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Article

Structural and Biochemical Basis for Intracellular
Kinase Inhibition by Src-specific PeptidicMacrocycles
Graphical Abstract

Highlights

d Peptidic macrocycles selectively inhibit Src kinase

d Compounds block ATP and peptide binding site

d Compounds inhibit activity of Src kinase in cells

d Migration of breast tumor-derived cells is inhibited

Aleem et al., 2016, Cell Chemical Biology 23, 1103–1112September 22, 2016 ª 2016 Elsevier Ltd.http://dx.doi.org/10.1016/j.chembiol.2016.07.017

Authors

Saadat U. Aleem, George Georghiou,

Ralph E. Kleiner, ..., W. Todd Miller,

David R. Liu, Markus A. Seeliger

[email protected] (D.R.L.),[email protected](M.A.S.)

In Brief

Aleem et al. show the structural basis for

the selective inhibition of c-Src kinase by

a macrocycle with activity in cells. This

demonstrates that synthetic macrocycles

can be engineered to become potent

inhibitors of intracellular enzymes.

Accession Numbers

5BMM

mailto:[email protected]


http://dx.doi.org/10.1016/j.chembiol.2016.07.017

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Please cite this article as: Aleem et al., Structural and Biochemical Basis for Intracellular Kinase Inhibition by Src-specific Peptidic Macrocycles, CellChemical Biology (2016), http://dx.doi.org/10.1016/j.chembiol.2016.07.017

Cell Chemical Biology

Article

Structural and Biochemical Basis for IntracellularKinase Inhibition by Src-specificPeptidic MacrocyclesSaadat U. Aleem,1,5 George Georghiou,2,5 Ralph E. Kleiner,3,4,5,6 Kip E. Guja,2 Barbara P. Craddock,1 Agatha Lyczek,2

Alix I. Chan,3,4 Miguel Garcia-Diaz,2 W. Todd Miller,1 David R. Liu,3,4,* and Markus A. Seeliger2,7,*1Department of Physiology and Biophysics, Stony Brook University, Stony Brook, NY 11794-8661, USA2Department of Pharmacological Sciences, Stony Brook University, Stony Brook, NY 11794-8651, USA3Department of Chemistry and Chemical Biology4Howard Hughes Medical Institute

Harvard University, Cambridge, MA 02138, USA5Co-first author6Present address: Department of Chemistry, Princeton University, Princeton, NJ 08544, USA7Lead Contact

*Correspondence: [email protected] (D.R.L.), [email protected] (M.A.S.)


SUMMARY

Protein kinases are attractive therapeutic targetsbecause their dysregulation underlies many dis-eases, including cancer. The high conservation ofthe kinase domain and the evolution of drug resis-tance, however, pose major challenges to the devel-opment of specific kinase inhibitors. We recentlydiscovered selective Src kinase inhibitors from aDNA-templated macrocycle library. Here, we revealthe structural basis for how these inhibitors retainactivity against a disease-relevant, drug-resistantkinase mutant, while maintaining Src specificity. Wefind that these macrocycles display a degree ofmodularity: two of their three variable groups interactwith sites on the kinase that confer selectivity, whilethe third group interacts with the universally con-served catalytic lysine and thereby retains the abilityto inhibit the ‘‘gatekeeper’’ kinase mutant. We alsoshow that these macrocycles inhibit migration ofMDA-MB-231 breast tumor cells. Our findings estab-lish intracellular kinase inhibition by peptidic macro-cycles, and inform the development of potent andspecific kinase inhibitors.

INTRODUCTION

Selective inhibition of protein kinases is an effective clinical strat-

egy for the treatment of diseases caused by aberrant kinase

signaling (Cohen and Alessi, 2013; Levitzki, 2013). However,

we lack selective small-molecule inhibitors for many disease-

associated protein kinases, and the limited kinase selectivity of

available inhibitors leads to dose-limiting off-target toxicity (Da-

vis et al., 2011).

The Src family of protein tyrosine kinases (SFKs) consists of

eight non-receptor tyrosine kinases that share high sequence

Cell Chemical Biolo

homology, domain architecture, and regulation (Parsons and

Parsons, 2004). SFKs regulate fundamental cellular processes

such as cell migration, differentiation, growth, and survival (Par-

sons and Parsons, 2004). Src kinase, the prototypical SFK, is

overexpressed or constitutively activated in many solid tumors

types (Summy and Gallick, 2003; Yeatman, 2004), and inhibition

of Src decreases metastasis and tumor growth in both cellular

and animal cancer models. Therefore, Src is considered a phar-

macological target for cancer therapy (Gargalionis et al., 2014;

Krishnan et al., 2012; Nagaraj et al., 2011; Tang et al., 2011;

Tsai et al., 2013; van Oosterwijk et al., 2013; Zhang and Yu,

2012). However, selective pharmacologic inhibition of Src kinase

is challenging because the eight members of the Src kinase fam-

ily are highly conserved, and few small-molecule kinase inhibi-

tors can distinguish between them (Anastassiadis et al., 2011;

Blake et al., 2000; Brandvold et al., 2012, 2015; Georghiou

et al., 2012; Gushwa et al., 2012; Kwarcinski et al., 2012).

Achieving specificity among different Src kinase family members

is crucial because off-target inhibition can create significant clin-

ical problems, such as immunosuppression and impaired T cell

function through inhibition of the hematopoietic SFKs Lck and

Hck (Lowell, 2004; Palacios and Weiss, 2004).

Most small-molecule kinase inhibitors were discovered in

high-throughput screens and their optimization was guided by

Lipinski’s ‘‘ruleof five’’ (RO5) thatdescribes featuresof someorally

bioavailable drugs (Lipinski et al., 2001). More recently, com-

poundssuchasmacrocycles that explore chemical spacebeyond

RO5-compliant compounds have received attention as selective

enzyme inhibitors (Driggers et al., 2008; Heinis, 2014; Villar et al.,

2014). Macrocycles are typically larger and can possess more

rotatable bonds than typical RO5 compounds. This plasticity is

balanced by the conformational restriction throughmacrocycliza-

tionandallowsmacrocycles toadoptconformations thatprecisely

complement a binding site (Villar et al., 2014). In addition, func-

tional groups can be displayed from the macrocycle backbone

and engage multiple interaction sites on the receptor indepen-

dently. Macrocyclic kinase inhibitors therefore have the potential

to be highly specific by exploiting multiple small differences in

the structure and sequence of the conserved kinase domain.

gy 23, 1103–1112, September 22, 2016 ª 2016 Elsevier Ltd. 1103




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Figure 1. Chemical Structures of the Sec-

ond-Generation Compounds MC4 and

MC25 Described in This Work


Approximately 70 macrocycles are currently used in the clinic.

Most of these are either natural products or natural product de-

rivatives, such as the immunosuppressant cyclosporine A and

the antibiotic vancomycin (Heinis, 2014). The challenges associ-

ated with synthesizing macrocycles and understanding their

pharmacological properties has deterred studies of these com-

pounds, and many questions concerning the molecular basis

of macrocycle-target interactions remain (Driggers et al., 2008;

Heinis, 2014; Rezai et al., 2006; Villar et al., 2014). In particular,

surprisingly little structural information is available for macro-

cycle-protein complexes. A recent comprehensive study listed

only 22 distinct X-ray cocrystal structures of macrocycles bound

to proteins (Villar et al., 2014); in contrast, 3,000 structures of

protein kinase domains have been determined in complex with

acyclic kinase inhibitors.

Recently, we discovered two families of Src-specific kinase by

in vitro selection of a 13,824-membered DNA-templated macro-

cyclic peptide library (Kleiner et al., 2010). The two families of

Src-specific kinase inhibitors are classified as nitrophenylala-

nine- or pyrazine-based macrocycles depending on the first

building block incorporated during synthesis (Figure 1). Mem-

bers of these families inhibit Src kinase with nanomolar potency

and more than 80-fold selectivity over the closely related Hck

and Abl kinase (Kleiner et al., 2010). This selectivity is remark-

able, as no clinically approved kinase inhibitor is selective for

Src over other SFKs or Abl kinase (Davis et al., 2011).

We previously reported the X-ray crystal structures of two pyr-

azine macrocycles, MC1 and MC4, bound to the Src kinase

domain (Georghiou et al., 2012). Our studies revealed that these

macrocycles bind to the Src/CDK-like inactive conformation of

the kinase, and occupy the ATP binding pocket while simulta-

neously disrupting substrate peptide binding. We showed that

three amino acid substitutions between Src and Hck determine

the specificity of macrocycles for Src over Hck. These structures

provide important insights into the binding mode of the pyrazine

macrocycles and how they achieve specificity for Src kinase.

However, the structural basis of the nitrophenylalanine macro-

cycles’ ability to inhibit Src kinase as well as the activating ‘‘gate-

keeper’’ mutant (Thr338Ile) of Src (Azam et al., 2008; Foda et al.,

2015) while maintaining selectivity has remained unknown. Phar-

macological inhibition of the Src gatekeeper mutant is of partic-

1104 Cell Chemical Biology 23, 1103–1112, September 22, 2016

ular interest, since the analogous muta-

tion in other kinases frequently underlies

drug resistance in the clinic, and few ki-

nase inhibitors are active against this mu-

tation. Moreover, inhibitors that are active

against the gatekeeper mutation typically

show reduced kinase selectivity (Kwar-

cinski et al., 2016; Zhao et al., 2014).

Here we present the X-ray cocrystal

structure of a nitrophenylalanine-based

macrocycle with Src kinase. This struc-

ture reveals the molecular basis of Src

kinase inhibition by the nitrophenylalanine macrocycles, and

identifies a novel interaction between the nitrophenyl group

and the catalytically essential and universally conserved Lys

295, thereby demonstrating how these compounds retain po-

tency against Src containing the gatekeeper T338I mutation.

We also demonstrate that themacrocycles retain activity against

disease-related inhibitor-resistance mutations in the activation

loop of the kinase. Finally, we show that the macrocycles inhibit

Src-dependent cell migration in a breast tumor-derived cell line.

Taken together, our findings establish that macrocycles can be

effective and highly selective kinase inhibitors in mammalian

cells and reveal the molecular basis of their activity against

wild-type and mutant forms of Src kinase. Our findings inform

the future development of macrocycles as research tools that

selectively modulate Src-dependent cell signaling without inhib-

iting hematopoietic SFKs.

RESULTS

The Nitrophenylalanine Group of MC25b Forms UniqueInteractions in the ATP Binding Pocket of Src KinaseTo elucidate the structural basis for the differences in potency,

selectivity, and response to kinase mutations observed between

the pyrazine (MC4) and nitrophenylalanine series of macrocycles

(MC25) (Figure 1), we determined the X-ray crystal structure of

MC25b bound to Src (Figures 2A and S1) and compared it

with our previously reported crystal structure of the Src,MC4b

complex (Georghiou et al., 2012). MC25 consists of a 19-atom

macrocycle, an ornithine scaffold and a trans-olefin, while MC4

consists of an 18-atom macrocycle, a diaminobutyric acid scaf-

fold, and cis-olefin (Figure 1). The site of DNA attachment in the

DNA-templated library is a carboxamide in MC4a/MC25a and a

carboxylate in MC4b/MC25b (Figure 1).

Despite their different A building blocks and different macro-

cyclic scaffolds, MC25b and MC4 bind to Src in similar confor-

mations and occupy the same three binding pockets in the active

site of Src (Figure 2C). The A, B, and C building blocks occupy

the adenine binding pocket, a hydrophobic pocket underneath

the b3-aC loop, and an amphipathic pocket near the DFG motif,

respectively (Figures 2A and 2B). The peptidic macrocycle back-

bones form almost identical hydrogen bonding patterns with the

Figure 2. X-Ray Cocrystal Structure of Src

and MC25b

(A) MC25b binds to the active site of the kinase,

located between the amino-terminal N lobe and

the carboxy-terminal C lobe. Src is in the Cdk/Src-

like inactive conformation, and the phosphate

binding P loop (red), helix aC (orange), and the

activation loop (blue) are highlighted.

(B) Details of the MC25b binding pocket (enlarged

from A). The nitrophenylalanine (A group) occupies

the adeninebindingpocket, the fluorophenylalanine

(B group) occupies a pocket underneath the b3-aC

loop, and the cyclohexylalanine (C group) occupies

a pocket near the peptide binding patch.

(C) Comparison of the structures of MC25b, MC1,

and MC4b when complexed with the Src kinase

domain. The macrocycle structures are shown

from a perspective that fixes the kinase domains

(not shown) in the same orientation.

(D) Interactions (dashed line) made between

MC25b and Src kinase domain. The nitrophenyl

group of MC25b (red) forms a salt bridge with

the catalytic lysine (Lys295). The ornithine back-

bone of MC25b forms hydrogen bonds with

Gln275, Cys277, and Gly279 of the P loop,

Asn391 in the C lobe, and a water-mediated

hydrogen bond with Arg388 in the activation loop

of Src.


kinase. MC25b forms five direct hydrogen bonds with the pep-

tide backbone of the kinase, and one water-mediated hydrogen

bond (Figure 2D) compared with the five direct and four water-

mediated hydrogen bonds between MC4b and Src. Four of the

five hydrogen bonds between themacrocycles and Src are iden-

tical. The water-mediated hydrogen bond between MC4b and

Met341 is replaced by a water-mediated hydrogen between

MC25b and Arg388. The importance of the backbone interac-

tions in MC25b is highlighted by the loss of potency seen in

several of the N-methylated amide variants of MC25 (MC28–

31; Table S2) (Georghiou et al., 2012). This loss is most likely

due to changes in backbone conformations caused by the

methyl groups, since only one of the hydrogen bonds is made

by an amide in MC25b, while six of the hydrogen bonds are

formed by carbonyl groups, including the C-terminal carboxylic

acid. We speculate that N-methylation results in the formation

Cell Chemical Biology

of cis peptide bonds instead of trans

peptide bonds (Chatterjee et al., 2008).

This altered conformation may impair

hydrogen bonding of macrocycle back-

bone carbonyls or interactions of macro-

cycle side chains with their respective

binding pockets (Chatterjee et al., 2008;

Georghiou et al., 2012).

The key difference in the interaction

between MC4 and MC25 with Src occurs

in the A building-block binding site. The

nitrophenylalanine occupies the adenine

binding pocket, but does not mimic the

adenine hydrogen bonding pattern

observed for MC4 and most ATP-

competitive kinase inhibitors (Krishna-

murty and Maly, 2010). When compared with the positioning of

the pyrazine group in MC4b, the nitrophenylalanine building

block does not bind as deeply into the pocket (Figure 2C).

Instead, the phenyl ring of this group occupies a hydrophobic

space formed by the side chains of Leu273, Val281, Ser345,

and Leu393.

The remaining interactions between the variable groups in the

B and C positions of themacrocycle are very similar between the

pyrazine family (MC4) and the nitrophenylalanine family (MC25).

The fluorophenylalanine in position B occupies the same hydro-

phobic pocket underneath the b3-aC loop as the phenylalanine

in MC4b. However, MC25b binds 1.1 A deeper into this hydro-

phobic pocket. Consistent with the structure, replacement of flu-

orophenylalanine (half maximal inhibitory concentration [IC50]%

0.004 mM) with tyrosine (IC50 = 0.79 mM) or methylphenylalanine

(IC50 = 0.37 mM) resulted in a 100- to 200-fold loss of potency

23, 1103–1112, September 22, 2016 1105

Figure 3. The Effect of Ionic Strength on the

Binding of Nitrophenylalanine and Pyrazine

Macrocycles to Src Kinase

(A) Fluorescence anisotropy was used to measure

the binding of the purified forms of the indicated

Src kinase domains with a fluorescein-labeled

macrocycle containing either a pyrazine (fluores-

cein-MC2) or nitrophenylalanine (fluorescein-

MC9) group at the A position at 0 mM NaCl (low

salt) or 500 mMNaCl (high salt). Experiments were

performed in triplicate. Error bars represent mean

values ± SD.

(B) KD values from (A) are plotted. Error bars

represent mean values ± SD.


(Table S2). The cyclohexylalanine at position C occupies an

amphipathic pocket in both structures of MC4 and MC25, which

is generated by the outward rotation of helix aC into the Cdk/Src-

like inactive conformation.

The Nitrophenylalanine Interacts with the UniversallyConserved Catalytic LysineThe crystal structure of MC25b suggests that the catalytic lysine

(Lys295) of Src is a key residue for MC25 binding, contributing

favorable van der Waals interactions and longer-range electro-

static interactions across 4.6 A with the nitrophenylalanine of

MC25 (Figures 2D and S2). These electrostatic interactions be-

tween the negatively charged oxygen of the zwitterionic nitro

group and Lys295 would not be expected if the nitrophenyl

group was replaced with pyrazine, as in MC4. Furthermore, the

importance of this interaction is supported by the observed

loss in potency when the nitrophenylalanine group was

substituted with other functionalities (in MC17–MC23) (Table

S2). Only the cyanophenylalanine containing MC21 retained its

potency for Src, possibly because it is capable of forming an

interaction between a negative partial charge at the cyano nitro-

gen and the positive charge of Lys295.

To probe the putative electrostatic interaction between the ni-

trophenyl group found inMC25b and the catalytic lysine (Lys295)

of Src, we used twomacrocycles that were fluorescein labeled at

the C terminus for direct binding studies: fluorescein-MC2, a

pyrazine macrocycle, and fluorescein-MC9, a nitrophenyl mac-

rocycle (Table S2). We anticipate that attaching the fluorophore

at this position does not interfere with macrocycle binding to

Src, since the C terminus was linked to DNA during the macro-

cycle library selection and our macrocycle Src structures indi-

cate that the C terminus is solvent exposed. We measured

how the ionic strength of the solvent andmutation of the catalytic

Lys295 affected binding affinity using fluorescence anisotropy.

We found that high salt concentrations lowered the affinity of

the nitrophenylalanine compound (fluorescein-MC9) to Src by


4-fold (Figures 3A and 3B). When the cat-

alytic lysine was mutated to methionine

(K295M), the binding affinity for the

nitrophenylalanine compound decreased

more than 100-fold and became indepen-

dent of ionic strength (Figures 3A and 3B).

In contrast, there were no significant dif-

ferences in the binding affinities of the

pyrazine compound (fluorescein-MC2) toward Src, or the

K295M mutant, at low or high salt concentrations (Figures 3A

and 3B). Taken together, these results suggest that the nitro-

phenyl group of MC25 forms an ionic interaction with the cata-

lytic lysine (Lys295) of Src kinase, whereas the pyrazine-based

MC4 does not. Since Lys295 is essential for kinase activity, resis-

tance mutations at this position that disrupt this ionic interaction

are unlikely.

MC25 and MC4 Have Differential Activity againstDrug-Resistance MutationsTyrosine kinases (such as Abl kinase) commonly present muta-

tions in the clinic that confer drug resistance. The gatekeeper

residue (Abl Thr315, Src Thr338) is the most common clinical im-

atinib-resistance mutation, followed by mutations in the P loop

and the activation loop (Bikker et al., 2009). Replacement of

the gatekeeper residue with a hydrophobic residue (Abl T315I,

Src T338I/M, EGFR T790M) activates the kinase (Azam et al.,

2008; Foda et al., 2015). A single nucleotide exchange in the hu-

man gene for Src kinase results in the T338Mmutation, while the

T338I mutation is present in approximately half of all chicken

Rous sarcoma strains of viral Src kinase. Since Src is currently

not a primary target of clinical kinase inhibitors, no clinical resis-

tance mutations have emerged.

The crystal structure of Src,MC25b presented here demon-

strates why the nitrophenylalanine family of macrocycles is

active against the T338I mutation (inhibition constant Ki =

0.016 mM) while the pyrazine family is not (Table S3) (Georghiou

et al., 2012). The distance between pyrazine and the Thr338 side

chain is only 4 A (Figure 2C). Replacement of threonine with the

larger isoleucine side chain would clash with the pyrazine (Geor-

ghiou et al., 2012). In contrast, the distance between the nitro-

phenyl of MC25b and Thr338 is 6.7 A (Figure 2C), and the

T338I mutation is not predicted to cause steric clashes (Fig-

ure S3). Introduction of the T338M mutation causes resistance

to MC25b (Ki = 5.3 mM) (Table S3), and the mutation is predicted

Figure 4. A Bcr-Abl Activation-Loop Inhibitor-Resistance Mutation

Modeled in Src(R419P) Results in a Loss of Potency for MC25b

(A) Inhibitory potency of macrocycles in biochemical kinase assay. Experi-

ments were performed in triplicate, and data represent mean values ± SD.

(B) Destabilization of Src/Cdk-like inactive conformation results in loss of

macrocycle potency. Src autophosphorylation (pTyr-416) or the L407G mu-

tation cause a loss of potency for both MC4b and MC25b. Experiments were

performed in triplicate, and data represent mean values ± SD.


to decrease the minimum distance to 4.3 A (Figure S3). Interest-

ingly, MC25b retains its selectivity for Src kinase (Ki = 0.010 mM)

and shows low activity against Hck (Ki = 1.4 mM), Fyn (Ki =

1.12 mM), and Lck (Ki = 1.0 mM) as well as moderate activity

against Yes kinase (Ki = 0.12 mM) (Table S3).

Since the backbone of MC25 extended further toward the

C-terminal end of the activation loop than MC4 (Figure S4), we

compared the potency of activation-loop mutations on macro-

cycle potency. We chose the most common imatinib-resistance

mutation found in the activation loop of BCR-Abl (H396P) (Corbin

et al., 2003; Young et al., 2006) and generated the analogous Src

mutant (R419P) to assess macrocycle potency (Seeliger et al.,

2007). Based on the crystal structure of the homologous muta-

tion in Abl (H396P), we hypothesized that substituting a proline

at this position altered the conformation of the activation loop

in the Src/Cdk-like inactive conformation, and would potentially

result in the loss of a hydrogen bond for MC25, but not for MC4

(Young et al., 2006). Indeed, MC4 was able to largely retain po-

tency (2.6-fold higher IC50 than wild-type Src) against this muta-

tion, while MC25 inhibited this mutant 26-fold less potently than

wild-type Src (Figure 4A). In the crystal structures of MC4 and

MC25, Arg419 faces away from the active site. A carbonyl group

from the backbone of MC25 forms a hydrogen bond with

Arg388, and the perturbation of the activation loop by R419P

would likely shift Arg388 too far to retain this hydrogen bond.

Collectively, these results show how the modularity in the

macrocycle architecture results in differential sensitivity to muta-

tions: the size and nature of the A group determines the sensi-

tivity to the gatekeeper mutation, while the size and conforma-

tion of the macrocycle backbone determines interaction with

the activation loop of the kinase.

Both Classes of Macrocycles Bind Selectively to theSrc-like Inactive ConformationThe crystal structures of MC4 and MC25 both demonstrate that

the macrocyclic inhibitors bind to the Src/Cdk-like inactive

conformation of Src. In particular, the binding pocket for the B

group underneath the b3-aC loop is enlarged by the outward

rotation of helix aC in the Src/Cdk-like inactive conformation.

To verify that the macrocycles are selective for the inactive

conformation, we tested their potency against the autophos-

phorylated form of Src that is locked in the active conformation.

We found that neither MC4b nor MC25b could inhibit Src when

the kinase was autophosphorylated (IC50 values >20 mM) (Fig-

ure 4B).We also tested the potency ofMC4b andMC25b against

a mutant form of Src (L407G) that is proposed to destabilize the

Src/Cdk-like inactive conformation (Seeliger et al., 2007). The

L407G mutation decreased the potency of MC4 and MC25 by

200–fold and 2,000-fold, respectively (Figure 4B).

These results emphasize that the macrocycle Src inhibitors

have a strong preference for a specific inactive kinase conforma-

tion, and that mutations or posttranslational modifications that

destabilize this conformation reduce the activity of the inhibitor.

MC25 Inhibits Migration of Breast Cancer CellsOur structural data explain how the macrocycles achieve po-

tency, kinase selectivity, and activity against certain mutations

while recognizing the Src/Cdk-like inactive conformation. We

next investigated how the in vitro inhibition data would translate

to the cellular context. We previously showed that high micro-

molar concentrations of MC25a decreased global tyrosine phos-

phorylation in NIH 3T3 Src�/� cells overexpressing a constitu-

tively activated form of Src (Y529F) (Georghiou et al., 2012).

While the modest cellular potency of MC25a against the consti-

tutively active Src Y529F mutant may be a result of limited mem-

brane permeability and cellular uptake, as has been observed for

other macrocycles (Bockus et al., 2015), we also speculated that

the Y529F mutation increases intracellular autophosphorylation

of Src on Tyr416 (chicken c-Src numbering) (Hunter, 1987), lock-

ing the kinase in the active conformation and disfavoring macro-

cycle binding. Indeed, our biochemical data indicate that MC4

and MC25a were more than 1,000-fold less potent against auto-

phosphorylated Src kinase (Figure 4B). We therefore tested

compound potency against endogenous levels of Src in breast

tumor-derived MDA-MB-231 cells. To quantify the effect of in-

hibitor of cellular kinase activity, we treated cells with inhibitor

or vehicle control for 24 hr, washed the cells, and immunopuri-

fied Src from cells. Kinase activity of the Src,MC25 complexes

was quantified in radioactive kinase assays. MC25a decreased

the activity of Src from MDA-MB-231 in a dose-dependent

manner with an IC50 of 0.06 mM (Figure 5A). Dasatinib as a control

at 1 mM inhibited Src kinase activity completely. The levels of im-

munopurified Src were unaffected by inhibitor concentration

(Figure 5B).

MDA-MB-231 cells depend upon Src activity for migration,

which is necessary for metastasis (Sanchez-Bailon et al., 2012,

Cell Chemical Biology 23, 1103–1112, September 22, 2016 1107

Figure 5. MC25a Inhibits Cell Migration in a Metastatic Breast Cancer Cell Line(A) Dose-dependent inhibition of MDA-MB-231 cells by MC25a. MDA-MB-231 cells were treated for 24 hr with MC25a or DMSO. Following cell lysis and

immunoprecipitation with Src antibody, radioactive kinase activity assays were performed, yielding an IC50 of 0.06 mM. Data represent mean values of four

experiments ±SEM.

(B) Western blot of immunoprecipitated Src from MC25a-treated cells as input control for radioactive kinase assays in (A).

(C) Wound-closure assay of MDA-MB-231 cells. Following serum starvation for 12 hr, cells were treated with either 20 mM of MC25a, or DMSO vehicle control.

(D) Following introduction of a sterile wound, wound closure was quantified by measuring the free space unoccupied by cells at the indicated time points.

Data represent mean values ±SEM. *p < 0.05.


2015; Tang et al., 2011; Tsai et al., 2013). Treatment of cells with

20 mMMC25 decreased the rate of cell migration and the closure

of an artificial wound. After 17.5 hr of treatment with MC25 (or

45 hr, when cells had been serum starved), the remaining wound

size doubled compared with vehicle control (Figures 5C and 5D).

Proliferation of MDA-MB-231 cells was not affected by MC25 as

observed previously for other Src inhibitors (Tsai et al., 2013).

These results show that the macrocycle Src inhibitors are active

in cells and can be used to inhibit a metastasis-related pheno-

type in a breast tumor-derived cell line.

DISCUSSION

Previously we reported two potent and unusually selective

macrocyclic inhibitors of Src kinase, MC4 and MC25, and eluci-

dated the structural and biochemical basis of Src-specific

inhibition by MC4 (Georghiou et al., 2012). While these two

macrocycles share structural similarities, MC25 demonstrated

several unique features over MC4 including cellular efficacy,

insensitivity to the activating and drug-resistant T338I gate-

keepermutation, and a higher degree of selectivity for Src kinase.

Here we have characterized the structural basis of Src kinase in-

hibition byMC25.Our structural data indicate that this compound

makes unique interactions in the adenine binding pocket that

enable retention of activity against T338I Src without compro-

mising inhibitor specificity. Furthermore, we demonstrate that

MC25 inhibits Src-dependent cell migration in a metastatic

breast tumor-derived cell line. These studies will be useful for

developing the next generation of macrocycle kinase inhibitors.

Comparison of the structures of pyrazine-based MC4 and ni-

trophenylalanine-based MC25 bound to Src kinase allowed us


to rationalize their Src selectivity and differential activity against

drug-resistant kinase mutations. We hypothesize that the excel-

lent kinase selectivity of these inhibitors is tied to their preference

for the inactive kinase conformation as well as the engagement

ofmultiple binding sites. Kinase inhibitors that target a distinct ki-

nase conformation are often more selective, since the relevant

conformational states may be more accessible to the target ki-

nase or may reveal target-specific structural features exploited

by the inhibitor (Muller et al., 2015; Tong and Seeliger, 2015;

Wang et al., 2014). For example, the highly selective kinase inhib-

itors imatinib and lapatinib bind to their targets in distinct inactive

kinase conformations. However, inhibition of specific kinase

conformations can also represent an Achilles heel for inhibitors,

as destabilization of the targeted conformation can render the in-

hibitor inactive. The macrocycles presented here and the Abl ki-

nase inhibitor, imatinib, target specific inactive conformations

that are less accessible upon phosphorylation of the kinase acti-

vation loop (Schindler et al., 2000). Consequently, phosphoryla-

tion of Abl kinase domain increases the dissociation constant

(KD) for imatinib 20-fold (Davis et al., 2011) and the enzymatic

Ki 100-fold (Seeliger et al., 2007). Despite this limitation, imatinib

is highly successful in the clinic, likely because activation-loop

phosphorylation is in a dynamic equilibrium mediated by phos-

phatases. Other examples of clinically successful kinase inhibi-

tors that lose potency against their activated kinase target

include the vascular endothelial growth factor receptor inhibitor

axitinib (50-fold increase in Ki) (Solowiej et al., 2009) and the

c-Kit inhibitor sunitinib (300-fold increase in IC50) (DiNitto et al.,

2010). The clinical success of imatinib, axitinib, and sunitinib in-

dicates that resistance of activation-loop phosphorylated ki-

nases to inhibitors can be tolerated and should not diminish


the potential of the macrocycles from being useful kinase

inhibitors.

MC25b buries almost 70% more solvent-accessible surface

area on the kinase (537 A2) than dasatinib (316 A2). Thus, the

larger size of MC25b compared with dasatinib may explain the

ability to connect multiple distinct binding sites and to spread

the binding energy over multiple and potentially lower-affinity

interactions.

Our macrocycle Src structures show that the main differences

between MC25 and MC4 interactions with Src occur in the

adenine binding site and the kinase activation loop. These two

regions are highly conserved among tyrosine kinases and are

of particular interest in the clinic as they are frequent sites of

drug resistance to small-molecule inhibitors. We find that while

MC25 extends toward the activation loop, the smaller scaffold

of MC4 does not extend into this region and MC4 binding is

therefore insensitive to the R419P mutation, which is analogous

to the clinically relevant H396P mutation in c-Abl that confers

resistance to imatinib in chronic myeloid leukemia patients.

Conversely, while MC4 inserts deep into the adenine binding

pocket and forms an adenine-like hydrogen bond with Thr338,

MC25 does not. Rather, our structural and biochemical data indi-

cate that MC25 resides 6.7 A from Thr338 and that its affinity for

Src derives from additional electrostatic interaction between the

nitrophenyl group and catalytic Lys295, thereby explaining how

MC25 binding is not perturbed by the T338I mutation. This

shallow positioning in the nucleotide binding site and interaction

with Lys295 are unusual, as most small-molecule kinase inhibi-

tors make a characteristic set of hydrophobic and hydrogen

bond interactions that mimic the adenine ring of ATP (Krishna-

murty and Maly, 2010). Exceptions are inhibitors such as the

50-fluorosulfonylbenzoate derivatives of the pan-SFK inhibitor

PP1 that covalently attach to the catalytic lysine (Gushwa

et al., 2012).

Notably, first- and second-generation small-molecule inhibi-

tors of c-Abl that are used in the clinic, such as imatinib and da-

satinib, respectively, form hydrogen bonds with Thr315 (the

analogous gatekeeper residue in c-Abl) and are inactive against

c-Abl containing a T315I mutation, the most common drug-

resistance mutation occurring after imatinib treatment (Soverini

et al., 2006). While c-Abl inhibitors that can inhibit this mutant

protein (e.g., ponatinib) have been described, these com-

pounds typically have compromised target specificity (Liu

et al., 2012). In this regard, MC25may serve as a promising tem-

plate for the development of future Abl/Src kinase inhibitors,

since it is both highly selective and insensitive to mutation of

the gatekeeper residue. Moreover, we predict that interactions

with Lys295 (or the analogous Lys290 in c-Abl), a residue

essential for catalysis, are less likely to be perturbed by resis-

tance mutations. Overall, the structural basis for the inhibition

of wild-type Src kinase and c-Abl-related drug-resistant mu-

tants by these two families of macrocycles provides a strategy

to develop specific and potent macrocyclic kinase inhibitors

that would be insensitive to mutations known to arise in the

clinic. Such an inhibitor would benefit from an aromatic group

with polar functionality in the A position to form long-distance

interactions with Lys295 while retaining distance from Thr338,

and a smaller MC4-based macrocycle backbone that would

be insensitive to mutations in the activation loop. We speculate

that such a compound would retain selectivity due to the B- and

C-position building blocks.

Finally, we show that MC25 inhibits endogenous Src activity in

the MDA-MB-231 breast tumor cell line (Tang et al., 2011; Tsai

et al., 2013) and that Src inhibition in MDA-MB-231 cells corre-

lates with a reduction of cell migration, a disease-relevant

phenotype of this metastatic breast tumor-derived cell line that

is known to depend on Src activity. The observation that MC25

inhibits migration of MDA-MB-231 cells, which depends on Src

activity but does not inhibit their proliferation, which depends

on Abl activity (Srinivasan and Plattner, 2006; Srinivasan et al.,

2008), can be attributed to MC25’s excellent selectivity for Src

over Abl kinase.

Metastasis is a marker of advanced stage disease clinically,

and represents an important target for drug inhibition Notably,

MC25 inhibited wound healing at concentrations not expected

to inhibit the hematopoietic kinases Hck and Lck. At present,

therapeutic strategies to inhibit Src are limited by off-target ef-

fects that cause immunosuppression and inhibitor toxicity (Lev-

itzki, 2013). Therefore, the biological potency and selectivity of

MC25 may provide a tool to help evaluate Src-dependent

signaling in tumor cell lines.

SIGNIFICANCE

Protein kinases are involved in virtually all cellular signaling

pathways, and their dysregulation underlies many diseases.

Kinases are therefore attractive drug targets if specific and

potent inhibition can be achieved. The high level of conser-

vation of structure and sequence within the catalytic kinase

domain complicates the development of specific kinase in-

hibitors. In addition, pharmacologic kinase inhibition is

made challenging by drug-resistant mutations in the target

that arise after treatment. All small-molecule kinase inhibi-

tors currently in clinical use bind to the conserved ATP bind-

ing site in the protein kinase domain. We have previously

identified a series of peptidicmacrocycles that are unusually

specific for Src kinase over other related Src family kinases,

and interfere both with ATP and peptide substrate binding.

Here we characterize the structural and biochemical mech-

anisms of Src kinase inhibition by one such compound,

MC25, which retains activity against a common disease-

relevant inhibitor-resistance mutation at the so-called gate-

keeper residue, T338I. Our study reveals a unique mode of

Src kinase inhibition and provides a mechanistic rational

for MC25’s unusual kinase selectivity as well as activity

against T338I Src. In addition, we show that this compound

is active in cells. Many natural product-derived drugs are

macrocycles, suggesting that the macrocyclic structure

imbues these molecules, which lie outside conventional

drug-like chemical space, with favorable properties such

as high specificity and bioavailability. However, the design

of synthetic macrocycles has been hampered by a paucity

of structural data and lack of macrocycles with cellular ac-

tivity. MC25 could serve as a tool compound informing the

future development of kinase inhibitors active against Src

kinase containing mutations at the gatekeeper residue,

including the T338Mmutation. Furthermore, the compounds



presented here could motivate future systematic studies to

derive empirical rules for bioactive macrocycles.

EXPERIMENTAL PROCEDURES

Constructs and Mutagenesis

Kinase domain constructs of chicken c-Src (residues 251–533) were purified

as previously described (Seeliger et al., 2005, 2007). Mutations were intro-

duced into Src kinase domain (K295M, L407G, and R419P) by site-directed

mutagenesis and were verified by DNA sequencing.

Protein Crystallization

The complex betweenMC25b (500 mM) and c-Src kinase domain (200 mM)was

formed in 20 mM Tris (pH 8.0), 125 mMNaCl, 2.5% DMSO, and 2.5% glycerol.

Src,MC25b crystallized in a hanging-drop format at room temperature when

mixed in a 1:1 ratio with mother liquor consisting of 14% polyethylene glycol

5000 monomethyl ether and 0.3 M NaH2PO4. Crystals were cryoprotected in

mother liquor supplemented with 20% glycerol, cryocooled, and stored in

liquid nitrogen. See Supplemental Experimental Procedures for details.

Data Collection and Processing

X-ray diffraction data were collected on beamline X29 at Brookhaven National

Laboratory at 100 K using a wavelength of 1.075 A. Phases were obtained by

molecular replacement using the kinase domain of c-Src (PDB: 3U4W) (Geor-

ghiou et al., 2012) in Phaser (McCoy et al., 2007). The diffraction data were

strongly anisotropic, and an anisotropic correction was carried out using the

Anisotropy Diffraction Server (Strong et al., 2006). These anisotropically scaled

data were then refined in Phenix (Adams et al., 2002). The model was built in

Coot. See Supplemental Experimental Procedures for further details and

refinement statistics.

Fluorescence Anisotropy

The change in fluorescence anisotropy of fluorescein-labeledMC2 andMC9 at

518 nm upon excitation at 492 nm was monitored with a Jobin Yvon Fluoro-

Max-4 spectrofluorimeter (Horiba). Src kinase domain (residues 251–533)

was titrated to 0.5 mM of the fluorescein-labeled macrocycle, in 100 mM Tris

(pH 8.0), 10mMMgCl2 at 25�C. For the salt titrations, 500mMNaCl was added

to the buffer prior to the addition of kinase. After equilibration, the increase in

anisotropy of the fluorescently labeled ligandwas recorded and fitted against a

quadratic binding equation utilizing the GraphPad Prism software to yield

the KD.

In Vitro Autophosphorylation of Src Kinase Domain

Src autophosphorylation experiments were performed by incubating 10 mM

Src kinase domain with 1 mM ATP in buffer containing 20 mM Tris (pH 8.0)

and 10 mM MgCl2 for 2 hr at 25�C.

Kinase Assays

For Src inhibition assays (Barker et al., 1995), 300 mM of an Src-optimal sub-

strate peptide (AEEEIYGEFAKKK) (Songyang et al., 1995) was combined

with 250 mM ATP. The concentrations of kinase used in these assays were

as follows: 0.0125 mM Src kinase domain, 0.025 mM R419P, 0.025 L407G. Ti-

trations of MC4b and MC25b (ranging from 0 to 83.3 mM) were performed at

30�Cas described previously for imatinib to determine the IC50 and the Ki (See-

liger et al., 2007).

Immunoprecipitation and Radioactive Kinase Assay

MDA-MB-231 cells were grown to 90% confluence and treated with dasatinib,

MC25a, or DMSO vehicle. Cells were harvested 24 hr following drug treatment,

washed once with PBS, and lysed with RIPA lysis buffer. Lysates were cleared

by centrifuging at 15,000 3 g for 10 min and then immunoprecipitated for Src

protein (32G6 antibody [Cell Signaling Technology]) overnight at 4�C. Thebeads were washed four times with ice-cold PBS supplemented with 1 mM

vanadate. The beads were divided into three tubes, two of which were used

in the radioactive kinase assay with a Src substrate peptide and [g-32P]ATP.

The remaining tube was analyzed by SDS-PAGE, transferred onto Hydro-

phobic Polyvinylidene Fluoride (PVDF) membrane (Millipore), and analyzed


by western blotting for total Src (32G6). See Supplemental Experimental Pro-

cedures for details.

Wound-Healing Assay

MDA-MB-231 cells were grown to confluence in 12-well plates in DMEM/

Ham’s F12 (50:50) supplemented with 10% fetal bovine serum at 37�C in

5% CO2. In some experiments, the cells were then serum starved for 12 hr

as indicated. Multiple wounds were generated in each well by scratching

with a sterile P200 pipette tip. The cells were then washed twice with PBS,

and treated with complete media supplemented with DMSO vehicle control

or 20 mM MC25a. Each condition was repeated in triplicate. Photographs

were taken per well at four different wound locations and at different time

points. The percentage of wound closure was calculated with ImageJ by

measuring the area unoccupied by cells at each time point, and normalizing

to the zero time point.

ACCESSION NUMBERS

Atomic coordinates and structure factors for Src,MC25b have been deposited

in the PDB with accession code PDB: 5BMM.

SUPPLEMENTAL INFORMATION

Supplemental Information includes Supplemental Experimental Procedures,

four figures, and three tables and can be found with this article online at

http://dx.doi.org/10.1016/j.chembiol.2016.07.017.

AUTHOR CONTRIBUTIONS

B.P.C., S.U.A., and W.T.M. performed all the cellular experiments. S.U.A. per-

formed the fluorescence anisotropy binding experiments. G.G., K.E.G., and

S.U.A. determined the structure of the Src,MC25b complex. G.G. performed

the in vitro kinase assays. R.E.K., A.I.C., and D.R.L. developed and synthe-

sized all the macrocyclic inhibitors utilized in this work. M.A.S. contributed

to the design of all the experiments and directed the study. All authors contrib-

uted to the writing of the manuscript.

ACKNOWLEDGMENTS

This work was supported by NIHGM119437 (M.A.S.), NIHCA058530 (W.T.M.),

NIH ES022930 (K.E.G.), NIH GM100021 (M.G.-D.), NIH GM118062 (D.R.L.),

NIH 2T32GM008444 (S.A., K.E.G., and A.L.), and HHMI (D.R.L.). Use of beam-

line X29 at the National Synchrotron Light Source of Brookhaven National Lab-

oratory (Upton, NY) was supported by the U.S. Department of Energy, Office of

Science, Office of Basic Energy Sciences under contract no. DE-AC02-

98CH10886. D.R.L. is a consultant for Ensemble Therapeutics, a company

that uses DNA-templated synthesis in drug discovery and development.

Received: July 22, 2015

Revised: July 4, 2016

Accepted: July 14, 2016

Published: September 1, 2016

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