Bcr-Abl inhibitors targeted to a domain interface 1 Allosteric Inhibition of Bcr-Abl Kinase by High

24 Pages·2016·8.62 MB·English

by  Shohei Koide

JBC Papers in Press. Published on February 24, 2016 as Manuscript M115.707901
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M115.707901
Bcr-Abl inhibitors targeted to a domain interface
Allosteric Inhibition of Bcr-Abl Kinase by High-Affinity Monobody Inhibitors Directed to the SH2-
Kinase Interface

John Wojcik,a Allan Joaquim Lamontanara,b Grzegorz Grabe,a,c Akiko Koide,a Louesa Akin,a
Barbara Gerig,b Oliver Hantschel,b* and Shohei Koidea*

From a Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL,
USA. b Swiss Institute for Experimental Cancer Research, School of Life Sciences, École polytechnique
fédérale de Lausanne, Lausanne, Switzerland. c Intercollegiate Faculty of Biotechnology, University of
Gdansk and Medical University of Gdansk, Gdansk, Poland.

Running title: Bcr-Abl inhibitors targeted to a domain interface

*Correspondence should be addressed to Prof. Shohei Koide, email: [email protected], or Prof.
Oliver Hantschel, email: [email protected].
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Keywords: ABL tyrosine kinase; enzyme inhibitor; protein-protein interaction; Src homology 2 domain nlo
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(SH2 domain); X-ray crystallography; PPI inhibitor; protein engineering; FN3 d fro
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ABSTRACT work provides strong evidence for the SH2-kinase w
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Bcr-Abl is a constitutively active kinase that interface as a pharmacologically tractable site for .jb
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causes chronic myelogenous leukemia (CML). allosteric inhibition of Bcr-Abl. .org
We have shown that a tandem fusion of two by/
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designed binding proteins, termed monobodies, Mutations conferring drug resistance are ue
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respectively directed to the interaction interface frequently observed in cancer and infectious t on
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pbhetowspeehno tythroe sSinHe-2b iannddin kgi nsaitsee odfo mthaei nSs Ha2n dd otom tahine dtairsgeeatseeds dfroulglso w(1in,2g) . Ttroea otmveerncto moef repsaitsiteanntcse , witi tihs pril 6, 2
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inhibits the of Bcr-Abl kinase activity. Because necessary to develop a therapeutic strategy that is 1
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the latter monobody inhibits processive based on a mechanism of action distinct from that
phosphorylation by Bcr-Abl and the SH2-kinase of the first-generation drug. Bcr-Abl is a fusion
interface is occluded in the active kinase, it protein of the breakpoint cluster region (BCR) and
remained undetermined whether targeting the the Abelson tyrosine kinase (ABL1) generated by
SH2-kinase interface alone was sufficient for Bcr- the Philadelphia chromosomal translocation (3).
Abl inhibition. To address this question, we Bcr-Abl is a constitutively active kinase that is
generated new, higher-affinity monobodies, with sufficient for the initiation and maintenance of
single nanomolar K values, targeting the kinase- CML. Although small-molecule ATP-competitive
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binding surface of SH2. Structural and Bcr-Abl inhibitors such as Imatinib (Gleevec) and
mutagenesis studies revealed the molecular its successors have dramatically improved overall
underpinnings of the monobody-SH2 interactions. survival of CML patients, the emergence of
Importantly, the new monobodies inhibited Bcr- resistance mutations renders kinase inhibitors
Abl kinase activity in vitro and in cells, and they ineffective in subsets of CML patients (4,5).
potently induced cell death in CML cell lines. This Importantly, a recently developed allosteric Bcr-
1
Copyright 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
Bcr-Abl inhibitors targeted to a domain interface
Abl inhibitor targeting the myristoyl binding Bcr-Abl dependent oncogenic transformation of
pocket can strongly limit the emergence of drug mouse bone marrow cells by inhibiting Bcr-Abl
resistance to ATP-competitive inhibitors and have kinase activity and induced apoptosis in human
entered clinical trials (6), highlighting the interest cells isolated from CML patients (9). These results
and strong demand for new therapeutic strategies supported the notion that the SH2-kinase interface
even in the case of a well-established target like is a potentially druggable site.
Bcr-Abl (5). Although the tandem monobody construct
Bcr-Abl has multiple protein domains was an effective allosteric Bcr-Abl inhibitor, it
including the SH2 and tyrosine kinase domains remained unclear whether targeting the SH2-
originating from Abl1 (Figure 1A) (7). Extensive kinase interface alone was sufficient for Bcr-Abl
mechanistic and structural studies have revealed inhibition, because the HA4 monobody in
the intramolecular interaction between the SH2 isolation also affected downstream signaling (13).
and kinase domains to be critical for full kinase HA4 inhibits the interaction of Abl SH2 with its
activity (Figure 1B). Consequently, a mutation that phospho-Tyr-containing ligands and consequently
disrupts this interface, I164E, inhibits enzyme processive phosphorylation of Bcr-Abl substrates
activity and Bcr-Abl mediated oncogenesis (8,9). (13). Furthermore, the long linker and second D
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These results suggest that the SH2-kinase interface monobody in the tandem monobody construct n
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is a potentially "druggable" site for allosteric substantially increased the complexity of the ad
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inhibition of the kinase activity. molecule, diminishing the benefit of monobodies fro
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In our previous work, to determine the as compact protein reagents. In this work, we h
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feasibility of disrupting the SH2-kinase interface aimed to develop high-affinity monobodies ://w
in trans, we utilized synthetic binding proteins, directed to the SH2-kinase interface to w
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termed "monobodies", to target this interface (9). unambiguously determine whether Bcr-Abl could .jb
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Monobodies are binding proteins developed from be inhibited by targeting solely this interface. We rg
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combinatorial libraries constructed on the generated new monobodies and characterized their y
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molecular scaffold of a fibronectin type III domain structure and recognition mechanism. These es
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(FN3) (10-12). Because monobodies are stable monobodies, used as single-domain reagents, n A
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and, unlike antibody fragments, do not contain a inhibited the in vitro and cellular activity of Bcr- ril 6
disulfide bond, they are well suited as genetically Abl and strongly decreased survival of CML cells. , 2
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encoded inhibitors of an intracellular target. We The results presented here establish the sufficiency 9
successfully generated a monobody termed "7c12" of pharmacologically targeting the SH2-kinase
directed to the kinase-binding surface of the Abl interface for the allosteric inhibition of Bcr-Abl.
SH2 domain (9). Because the 7c12 monobody had
only moderate affinity and hence moderate EXPERIMENTAL PROCEDURES
biological effects in vitro and in cells, we needed Protein expression and purification––The
to fuse it with another monobody, termed HA4, Abl SH2 domain and monobodies were produced
binding to a different surface of the SH2 domain, with an N-terminal tag containing His10, FLAG
namely the binding site for phospho-Tyr- and TEV-recognition motifs using the pHFT2
containing ligands (13). This tandem fusion vector (14). The I164E mutation was introduced
approach enhanced the effective affinity so that using the Kunkel mutagenesis method (15).
the HA4-7c12 fusion could successfully compete Proteins were expressed in E. coli BL21(DE3) and
with the intramolecular interaction between the purified to apparent homogeneity using nickel
SH2 and kinase domains (Figure 1C). Expression affinity chromatography and size exclusion
of this tandem monobody construct suppressed
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Bcr-Abl inhibitors targeted to a domain interface
chromatography (Superdex 75, GE Healthcare GG3/Abl SH2 complexes were purified with a
Life Sciences). Superdex 75 column (GE Lifesciences). The
cDNAs encoding for human ABL1 (Abl complexes were concentrated to ~7.5mg/ml
kinase domain (KD): residues 248-534, Abl SH2- (AS25/Abl SH2) and 10 mg/ml (GG3/AblSH2),
kinase domain (SH2-KD): residues 138-534; mixed 1:1 with a well solution containing 0.1M
spliceform 1b numbering) were cloned into the Imidazole pH 7.8 and 3.5M NaCl (k7_4), 0.1M
NheI and XhoI restriction sites of pET-21d (Merck Imidazole pH 8.5 and 3.4M NaCl (k7_8) or 0.1M
Millipore). Proteins were co-expressed with the sodium tartrate pH=8 and 25% w/v polyethylene
YopH phosphatase in E. coli BL21(DE3). Protein glycol 3350 (GG3/Abl SH2), and crystallized
purification was carried using the C-terminal using the hanging-drop vapor-diffusion method.
hexahistidine tag by nickel affinity Glycerol (20%) was used as a cryoprotectant in all
chromatography and further purified by anion cases. X-ray diffraction data were collected at the
exchange chromatography in 150 mM NaCl, 20 Advanced Photon Source, beamlines 24 ID-C
mM Tris-HCl pH 7.5, 5% Glycerol and 1 mM (AS25/AblSH2 complexes) and 24 ID-E
DTT as described (16). (GG3/Abl SH2) at a wavelength of 0.97872 Å, a
Monobody generation and temperature of 100 K. Data collection and D
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characterization––General methods for phage- structure determination statistics are given in n
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and yeast-display library sorting and gene Table 1. Diffraction data were processed and ad
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shuffling have been described (11-13). The scaled with the HKL2000 package (18). The fro
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monobody libraries used have been reported (11). structures were solved by molecular replacement h
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In phage-display library sorting, target proteins using Phaser in the CCP4 program suite (19,20). A ://w
were immobilized using a high-affinity ligand for multicopy search was performed with the Abl SH2 w
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the His tag (17). The GG3 and GG10 domain and the FN3 scaffold, without the loop .jb
10 c
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monobodies were isolated after four rounds of regions, as the search models (PDB ID's 2ABL rg
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phage-display selection using the target and 1FNA, respectively). Simulated annealing, y
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concentrations of 250, 100, 100 and 100 nM for energy-minimization, B-factor refinement and es
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the first through fourth rounds in the presence of a map building were out using CNS (21,22). Rigid- n A
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ten-fold excess of the I164E mutant SH2 domain body refinement was carried out using REFMAC5 ril 6
so that monobodies binding to the wild type but in the CCP4 program suite. TLS , 2
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not the mutant are predominantly retained. (translation/libration/screw) groups were defined 9
Isolation of AS25 and AS27 involved additional using the TLSMD server (23), and TLS
steps utilizing yeast surface display and has been refinement, B-factor refinement, bulk solvent
reported (11). Combinatorial libraries for affinity parameters, final positional refinement, and the
maturation of AS25 were constructed in the yeast search for and refinement of water molecules was
surface display format. Binding measurements in carried out using REFMAC5. Model building and
the yeast display format were performed using a evaluation were carried out using the Coot
Millipore Guava flow cytometer as described program (24), and molecular graphics were
previously (11,12). The dissociation constants generated using PyMOL (DeLano Scientific).
determined from the yeast display format agreed Surface area calculations were performed using
closely to those determined using purified the PROTORP protein–protein interaction server
monobody samples with surface plasmon (25). Shape complementarity was determined
resonance (11). using the Sc statistic (26).
Crystallization, data collection and NMR spectroscopy––Chemical shift
structure determination––The AS25/Abl SH2 and assignments for the Abl SH2 domain were from a
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Bcr-Abl inhibitors targeted to a domain interface
previous work (11). 15N HSQC spectra were taken cell signaling); anti-Myc (Rockland, Rabbit) and
with15N-enriched Abl SH2 domain (~90 µM) anti-pY 4G10 (in-house, Mouse).
alone and in the presence of unlabeled Autophosphorylation levels in the activation loop
monobodies (AS25, AS27, GG3, GG10). All (Y412) were quantified using a Li-Cor Odyssey
assigned peaks from the Abl SH2 domain were system.
classified based on the degree of peak shift Retroviral transduction and FACS
according to a procedure described previously analysis of K562 cells stably expressing
(14). monobodies––K562 cells stably-expressing N-
In vitro kinase assays––Five nanograms of TAP fused monobodies (AS25, AS27, HA4 and
recombinant Abl KD or SH2-KD protein were the non-target binding monobody HA4Y87A)
pre-incubated with the recombinant AS25 and were generated by retroviral gene transfer, as
AS27 monobodies at various concentrations or described previously (13). For six consecutive
buffer controls for 10 minutes at room temperature days starting 24 hours after the second retroviral
in a volume of 10 µL. Kinase activity was infection of the target K562 cells and every 24
determined by the addition of 20 µM ATP, 7 µCi hours, aliquots of 106 cells were removed from the
γ-32P-ATP and 75 µM of an optimal Abl substrate culture and were analyzed by flow cytometry D
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sequence carrying an N-terminal biotin (biotin- (Accuri C6, BD Biosciences). Cells were analyzed n
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GGEAIYAAPFKK-amide) in kinase assay buffer for GFP expression (that is co-expressed with the ad
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(20 mM Tris-HCl pH 7.5, 5 mM MgCl2, 1 mM TAP-tagged monobodies) and in parallel stained fro
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DTT, 30 µM BSA) for 20 minutes at room with Annexin V Cy5 (BD PharmingenTM, h
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temperature in a final assay volume of 20 µL. The product number 559933) and 7-AAD (BD ://w
terminated reaction (10 µL 7.5 M PharmingenTM, product number 559925). w
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guanidinhydrochlorid) was spotted onto a SAM2 .jbc
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Biotin Capture membrane (Promega) and further RESULTS rg
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treated according to the instructions of the Monobodies to the Abl SH2-kinase y g
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manufacturer. interaction interface––We identify monobodies es
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HEK293 cells transfection––HEK293 from combinatorial libraries in which positions in n A
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cells were cultured in Dulbecco’s Modified the FN3 scaffold are diversified. The goal is to ril 6
Eagle’s Medium supplemented with 10% fetal calf achieve both shape and chemical complementarity , 2
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serum and 1% penicillin/streptomycin. Cells were with the intended surface of the target molecule 9
co-transfected with Abl PP (27) or Bcr-Abl and (28). We have recently developed monobody
6xMyc tagged monobody expression vectors using libraries using a new design that presents
the Polyfect transfection reagent (Qiagen) and diversified amino acid positions in a concaved
following the supplier protocol. 48 hours after topography (Figure 2A and 2B) (11). This new
transfection, cells were harvested and lysed in IP library design complements our original design in
buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, which diversified residues are presented on a
1% NP-40, 5mM EDTA, 5mM EGTA, 25 mM convex surface made with three loops (BC, DE
NaF, 1mM orthovanadate, 1mM PMSF, 10 mg/ml and FG) located on one end of the scaffold
TPCK and protease cocktail inhibitor from Roche) (10,13). We reasoned that monobodies from the
and cleared by centrifugation. For immunoblot new design are better suited for targeting the
analysis 100 µg of cellular lysate was used. kinase-binding surface of the SH2 domain that is
Western blotting and quantification–– mostly flat and slightly convex.
Antibodies used for Western blotting include: anti- To enrich monobodies targeting the
Abl (Mouse, Sigma); anti-phospho-Y412 (Rabbit, intended surface of the SH2 domain, we first
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Bcr-Abl inhibitors targeted to a domain interface
employed a strategy that utilizes a ‘decoy’ CD loop in addition to the FG loop and the β-sheet
competitor, the I164E mutant in this case, for region (11) (Figure 2A and 2B). We reasoned that
eliminating those clones binding to surfaces the additional diversified residues in the CD loop
outside the desired epitope (Figure 1D). The I164E would provide increased surface area for
mutation is located in the kinase-interaction interaction with the targeted site. We also
surface of the SH2 domain and disrupts the SH2- incorporated gene shuffling and additional library
kinase interface thereby inhibiting kinase activity sorting using yeast surface display as reported
(Fig 1B). . The library used in this selection was previously (11), in an effort to increase the
an earlier version of the "loop-and-side" library coverage of the sequence space and improve the
reported previously (11), in which amino acid identification of high-affinity clones. We have
diversity was introduced into the FG loop and the produced high-affinity monobodies with K values
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β-strands of the scaffold (Figure 2A and 2B). in the low nanomolar range to several targets using
Two monobodies, GG3 and GG10 this strategy, including two to the Abl SH2
identified using this method (Figure 2A) bound to domain, AS25 and AS27 (11,30). Therefore, we
wild-type SH2 but not to the I164E mutant, as characterized these monobodies that had been used
expected. To better define the targeted epitope, we only for the purpose of assessing the performance D
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conducted epitope mapping by 15N-HSQC of the library in generating high-affinity n
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chemical shift perturbation using uniformly 15N- monobodies but had otherwise remained ad
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labeled Abl SH2 domain. This method detects uncharacterized (Figure 2A). AS25 and AS27 fro
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changes in the 15N-HSQC resonances of the 15N- bound the Abl SH2 domain with KD values of 35 http
labeled component, i.e. Abl SH2 in this case, that nM and 7.7 nM, respectively, as assessed by ://w
are affected by the binding of another component, binding titration performed in the yeast surface w
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i.e. a monobody (29). The effects of GG3 and display format (Figure 3A). Note that KD values .jbc.o
GG10 on Abl SH2 backbone amide resonances determined with the yeast surface display titration rg
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were nearly identical (Figure 3B). The residues generally agree with those from biophysical y g
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most greatly affected were located adjacent to measurements of purified proteins, such as SPR, es
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I164 and within the kinase-interacting surface, which is the case for AS25 and AS27 (11). n A
confirming that these monobodies interact with the 15N HSQC NMR epitope mapping pril 6
targeted epitope. While we were successful in revealed that AS25 and AS27 also bound to an , 2
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identifying monobodies interacting with the epitope predicted to overlap substantially with the 9
kinase-activating interface, the affinity of these kinase-interacting interface. The 15N HSQC
monobodies, as assessed by isothermal titration spectra of [U-15N]-SH2 domain in complex with
calorimetry was modest (K = 2.54 and 0.90 µM either monobody were largely superimposable
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for GG3 and GG10, respectively; data not shown) (Figure 3B), consistent with a highly similar
and weaker than that of the 7c12 monobody (K = epitope. This similarity was expected given their
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~50 nM) that failed to show strong biological closely related sequences including identical FG
effects on its own (9). This comparison suggested loops (Figure 2A). The HSQC peak of I164 of the
that the new monobodies would not be sufficiently Abl SH2 was greatly affected by the binding of
potent, though binding to the intended surface of either monobody. In binding assays, AS25 and
the SH2 domain. AS27 interacted with the I164E mutant >100-fold
Rather than engaging in extensive affinity more weakly than the wild type SH2 domain
maturation of GG3 and GG10, we next proceeded (Supplementary Figure 1E and data not shown),
with the selection of monobodies from a next- providing independent verification of the locations
generation library that incorporated diversity in the of these epitopes. Together, these data
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Bcr-Abl inhibitors targeted to a domain interface
demonstrate that we were able to identify high- Whereas GG3 and AS25 use similar
affinity monobodies that bound to the kinase- surfaces to interact with similar surfaces of the Abl
interaction surface of the Abl SH2 domain. SH2 domain, there are notable differences
Crystal structures of monobodies in between the two complexes. The positions of the
complex with Abl SH2––To better understand the β-sheet core of the monobody scaffold in these
mode of interaction of monobodies targeting the structures are related by an approximately 30°
kinase-interaction interface of Abl SH2, we rotation with I88, located at the N-terminal end of
determined crystal structures for the GG3/SH2 and the G strand, as the pivot point (Figure 5A). AS25
AS25/SH2 complexes and compared them with buries substantially larger surface areas in the
the structures of the Abl SH2-kinase domains and interface than GG3 (Table 2). This difference
the 7c12/SH2 complex (Figure 4; Table 1). The comes mainly from the positioning of the CD loop
AS25/SH2 complex was crystallized in two and the β-sheet region of the monobody scaffold.
distinct space groups (Henceforth referred to as It appears that the interactions afforded by the
crystals “A” and “B”). The Abl SH2 domain additionally mutated residues of AS25 brought the
maintained its structure in the GG3 and AS25 CD loop and the β-sheet region closer to the SH2
monobody complexes (RMSD <0.4Å for the domain in the AS25 complex than the GG3 D
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backbone atoms) and was also close to that in a complex (Figure 5A). The shape complementarity nlo
previously determined monobody complex (PDB: assessed with the Sc value (26) was similarly good ade
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3K2M) and multi-domain Abl segments for both structures (Table 2), indicating that the fro
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containing the SH2 domain (PDB: 1OPL, 1OPK interfaces are well packed. The GG3 complex had h
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and 2FO0). The two AS25/SH2 complex the highest Sc value, suggesting a better-packed, ://w
structures are nearly identical (RMSD of 0.567 Å concentrated binding interface. These interface ww
for all Cα atoms), with the only substantial characteristics are consistent with the different .jbc
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difference in the position of the CD loop, which is designs of monobody libraries from which the two brg/
folded inward in the "A" structure and reflected monobodies are derived. y g
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outward in the "B" structure. Both GG3 and AS25 In both structures the FG loop contributed est o
monobodies form a cradled binding surface substantially to the buried surface areas in the n A
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utilizing positions in both β-sheet and loop regions interface, with 311 Å2 and 303 Å2 of buried ril 6
(Figure 4C and 4D), as intended by the design of surface areas for GG3 and AS25, respectively , 2
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the library (Figure 2B). The epitopes of the (Table 2). However, except for a Val residue 9
monobodies are approximately centered on I164 of inserting its side chain into a small cavity of the
the Abl SH2 domain, the residue within the SH2- SH2 domain (Figure 5B and 5C), the locations and
kinase interface that we targeted in our “decoy” modes of interaction of the FG loops were
selection strategy (Figure 4C and 4D). I164 different between the two structures (Figure 5A).
interacts with the β-sheet regions of both In the AS25 complexes, the FG loop forms a
monobodies. These epitopes overlap substantially broad, flat surface in which all but one residue
with the epitope of the kinase domain (Figure 4A) (Y84) make contact with the SH2 domain (Figure
as well as that of 7c12 (Figure 4E), suggesting that 5B). By contrast, in the GG3 structure only five
these monobodies would be competitive inhibitors residues located in the C-terminal half of the FG
of the interaction between the SH2 and kinase loop make contact with the SH2 domain, but each
domain. These results also rationalize the strongly buries a large surface area (>50 Å2; Figure 4C).
decreased interaction of the I164E mutation with Together, the crystal structures confirm
these monobodies. that the monobodies target the intended surface of
the SH2 domain, and demonstrate that there are
6
Bcr-Abl inhibitors targeted to a domain interface
multiple, distinct solutions to the problem of between the two monobody/Abl SH2 complexes
recognizing the same surface of a target protein and thus the mutations did not form productive
even using a single scaffold. interactions in AS27. Although our structure-
Interface Energetics and affinity guided designs were successful in enhancing the
maturation of AS25––To assess the energetic affinity of AS25, the highest affinity achieved did
contributions of individual monobody residues not substantially exceed that of AS27. Thus, we
within the AS25/Abl SH2 interface, we performed used AS25 and AS27 in the functional assays
alanine-scanning mutagenesis. We constructed described below.
individual point mutants of a total of nine residues AS25 and AS27 bind and inhibit kinase
in AS25 that each bury more than 20 Å2 of surface activity of Bcr-Abl––To examine the effects of the
area in both crystal structures. The mutated monobodies on Bcr-Abl kinase activity, we first
residues are together responsible for >55% of total performed binding experiments to determine if
monobody surface area burial. We measured their AS25 and AS27 are able to compete with the SH2-
affinity to the Abl SH2 domain by SPR. The kinase domain interaction 'in trans'. We expressed
experiment identified four hot-spot residues, Y45, and purified fragments of Bcr-Abl, one containing
Y79, V80 and N83, where mutation to alanine the SH2-kinase domain unit (SH2-KD) and the D
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markedly reduced binding, corresponding to a other containing only the kinase domain (KD) as n
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ΔΔG ≥ 3.0 kcal mol–1 (Figure 5D). In addition, described recently (16). In addition, SH2-KD ade
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two residues (G76A and P87A) contributed proteins carrying a mutation that either disrupts or fro
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modestly to binding (ΔΔG = ~0.9 and 1.0 kcal increases the SH2-KD interaction, I164E in the h
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mol–1, respectively). These results provide further SH2 domain or T231R in the kinase domain, ://w
validation of the contacts observed in the crystal respectively, were purified (31,32). Size exclusion ww
structure and highlight energetic contributions chromatography experiments showed efficient .jbc
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from both the CD and the FG loops, consistent formation of AS25 complexes with the wild-type rg
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with the cradle-like design of the library (Figure and T231R-containing SH2-KD proteins, whereas y g
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2B). no binding of AS25 to KD or SH2-KD(I164E) was es
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We next enhanced the affinity of AS25 observed (Figure 3C-F). AS25 and AS27 bound n A
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using structure-guided design. We found that the SH2-KD protein with KD values of ~10 nM, as ril 6
R239 of the Abl SH2 domain located in the tested using monobodies displayed on yeast , 2
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interaction interface had no negative charges in its surface (Figure 3B). 9
vicinity (Figure 5E). The T39D mutation in the C- To determine whether AS25 or AS27
strand of AS25 enhanced the affinity ~8-fold to a binding to the SH2-kinase interface also results in
K value of 2.5 (±2) nM (Figure 5E), probably by inhibition of kinase activity, we performed in vitro
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forming electrostatic interactions with R239. kinase assays with recombinant SH2-KD and KD.
Similarly, from a small combinatorial library, we We observed substantial decreases in the in vitro
identified a double mutant, S89Q/N91D, that had kinase activity of SH2-KD with these monobodies
even higher affinity with a K value of 1.3 (±0.2) in a dose-dependent manner, whereas KD alone
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nM (~15-fold enhancement). Interestingly, the was not inhibited (Figure 6A). At the highest
S89Q/N91D mutations only marginally enhanced monobody concentrations tested, the level of SH2-
(~4 fold) the affinity of AS27 and T39D had no KD activity was the same as that observed for KD.
significant effect (data not shown). The inability to This degree of decrease in kinase activity is
transfer the positive effects of these mutations on similar to that by the I164E mutation on full-
AS25 affinity to AS27 suggest that the local length Bcr-Abl, a mutation that disrupts the SH2-
structure near R239 is substantially different kinase interface (9). In line with our previous data
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Bcr-Abl inhibitors targeted to a domain interface
with the HA4-7c12 fusion monobody and other the phosphorylation of Abl Y412 located in the
allosteric Bcr-Abl inhibitors (9,33), higher activation loop of the kinase, a marker for the
concentrations of these inhibitors are needed to activated Bcr-Abl kinase (Figure 6B). The degree
impact on full-length Bcr-Abl in vitro kinase of inhibition was greater than that achieved with
activity than would be expected from binding the previous monobody, 7c12, and approached that
affinity measurement to the allosteric by the interface-disrupting mutation, I164E. These
pocket/domain. These data strongly suggest that results strongly suggest that these new
the AS25 and AS27 monobodies inhibit Abl monobodies potently inhibit Bcr-Abl in cells.
kinase by binding to the targeted surface of the Similar results were obtained with constitutively
SH2 domain and thereby disrupting the SH2- active full-length Abl (Figure 7C)
kinase interface, as designed. Finally, we tested if the inhibition of Bcr-
AS25 and AS27 inhibit Bcr-Abl activity in Abl kinase activity that we observed in vitro and
cells and inhibit CML cell survival––To determine in cells impacted on survival of the CML cell line
whether AS27 is sufficiently specific to be a useful K562. Growth and survival of K562 cells is
tool in the cellular context, we first examined its exquisitely dependent on Bcr-Abl kinase activity
ability to capture endogenous Bcr-Abl from cell and signaling (34). We attempted to constitutively D
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lysates. In this assay, a biotinylated recombinant express AS25 or AS27 in K562 cells by retroviral n
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AS27 protein was incubated with lysate from the gene transfer. Despite our repeated trials and in ad
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Bcr-Abl expressing cell line K562, and, following contrast to 7c12 and several other monobodies fro
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the addition of streptavidin-coated beads and serial (9,13,30), we were unable to detect AS25 or AS27 h
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washing, bound Abl and Bcr-Abl was detected monobody expression by immunoblotting or by ://w
using a fluorescently-labeled monobody, HA4, flow cytometry analysis for a co-expressed GFP w
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that is specific to the phosphopeptide binding six days after retroviral infection (data not shown). .jb
c
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interface of the Abl SH2 domain (13), or with an In contrast, the Bcr-Abl-negative cell lines UT-7 rg
b/
anti-Bcr antibody (Figure 7A). AS27 captured and HEK293 could be transduced to express AS25 y
g
u
Bcr-Abl kinase while a control, non-binding or AS27 constitutively, stable over time and at es
t o
mutant, AS27(Y45K;V80K) did not. Moreover, comparably high expression levels as several other n A
p
the capture of Bcr-Abl by AS27 was eliminated by monobodies (data not shown). This difficulty ril 6
pre-incubating AS27 with purified Abl SH2 prompted us to hypothesize that AS25 and AS27 , 2
0
1
domain. Likewise, co-immunoprecipitation expression might not be compatible with K562 9
experiments showed strong binding of AS25 and cell proliferation or survival. To test this
AS27 to constitutively activated full-length Abl possibility, we monitored the relative population
protein ("Abl PP") (27) in mammalian cell lysates, of GFP positive, i.e. monobody-expressing, cells
whereas the I164E mutation strongly decreased to non-expressing cells starting 24h after retroviral
binding to background levels observed with a non- infection of K562 cells. The cells expressing AS25
binding control monobody (Figure 7B). Together, or AS27, but not HA4 or a nonbinding mutant,
these results demonstrate that AS25 and AS27 are were rapidly depleted from the culture (Figure 6C)
sufficiently specific to recognize the SH2 domain and the AS25- or AS27-expressing cell population
of full-length Abl and Bcr-Abl in cell lysates. contained much more early and late apoptotic
We next assessed the effects of the AS25 cells, as respectively detected using Annexin V
and AS27 on Bcr-Abl activity in cells by and 7-amino-actinomycin D (7-AAD) staining
examining the activation state of Bcr-Abl by co- (Figure 6D). These results indicated that AS25 and
expressing Bcr-Abl with different monobodies in AS27 indeed strongly inhibit proliferation of K562
HEK293 cells. AS25 and AS27 strongly decreased and induces apoptosis. Taken together, these
8
Bcr-Abl inhibitors targeted to a domain interface
results from cellular studies strongly suggest that (Y79, V80 and N83) bound to a contiguous
AS25 and AS27 potently inhibit the kinase activity surface including this cavity. It is tempting to
of Bcr-Abl (and Abl) that is critical for speculate that one might be able to use this
downstream signaling pathways that mediate cell structural information for generating small
survival of the Bcr-Abl oncogene-addicted K562 molecule inhibitors, either by directly designing
cell line. small molecule mimics of the hot-spot residue or
by performing site-directed screening of small
DISCUSSION molecules utilizing, for example, the tethering
This work establishes the feasibility of approach (35).
targeting only the SH2-kinase interface for It is notable that AS25 and AS27 have
pharmacologically inhibiting Bcr-Abl. Although been generated in an unbiased manner, i.e. without
one might have considered that the original the use of a competitor but they bound to the
tandem monobody was successful only because kinase-interaction surface of the Abl SH2 domain.
the binding of the HA4 monobody to a readily In contrast, our first attempt using the "loop only"
accessible effectively anchored the 7c12 library produced monobodies predominantly
monobody to the vicinity of the SH2-kinase targeting to the phosphopeptide-binding cleft of D
o
w
interface (Figure 1B), results in this work show the Abl SH2 domain, such as the HA4 monobody n
lo
that such anchoring is not a prerequisite for (13). These two epitopes are involved in natural ad
e
d
disrupting the interface. They also show that the protein-protein interactions, but their shapes are fro
m
blockage of the SH2-pY ligand interactions by the distinct. The kinase-binding surface is flat and h
ttp
HA4 monobody was not required for Bcr-Abl slightly convex, whereas the peptide-binding site ://w
inhibition when the SH2-kinase interface is forms a cleft. These observations suggest that the w
w
potently inhibited. Interestingly, these monobodies new library include monobody clones that are .jb
c
.o
bound to the SH2-kinase fragment as tightly as particularly suited for binding to a flatter surface. rg
b/
they did to the SH2 only fragment (Figure 3). The These observations further support the importance y
g
u
negligible effect of the kinase domain is of controlling interface topography in designing es
t o
surprising, as we anticipated that the kinase binding proteins (28). The results also illustrate the n A
p
domain should sequester the monobody binding importance of utilizing complementary libraries in ril 6
site. The fact that AS25 and AS27 with low nM generating monobodies toward a new target or a , 2
0
1
K values achieved effective inhibition suggests new site. 9
D
that the SH2-kinase interface is dynamic and Whereas the focus of this study is to
exhibit substantial levels of transient opening determine the sufficiency of targeting a particular
during which these monobodies can bind to the surface of a target protein for inhibition, our
epitope. This is also in line with the very low results clearly illustrates a general approach that
affinity between the separated SH2 and kinase can identify a potentially druggable site and define
domains, i.e. in trans binding (Hantschel lab, cellular effects of perturbing the site.
unpublished observation). These results are Technological advances have made it possible to
encouraging in terms of developing therapeutic rapidly generate synthetic binding proteins, such
agents targeted to the SH2-kinase interface. as monobodies, with high affinity and high
Structural analyses of the two monobody- specificity suitable for assessing cellular effects.
SH2 complexes revealed a small cleft of the SH2 These attractive features should make synthetic
domain that both monobodies exploited (Figure binding proteins broadly useful in drug target
5B and 5C). Three hot spot residues of AS25 discovery and validation.

9
Bcr-Abl inhibitors targeted to a domain interface
Acknowledgements: We thank S. Georgeon for the gel filtration data and technical support throughout
the project.

Conflict of interest: JW, AK and SK are listed as are inventors on a patent application filed by the
University of Chicago that covers a design of monobody libraries (US 13/813,409). The other authors
declare no conflicts of interest with the contents of this article.

Author Contributions: JW, OH and SK designed the project. JW, GG, AK and SK generated
monobodies and performed in vitro binding experiments. JW and GG performed crystal structure
determination. JW performed NMR characterization. LA and AK performed structure-guided affinity
maturation. AJL performed in vitro and cellular kinase inhibition assays. BG performed assays on CML
cells. JW, OH and SK wrote the paper with contributions from the other authors. All authors commented
on the manuscript.

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