systems is
underway.
Acknowledgment. We acknowledge financial support by the
NIH (CA079954).
Supporting Information Available: Experimental details of peptide
synthesis, structure, characterization, and photolytic behavior. This
material is available free of charge via the Internet at http://pubs.acs.org.
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