Tethering small molecules to a phage display library: Discovery of a selective bivalent inhibitor of protein kinase A

Article abstract of DOI:10.1021/ja076197d

We report a noncovalent tethering methodology for the fragment-based selection of bivalent ligands targeting protein kinases. In this approach, a small-molecule warhead, staurosporine, directs a phage display cyclic peptide library to the active site of cAMP-dependent protein kinase (PKA), allowing for targeted library enrichment. A cyclic peptide discovered through this selection, when covalently attached to a staurosporine derivative, displayed a 90-fold increase in affinity for PKA. Moreover, the bivalent inhibitor was shown to be significantly more selective than the starting warhead when tested against a small panel of kinases. Thus our general methodology allows for covalent linkage of known small-molecule ligands to biological libraries for discovering potent bivalent inhibitors of biological targets. Copyright

Full text of DOI:10.1021/ja076197d

Published on Web 10/18/2007
Tethering Small Molecules to a Phage Display Library: Discovery of a
Selective Bivalent Inhibitor of Protein Kinase A
Scott C. Meyer, Carolyn D. Shomin, Thomas Gaj, and Indraneel Ghosh*
Department of Chemistry, UniVersity of Arizona, Tucson, Arizona 85721
Received August 16, 2007; E-mail: ghosh@email.arizona.edu
There is much current interest in developing fragment-based
strategies for targeting protein surfaces and active sites.1 Toward
this goal, we describe a new fragment-based bivalent ligand
selection methodology that allows for the discovery of protein
surface-targeted cyclic peptides that are steered by an active site
binding small-molecule ligand (Figure 1). This approach allows
for coupling high-affinity but promiscuous ligands to the large
chemical space encompassed by biological libraries. Here, we
demonstrate the feasibility of this approach by converting the
nonselective kinase inhibitor, staurosporine, into a selective inhibitor
of the cAMP-dependent protein kinase (PKA).
With over 500 members in the human genome, protein kinases
comprise an important class of enzymes, and their deregulation is
often implicated in various disease states.2 Thus, selective inhibition
of protein kinases has the potential to provide useful reagents and
therapeutic leads.3 Most small-molecule inhibitors of kinases target
the conserved ATP binding pocket, often displaying high affinity.
While this has produced therapeutically useful inhibitors, they are
often promiscuous.4
Figure 1. Bivalent inhibitor selection strategy: (A) Noncovalent tethering
of staurosporine with a phage-display peptide library through a coiled-coil
heterodimer for targeting a kinase, PKA. The selected peptide can be
subsequently conjugated to staurosporine to provide a bivalent kinase
inhibitor. (B) Staurosporine derivative 2 conjugated to the Jun. (C) Phage-
displayed cyclic peptide library attached to Fos (X ) any of the 20 natural
amino acids). (D) Selected cyclic peptide 3.
An elegant strategy for increasing affinity and selectivity is the
development of bisubstrate analogue inhibitors,5a where a peptide
analogue of the kinase’s protein substrate is covalently attached to
an ATP-competitive small molecule.5b-f Cole and co-workers
demonstrated the feasibility of this approach against the insulin
receptor protein tyrosine kinase (IRK).5c In another example,
Schepartz and co-workers5f improved selectivity by utilizing
structurally constrained peptide substrates conjugated to the stau-
rosporine-like natural product, K252a. Though elegant, a potential
drawback of the aforementioned bisubstrate design approaches is
that they necessarily rely on structural information as well as prior
knowledge of a specific peptide substrate for the targeted kinase.
Many kinases of biological or pharmacological interest do not
satisfy the above criteria and therefore lie outside of the scope of
these design strategies. We envisioned that an in vitro bivalent
selection approach that combined small-molecule targeting with
biological selection would have the potential to complement design
strategies and provide selective bivalent inhibitors without prior
structural or peptide substrate information.
Two previous selection efforts have coupled large biologically
derived peptide libraries to synthetic molecules utilizing the
orthogonal chemistry of cysteine residues.6 This chemistry, though
very promising, can have low yields and potentially suffers from
nonspecific reactivity in the presence of the library platform (such
as phage particles or ribosomes) as well as cysteine residues within
the library being interrogated. With this in mind, we describe a
noncovalent tethering strategy that would select for cyclic peptides
from a phage display library7 directed toward the active site of a
protein kinase by an ATP-competitive small molecule. The small
molecule would be functionally tethered to the library by non-
covalent self-assembly mediated by a coiled-coil heterodimer that
has been previously utilized for numerous designs8 (Figure 1). This
warhead-guided phage display selection would potentially afford
cyclic peptide motifs with affinity for the kinase surface adjacent
to the active site. The selected peptides could be subsequently
conjugated to the small-molecule warhead to afford a bivalent
inhibitor with the potential for increased affinity and enhanced
selectivity for the targeted kinase.
To test this new approach, we chose the much studied ATP-
competitive kinase inhibitor, staurosporine 1, as our promiscuous
kinase targeting warhead. The carboxylated derivative of stauro-
sporine 2 (Figure 2A) with a reported IC50 value 47-fold weaker
than staurosporine9 1 was tethered to Jun (Figure 1B) and retained
its ability to inhibit PKA (Supporting Information, Figure S1). Next,
a phage-displayed peptide library7 was constructed adjacent to the
Fos domain, which heterodimerizes with Jun. The library incor-
porated two conserved Cys residues flanking the six positions of
diversity, each incorporating all 20 amino acids (Figure 1C).7b
With the library in hand, selection was carried out against PKA.
In each round of selection, the 2-Jun conjugate was mixed with
the phage-displayed library conjugated to Fos and exposed to
immobilized PKA. After six rounds of selection, several peptides
were isolated and characterized for inhibiting PKA. The cyclic
peptide CTFRVFGC was the most abundant sequence identified
and was also found to be the most active in initial assays against
PKA, with IC50 values in the mid-micromolar regime (Supporting
Information, Figure S5). The successful selection of the cyclic
peptides highlights the important distinction from previous ap-
proaches in that our bivalent tethering approach required no prior
knowledge of the structure of the kinase or the chemical structure
of the protein substrate.5
Having established that active-site-directed selection was indeed
possible, we turned toward testing our central hypothesis: that our
strategy could be used to discover covalently linked bivalent
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J. AM. CHEM. SOC. 2007, 129, 13812-13813
10.1021/ja076197d CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
sporine: ASK1 ) 120 nM, CaMKIIâ ) 3 nM, cSrc ) 100 nM,
EphA5 ) 150 nM, and Mnk2 ) 22 nM), their availability, and
their wide distribution across the human kinome.2 Selectivity assays
were carried out for 2 and 4 at a fixed concentration of 100 nM.
The inhibition results clearly demonstrate that the bivalent inhibitor
4 significantly reduces kinase activity only for PKA (Figure 2C)
when compared to the parent inhibitor 2. Structural alignments of
the five kinases mapped onto the crystal structure of PKA indicate
that, though the active sites are quite similar, there are a number
of dissimilar surface sites that could be responsible for imparting
specificity to the bivalent inhibitor (Supporting Information, Figure
S7). Interestingly, the attachment of our cyclic peptide was observed
to decrease the activity of the parent inhibitor 2 in the case of
CaMKIIâ and cSrc.
Thus, our results confirm that the phage-display-selected peptide
3 can impart increased affinity and selectivity to the small-molecule
ligand 2. This tethering strategy therefore holds the potential to
identify inhibitors of protein kinases with increased selectivity, while
relying minimally on structural or substrate information for a given
target. Future studies will aim to clarify mode of inhibition, obtain
structural information, study methods for cellular delivery, and apply
this approach to pharmacologically relevant kinase targets. More
generally, the ability to incorporate user defined synthetic warheads
during in vitro selection may extend this bivalent ligand discovery
strategy to numerous biological targets as well as selection platforms
for increasing ligand affinity, and more importantly, selectivity.
Acknowledgment. The authors thank the NSF (CHE-0548264)
and NIH (R01AI068414) for partial support of this research. We
thank Ghosh lab members for helpful discussions.
Supporting Information Available: Experimental details and the
complete citations for refs 1a and 4b. This material is available free of
charge via the Internet at http://pubs.acs.org.
Figure 2. Inhibitory activity and selectivity of bivalent PKA inhibitor: (A)
Chemical structures of warhead 2, selected cyclic peptide 3 conjugated to
2 to afford bivalent inhibitor 4. (B) Inhibition of PKA (2 nM) with 2, 3,
and 4. (C) An activity screen (PKA, ASK1, CaMKIIâ, cSrc, EphA5, and
Mnk2) shows significantly increased inhibition at 100 nM by the selected
bivalent inhibitor 4 compared to 2 only for PKA.
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