Structural Analysis of DFG-in and DFG-out Dual Src-Abl Inhibitors Sharing a Common Vinyl Purine Template

Structural Analysis of DFG-in and DFG-out DualSrc-Abl Inhibitors Sharing a Common Vinyl PurineTemplate

Tianjun Zhou, Lois Commodore, Wei-ShengHuang, Yihan Wang, Tomi K. Sawyer,William C. Shakespeare, Tim Clackson,Xiaotian Zhu and David C. Dalgarno*

ARIAD Pharmaceuticals Inc, 26 Landsdowne St., Cambridge, MA02139, USA*Corresponding author: David C. Dalgarno,[email protected]

Bcr-Abl is the oncogenic protein tyrosine kinaseresponsible for chronic myeloid leukemia (CML).Treatment of the disease with imatinib (Gleevec)often results in drug resistance via kinase muta-tions at the advanced phases of the disease, whichhas necessitated the development of new muta-tion-resistant inhibitors, notably against the T315Igatekeeper mutation. As part of our efforts to dis-cover such mutation resistant Abl inhibitors, wehave focused on optimizing purine template kinaseinhibitors, leading to the discovery of potent DFG-in and DFG-out series of Abl inhibitors that are alsopotent Src inhibitors. Here we present crystalstructures of Abl bound by two such inhibitors,based on a common N9-arenyl purine, and that rep-resent both DFG-in and -out binding modes. In eachstructure the purine template is bound deeply inthe adenine pocket and the novel vinyl linker formsa non-classical hydrogen bond to the gatekeeperresidue, Thr315. Specific template substitutionspromote either a DFG-in or -out binding mode, withthe kinase binding site adjusting to optimize molec-ular recognition. Bcr-Abl T315I mutant kinase isresistant to all currently marketed Abl inhibitors,and is the focus of intense drug discovery efforts.Notably, our DFG-out inhibitor, AP24163, exhibitsmodest activity against this mutant, illustratingthat this kinase mutant can be inhibited by DFG-outclass inhibitors. Furthermore our DFG-out inhibitorexhibits dual Src-Abl activity, absent from the pro-totypical DFG-out inhibitor, imatinib as well as itsanalog, nilotinib. The data presented here providesstructural guidance for the further design of novelpotent DFG-out class inhibitors against Src, Abland Abl T315I mutant kinases.

Key words: Abl, CML, crystal structure, drug resistance, inhibitor,mutation, Src, T315I

Received 16 September 2009, revised 16 September 2009 and acceptedfor publication 23 September 2009

Introduction

Bcr-Abl, the constitutively activated fusion product of the Philadel-phia chromosome (Ph), is the oncogene product driving the pathologyof chronic myelogenous leukemia (CML) (1–3). The discovery of theAbl kinase inhibitor imatinib (STI-571, Gleevec) has transformedtreatment of CML; however, despite early successes with imatinib,resistance to treatment has occurred, particularly in the moreadvanced phases of the disease (4–6). This resistance arises fromthe appearance of mutated forms of the Bcr-Abl kinase which exhibitmuch weaker imatinib binding, either by direct occlusion of thekinase binding site or by destabilization of the kinase conformationrequired to bind imatinib (7). Consequently, the development ofsecond and third generation Bcr-Abl inhibitors which are capable ofovercoming clinically relevant resistant mutations has continued(8–10). Since the Abl T315I mutant has proven resistant to allcurrently marketed Abl kinase inhibitors, a critical clinical need existsfor new inhibitors capable of overcoming this key mutant. Currentefforts are focused on developing inhibitors targeting Bcr-Abl with aT315I gatekeeper residue mutation in the kinase domain (11–15).

Bcr-Abl inhibitors can be divided into either DFG-in or DFG-out clas-ses, dependent on their binding interactions with the kinasedomain. Imatinib, for example, is the prototypical example of theDFG-out class of inhibitors which bind the kinase domain in aninactive conformation associated with an inhibitor promoted confor-mational change in the activation loop Asp-Phe-Gly (DFG) residues(7,16,17). Conversely, dasatinib (BMS-354825), a second generationDFG-in class inhibitor, binds the kinase domain in a near active con-formation, typically seen with ATP or mimics (18). Since Abl andSrc family kinases (SFKs) exhibit high binding site homology, manysuch DFG-in class Abl inhibitors exhibit SFK inhibitory activity, andtypically retain both activities even after optimization for a singlekinase (19,20). Compared to imatinib, these DFG-in inhibitors typi-cally exhibit a different, narrower spectrum of resistant mutations

Editor’s invited manuscript to celebrate the 4th Anniversary of Chemical Biology & Drug Design.

18

Chem Biol Drug Des 2010; 75: 18–28

Research Article

ª 2009 John Wiley & Sons A/S

doi: 10.1111/j.1747-0285.2009.00905.x

which are normally associated with direct protein-inhibitor contactpoints, most notably the gatekeeper residue T315I mutation (21).

Nilotinib (AMN-107), a second-generation DFG-out inhibitor wasdeveloped to enhance the potency of imatinib-like moleculesagainst Abl, and consequently to overcome a number of the imatinibresistant mutations (22,23). Imatinib itself is a poor Src inhibitor,IC50 > 100 lM, hence inhibitors closely derived from this chemotyperetain weak Src activity, although they do exhibit other kinaseselectivity. The initial supposition that Src was unable to adopt aDFG-out conformation has been disproven, since imatinib has beencrystallized with Src in DFG-out binding mode, despite its weakinhibitory activity (24). Recent reports have also focused on theoptimization of DFG-out Src inhibitors, yielding compounds whichalso possess Abl activity (12,25). Although the energetic cost of pro-moting a DFG-out conformation for Src versus Abl is unknown, opti-mized inhibitors can bind both Src and Abl in a DFG-outconformation.

It is noteworthy that the Abl inhibitors discussed above all formhydrogen bonds to the side chain of the gatekeeper residue Thr315(16,18,22). These hydrogen bonds not only enhance binding potency,but also enhance kinase specificity since most kinases do not pos-sess a threonine residue at this position (26). While it is advanta-geous to maintain this hydrogen bond when targeting wild type(wt) Abl, its presence is detrimental to inhibitors when the gate-keeper residue is mutated. The occurrence of a steric clashbetween the Ile315 side chain of Abl T315I and the amino group inimatinib, nilotinib or dasatinib, each of which forms this hydrogenbond in the wt kinase, is believed to be the major reason why noneof these inhibitors is active against the Abl T315I. Consequently,one strategy to inhibit both the wt and the T315I mutant Abl kinas-es is to design inhibitors that do not form a hydrogen bond to thegatekeeper residue. Meanwhile, to compensate for the loss of thishydrogen bond, other regions of the protein may be targeted forincreased molecular recognition regaining binding potency.

As part of our Abl kinase drug discovery efforts, we have designeda novel N9-arenyl purine template targeting the hinge and the gate-keeper residue. Modeling results indicated that the vinyl linkageavoids steric clash with Thr315 and also provides the correct trajec-tory for further chemical substitutions targeting other regions of Ablkinase. Based on a common template of N9-arenyl purine template,two chemical series, both DFG-in and -out inhibitors, were opti-mized for potency against Abl kinase. The detailed structure-activityrelationship (SAR) of both series has been described elsewhere,and reveals that both classes of inhibitor possess dual Src and Ablactivity (27,28).

Here we report crystallographic co-structures of N9-arenyl purineinhibitors bound to Abl kinase in both DFG-in and DFG-out bindingmodes. In the Abl:AP24283 co-structure, the inhibitor is bound inDFG-in mode, whereas in the Abl:AP24163 co-structure, the inhibitoris bound in DFG-out mode. For each co-structure, we describe keyinhibitor-protein interactions that drive molecular recognition, com-paring these binding interactions with those of other relevant Ablinhibitors; dasatinib for AP24283 and nilotinib for AP24163. In addi-tion, the DFG-out inhibitor, AP24163, exhibits moderate activity

against the Abl T315I mutant kinase, and we discuss this activity instructural context. Finally, both our DFG-in and DFG-out moleculespossess potent Src activity in addition to their Abl activity. Suchdual activity was seen previously in the case of DFG-in molecules,but not in the case of the DFG-out Abl inhibitors imatinib and niloti-nib. We examine the Abl:AP24163 structure to propose a structuralrationale for this unusual dual activity.

Results and Discussion

Structure of Abl in complex with AP24283The kinase domain of murine c-Abl was co-expressed in Escherichiacoli with the protein tyrosine phosphatase Yop H (See ExperimentalProcedures). The amino acid sequence of murine c-Abl is identicalto that of human c-Abl in the kinase domain except for the substi-tution of Asn to Ser at position 336, located in a loop followinghelix aD and distant to the ATP binding site. Abl kinase is inhibitedpotently by AP24283 with an IC50 of <0.5 nM, and with a wt AblBa ⁄ F3 cellular IC50 = 11 nM (Table 1) (28).

The structure of Abl bound by AP24283, solved at a resolution of1.90 � (Table 2), shows a typical bilobal kinase architecture withthe inhibitor bound at the ATP site between the NH2- and COOH-terminal lobes (N-lobe and C-lobe, respectively) (Figure 1A).Although the protein is unphosphorylated on Tyr393 in the activa-tion loop, Abl kinase bound with AP24283 adopts an active confor-mation similar to that of phosphorylated Lck bound with AMP-PNP,with the exception of the P-loop (29). The highly conserved Asp-Phe-Gly (DFG) motif located at the base of the activation loopadopts a DFG-in conformation while the remainder of the loopadopts an open and extended conformation which is typically seenin phosphorylated kinases (30,31). Helix aC in the complex structurealso adopts a rotated-in conformation allowing the formation of ahydrogen bond between Glu286 of aC and Lys271 of strand b3, afeature regarded as a hallmark of active kinases. The P-loop, con-necting strands b1 and b2 and spanning residues from Lys247 toVal256 in the N-lobe, collapses down towards the C-lobe, makingtight contact with AP24283 and burying the inhibitor inside the pro-tein (Figure 1A). This collapsed conformation of the P-loop is notcompatible with the conformation of the active kinase bound withATP due to a clash with the a-phosphate of ATP, and is likely pro-moted by the bound inhibitor.

AP24283 contains an N9-arenyl disubstituted purine bearing a 4-dimethylphosphinoxide (DMPO) substituted aniline at the purine C6position, and a vinyl-linked methylindazole group at the purine N9position (Figure 1A). In the complex structure, the DMPO-phenylring is orientated almost co-planar to the purine core whereas theplane of the bicyclic indazole is nearly perpendicular to the core(Figure 2A). The purine template occupies the adenine binding siteand is hydrogen bonded to the hinge of the kinase and formsnumerous interactions with residues from both N- and C-lobes ofthe kinase. The aromatic substituents on the purine templateoccupy hydrophobic pockets adjacent to the adenine binding site;the methylindazole ring binds in the hydrophobic selectivity pocket(herein referred to as selectivity pocket) located behind the gate-keeper residue Thr315 whereas the DMPO-phenyl group binds in

Structures of Abl Kinase Domain Bound by Vinyl-purine based inhibitors

Chem Biol Drug Des 2010; 75: 18–28 19

a hydrophobic pocket close to the solvent front. Most of AP24283is buried inside the protein except for the DMPO moiety, which islargely solvent-exposed and makes almost no contact with theprotein.

A total of four hydrogen bonds are formed between AP24283 andAbl (Figure 2A). Two hydrogen bonds are made to the hinge region,

from N7 of the purine ring and the bridging amino nitrogen to thebackbone amide nitrogen and carbonyl oxygen of Met318, respec-tively. The remaining hydrogen bonds are made from the two nitro-gen atoms in the pyrazolo ring of the methylindazole substituent,one to the side chain carboxylate oxygen of Glu286 in helix aC andthe other to the main chain amide nitrogen of Asp381 in the DFG-motif. A similar hydrogen bonding pattern is observed in imatinib

Table 1: Enzymatic and cellular data summary (IC50 in nM)

Compounds Structure Abl kinase Abl T315I Src kinase Wt Abl Ba ⁄ F3 Parental Ba ⁄ F3

AP24283

N N

NNH

N

PO

NHN <0.5 >10 000 <0.5 11 >10 000

AP24247

N N

NNH

N

PO

NHN 2.4 >50 000 17 850 >10 000

AP24149

N N

NNH

N

PO

3.6 8900 9.1 190 >10 000

AP24245

N N

NNH

N

PO

NH17 >5000 198 607 >10 000

Dasatinib

S

N

NH

N N

NN

HOHN

O

Cl

<0.5 >5000 <0.5 1.1 >10 000

AP24163

NN

N

N

HN

CF3

HN

O

N

N 25 478 7.6 7.3 6455

AP24348

NN

N

N

HN

CF3O

N

N 48 10 000 84 7 >10 000

Nilotinib

NHHN

CF3O

N

N

N

N

N

120 >5000 >700 21 >10 000

Abl kinase: Abl kinase enzyme assay; Abl T315I: Abl T315I mutant kinase assay; Src kinase: Src kinase enzyme assay; wt Abl Ba ⁄ F3: Ba ⁄ F3 cells transformedwith wt Abl; Parental Ba ⁄ F3: parental Ba ⁄ F3 cells.

Zhou et al.

20 Chem Biol Drug Des 2010; 75: 18–28

and other DFG-out molecules, albeit through an amide group inthose inhibitors (16,22). In addition to these four hydrogen bonds,AP24283 also forms a C-H-O non-classical hydrogen bonding inter-action (32) with the gatekeeper residue Thr315, making close con-tact between the olefinic proton in the vinyl linker (a to purine N-9)of AP24283 and the side chain of Thr315 with a carbon-to-oxygendistance of approximately 3.2 � (Figure 2A).

AP24283 also makes extensive van der Waals (vdw) contacts to thekinase. The purine template is sandwiched between Ala269 in b3and Leu370 in b7. Additionally, strong vdw contacts are madebetween purine and the P-loop through edge-on interactions withTyr253 located in the middle of the loop. The specific conformationof Tyr253 is stabilized by a network of hydrogen bonds withAsn322 through the OH group of Tyr253 (Figure 3C) as well ashydrophobic packing interactions with the aliphatic portion ofGln252. The phenyl moiety of the DMPO-phenyl group binds in ahydrophobic pocket in the vicinity of ATP ribose binding site, makingvdw interactions with the side chain of Phe317 in the hinge, andthe main chain atoms of Thr319 and Gly321 (Figure 2A). The P-loopalso contributes to the vdw contacts with DMPO-phenyl groupmainly through the side chain of Tyr253 and the main chain ofGly249. Finally, the methylindazole ring is bound in the selectivitypocket located behind the gatekeeper residue. The methylphenylportion of the methylindazole is buried completely inside the pocketnear the N-lobe and is in close contact with the side chains ofIle313 and Met290, and the aliphatic part of Lys271. The pyrazoloring of the methylindazole, on the other hand, is orientated towardsthe C-lobe, binding in a sub-pocket that extends from the selectivitypocket which consists of residues including Lys271, Glu286, Met290and Val299 from the N-lobe and Leu370, Ala380, Asp381 andPhe382 from the C-lobe (Figure 2A). A small but deeply buriedhydrophobic sub-pocket is also observed in the N-lobe, consistingof Val256, Ala269, Lys271, Ile313 and Thr315, which is occupiedfully by the methyl group of the methylindazole in AP24283.

The interactions of AP24283 with Abl observed in the crystal struc-ture are largely consistent with the kinase inhibitory activities seenin the vinyl purine chemical series. One critical element contributingto the Abl activity of AP24283 is the single methyl group substitu-tion on the indazole ring. While the IC50 of AP24283 was estimatedto be <0.5 nM in the Abl kinase assay, AP24247, an analog ofAP24283 without this methyl group suffered a fivefold loss in activ-ity with an IC50 = 2.4 nM (Table 1). The loss of potency was evenmore dramatic in a Ba ⁄ F3 Abl cellular proliferation assay withabout an 80-fold difference between the two inhibitors. Both thestructural and assay data therefore underscore the importance offilling this small N-lobe hydrophobic sub-pocket with an appropriatesubstituent. Other important features revealed in the crystal struc-ture are the hydrogen bonds contributed by the methylindazolegroup. Potent inhibition is observed only when both the NH (hydro-gen bond donor) and N (acceptor) are present (Figure 1A). Whenboth of these two hydrogen bond formers are absent, as exempli-fied in AP24149, mimicking dasatinib, or only one hydrogen bondformer is available, as in AP24245, the inhibitory activity of thesecompounds shows a 10–30 fold loss compared to AP24283(Table 1).

Comparison of AP24283 and dasatinib Ablco-structuresDasatinib is a second generation DFG-in Abl inhibitor whose Abl co-structure has been described (18). In our kinase assays, dasatinibwas found to inhibit wt Abl with an IC50 of <0.5 nM (Table 1). Astructural overlay of Abl:AP24283 and Abl:dasatinib reveals overallsimilarities in protein conformation including a DFG-in conformationof the activation loop (Figure 3A). Moreover, bound AP24283 islargely superimposable with dasatinib. Both inhibitors share twohydrogen bonds to the kinase hinge, and the DMPO group inAP24283 occupies approximately the same space as the hydroxyeth-ylpiperazine group in dasatinib. Additionally, both AP24283 and

Table 2: Data collection and refinement statistics

Abl:AP24283 Abl:AP24163

Diffraction dataSpace group P21 P21

Unit cell dimensions a = 71.83 �, b = 58.72 �, c = 76.17 �, b=118.32� a = 43.47 �, b = 59.98 �, c = 123.55 �, b=89.99�Resolution range (�) 50–1.90 (1.97–1.90) 50–1.22 (1.26–1.22)Total observation 1 270 718 2 805 408Unique reflections 44 725 189 032Data completeness (%) 99.1 (96.7) 98.5 (87.8)Rmerge (%)a 7.3 (51.7) 5.5 (48.1)Intensities I ⁄ r (last shell) 16.2 (1.7) 26.8 (1.5)

RefinementRwork

b ⁄ Rfreec (%) 20.1 (24.1) 18.7 (20.3)

No. of non-H protein atoms 4450 4598No. of heterogen atoms 64 82No. of waters 362 826RMS deviations in bond length (�) 0.009 0.017RMS deviations in bond angle (�) 1.07 1.48Average protein B values (�2) 36.9 59.8

aRmerge =P

hkl[(P

j|Ij ) <I>|) ⁄P

j|Ij|].bRwork =

Phkl |Fo ) Fc| ⁄

Phkl |Fo̧ where Fo and Fc are the observed and calculated structure factors, respectively.

cRfree is the R factor calculated for a randomly selected 5% of the reflections that were omitted from the refinement.



dasatinib bind to the selectivity pocket. Despite such similarities, dif-ferences in binding interactions are present between AP24283 anddasatinib. Significant differences are seen in the hydrogen bondinginteractions with the non-hinge kinase regions. While the aminogroup in dasatinib makes a hydrogen bond with Thr315, the corre-sponding vinyl moiety in AP24283 forms a close contact with this res-idue. In addition, in Abl:AP24283, two hydrogen bonds are formedbetween the inhibitor and the DFG-motif and helix aC, while suchinteractions are missing in dasatinib. A critical element of both

AP24283 and dasatinib binding to Abl is the aromatic substituent thatfills the selectivity pocket behind Thr315. Although the methyl groupin AP24283 and the chlorine group in dasatinib occupy similar loca-tion in the small and deeply buried hydrophobic sub-pocket in Abl,discussed above, the methylindazole moiety in AP24283 is rotatedabout 30� toward the DFG-motif compared to the 2,6 disubstitutedphenyl group in dasatinib (Figure 3A). This rotation allows optimalinteractions of the indazole ring of AP24283 with the DFG-motif andGlu286 in helix aC to form two coordinated hydrogen bonds.

Abl:AP24283 (DFG-In)

AP24283

Abl:AP24163 (DFG-Out)

AP24163

T315

F382

E286

M290

D381

AP24163

3.2Å

A B

C

Figure 1: Crystal structures of Abl:AP24283 and Abl:AP24163. (A) Top, chemical structure of AP24283. The purine template is colored inblue with atom numbering, dimethylphosphinoxide (DMPO) phenylamine in orange, vinyl linker in red and methylindazole in green; bottom:overview of Abl:AP24283 complex. (B) Top, chemical structure of AP24163. The purine template is colored in blue, cyclopropylamine in orange,vinyl linker in red, methylphenylamide in green, trifluoromethyl (CF3) phenyl in cyan and methylimidazole in black; bottom: overview ofAbl:AP24163 complex. (C) 2fo-fc electron density in the vicinity of the gatekeeper Thr315 in Abl:AP24163, calculated at 1.22 � resolution andcontoured at 1.0r. The distance between the Oc of Thr315 and the carbon atom of the vinyl linker a to purine N-9 is shown.

Zhou et al.


Analysis of the two co-structures also shows that the Abl proteinsdiffer in P-loop conformation. While the P-loop adopts a more openstructure in Abl:dasatinib, it is folded down over AP24283. ForAbl:AP24283, multiple vdw contacts are made between Tyr253 fromthe P-loop and the purine template as well as the DMPO-phenylmoiety of AP24283 via aromatic edge-on interactions (Figure 3A).The same type of interaction is not possible in dasatinib becausethe methyl substitution on the pyrimidine ring of dasatinib displacesTyr253 from its location in the Abl:AP24283 co-structure. AlthoughTyr253 is still located in the vicinity of dasatinib, interactionsbetween the two moieties are very limited. The additional vdwinteractions between the P-loop and AP24283 may explain the highbinding potency of this inhibitor even though AP24283 forfeits ahydrogen bond to Thr315 compared to dasatinib.

Structure of Abl in complex with AP24163Although AP24163 shares the same purine template and vinyl linkeras AP24283, AP24163 was designed to be a DFG-out class inhibitorthrough incorporation of a privileged DFG-out binding element,namely attachment of an aryl amide to the methylphenyl group (Fig-ure 1B). In AP24163, the purine template is substituted at C6 witha cyclopropylamine group and by a vinyl-linked methylphenyl groupat N9. This methylphenyl group is further substituted by an amide

linked, methylimidazole and trifluoromethyl (CF3) substituted phenylmoiety. AP24163 is a potent Abl kinase inhibitor with anIC50 = 25 nM (Table 1), however due to the activated form of Ablkinase used in the kinase assay (phosphorylated on Tyr393), theIC50 of AP24163 is likely to be underestimated, e.g. compared to aDFG-in inhibitor (16). The cellular Abl Ba ⁄ F3 potency of AP24163,IC50 = 7.3 nM, is comparable to that of AP24283, IC50 = 11 nM. Fur-thermore, AP24163 has been demonstrated to be metabolically sta-ble and has good oral bioavailability in rats and mice (27), and hasbeen subjected to in vitro profiling for mutant Abl drug resistance(Azam M, unpublished results).

Similar strategies in protein purification and crystallization were usedto obtain the Abl: AP24163 co-structure, which was solved at veryhigh resolution (1.22 �) (Figure 1C, Table 2). As illustrated in Fig-ure 2B, AP24163 binds to Abl in an extended conformation. Whilethe purine template and the methylphenyl group are largely co-planarwith the vinyl linker and the trifluoromethylphenyl group, respectively,the two chemical groups themselves lie almost perpendicular to eachother. As with AP24283, the purine template of AP24163 occupiesthe adenine binding pocket and the methylphenyl ring binds in theselectivity pocket. The trifluoromethylphenyl moiety is bound in thehydrophobic pocket vacated by Phe382 of the DFG-motif as thekinase conformation switches from DFG-in to DFG-out conformation

A

B

Figure 2: Binding modes of AP24283 and AP24163 in Abl Kinase. (A) Stereo view of Abl:AP24283 interactions. (B) Stereo view ofAbl:AP24163 interactions. Hydrogen bonds are depicted as dashed red lines.



(Figure 3C). Finally, the methylimidazole group binds in a hydrophobicpocket near helix aC, and the cyclopropyl group binds in a pocketnear the hinge, both of which are partially exposed to solvent.

A total of four hydrogen bonds are formed between AP24163 andAbl. Two hydrogen bonds are formed to the hinge of the kinasethrough the cyclopropylaminopurine corresponding to those observedin Abl:AP24283. The other two hydrogen bonds are made betweenthe amide group of the inhibitor and the side chain carboxylate oxy-gen of Glu286 in helix aC and the main chain amide nitrogen ofAsp381, respectively (Figure 2B). In addition, as in AP24283, a closeinteraction is observed between the vinyl group and the gatekeeperresidue Thr315 with a carbon-to-oxygen distance of approximately3.2 � (Figure 1C). The high resolution structure reveals clearly thatthe vinyl group is in a trans-conformation with the carbon atomadjacent to the purine core located close to Thr315. The remainingbinding interactions are non-polar. With the purine template, vinyllinker and the methylphenyl group binding similarly to AP24283, thetrifluoromethylphenyl group of AP24163 occupies a pocket nearhelix aC created by the DFG-motif flip, previously mentioned. TheCF3 substituent is buried deep inside the protein and is in closecontact with residues Val379, Ala380 and Asp381 from the activa-tion loop, Ile293 from helix aC, Leu298 from a nearby loop afterhelix aC, and Leu354, Phe359 and His361 preceding the HRD-motifin the catalytic loop (Figure 2B). The methylimidazole substituent,on the other hand, is partially solvent-exposed. It fills the rest ofthe pocket created by the DFG-motif flip and interacts with residuessuch as Glu282, Glu286 and Val289 from helix aC, Phe359 from anearby loop and Asp381 from the DFG-motif. As a consequence ofthe DFG-motif flip, Phe382 binds near the adenine binding site andis involved in multiple vdw interactions with the purine template,vinyl linker and part of the methylphenyl group. Finally the P-loopadopts a bent conformation toward AP24163 and makes favorablevdw interactions to the inhibitor with Leu248 and Val256. Surpris-ingly, Tyr253 located at the tip of the P-loop makes no direct con-tacts with the bound inhibitor.

Comparison of AP24163 and nilotinib Ablco-structuresThe DFG-out binding mode of Abl kinase inhibitors was first reportedin the crystal structure of Abl bound to imatinib (7,16). Nilotinib, is aderivative of imatinib with improved potency, Abl kinaseIC50 = 120 nM (Table 1), furthermore the Abl co-structure of nilotinibwas recently described (22). AP24163 and nilotinib share commonfunctionalities that bind in the pocket induced by the DFG-motif flip(Figure 3B). These include a reversed amide relative to that in imati-nib, and a methylimidazole substituted trifluoromethylphenyl group.The main differences between AP24163 and nilotinib, therefore, aretheir hinge binding elements, for nilotinib, a pyridinyl group, and thelinker between the hinge binder and the methylphenyl group.

Comparison of the Abl:AP24163 and Abl:nilotinib co-structuresreveals Abl adopts a similar conformation when bound to eitherAP24163 or nilotinib (Figure 3B). A slight N- and C-lobe twist isobserved with AP24163 resulting in a more open conformation rela-tive to the of Abl:nilotinib structure. In both structures the methyl-

A

B

C

Figure 3: Structural comparison of Abl and inhibitor co-structures.(A) Overlay of Abl (light blue):AP24283 (gray) with Abl (purple):dasati-nib (green). (B) Overlay of Abl (light blue):AP24163 (gray) with Abl(purple):nilotinib (green). (C) Overlay of Abl (light blue):AP24283(gray) with Abl (purple):AP24163 (green). Hydrogen bonds withinAbl:AP24283 and Abl:AP24163 are depicted as dashed red lineswhereas those in Abl:dasatinib and Abl:nilotinib black. The pseudohydrogen bond formed between the Oc of Thr315 and the carbonatom of the vinyl linker a to purine N-9 in both Abl:AP24283 andAbl:AP24163 is shown in dashed purple line. Structural superpositionswere performed against the hinge of the kinase, using PDB code2GQG for human Abl:dasatinib and 3CS9 for human Abl:nilotinib.

Zhou et al.


phenyl and trifluoromethylphenyl groups occupy the selectivitypocket behind the gatekeeper residue and the hydrophobic pocketinduced by the DFG-out binding mechanism, respectively. Whilethese two chemical groups are largely superimposable, there is atwist of �75� in the methylimidazole ring orientation between thetwo inhibitors (Figure 3B). The differences in the binding of methy-limidazole appear to be caused by crystal lattice contacts in Abl:nil-otinib structure which influence the shape of nearby helix aC (22).The major structural differences in binding Abl are seen in locationsaround the hinge region. While AP24163 makes two hydrogen bondsto the hinge of Abl, nilotinib forms only one. The hydrogen bondbetween the amino linker of nilotinib and the side chain of the gate-keeper Thr315 is absent in AP24163, instead, a pseudo hydrogenbond is formed between the vinyl linker of AP24163 and Thr315.

Additional differences are observed in the ligand interactions withthe P-loop as well as Phe382 from the DFG motif (Figure 3B). WhileTyr253 of the P-loop forms favorable vdw interactions with both thepyridinyl and pyrimidinyl rings of nilotinib, it does not make anydirect contacts with AP24163. In Abl:AP24163, Phe382 of the DFG-motif adopts a different side chain rotamer conformation than thatin Abl:nilotinib and maintains close vdw contacts with the purinetemplate (3.4 � to N3 of purine) and the vinyl linker (3.6 � to thecarbon b to purine N-9). Although Tyr253 does not contact AP24163directly, it plays a facilitating role by stabilizing the side chain con-formation of Phe382 with perpendicular edge-to-face interactions(Figure 3B). This particular conformation of Tyr253 is further stabi-lized by formation of a hydrogen bond to the main chain of Arg367(Figure 3C).

Abl T315I inhibitory activity of AP24163While both AP24163 and nilotinib exhibit potent inhibition againstwt Abl kinase, AP24163, unlike nilotinib (or imatinib), also inhibitsthe T315I mutant kinase with modest potency (Abl T315I kinaseIC50 = 478 nM and Ba ⁄ F3 Abl T315I cell proliferation assayIC50 = 422 nM) (27). Inhibitors such as nilotinib are optimized toform a hydrogen bond with the gatekeeper Thr315, so maintainingthe same binding pose in the T315I mutant inevitably leads tosevere steric clashes with these inhibitors due to the increased bulkof an isoleucine side chain. Such a clash appears to be alleviatedin part by deploying the less sterically demanding vinyl linkage uti-lized in AP24163. Incorporation of a vinyl linkage alone is not suffi-cient to gain Abl T315I activity, since AP24283, like other DFG-inAbl inhibitors is devoid of this activity (20,21). To gain Abl T315Iactivity, as seen in the case of AP24163, both a vinyl linkage andprivileged DFG-out binding element appear to be necessary.

In a previous manuscript describing the crystal structure of AblT315I kinase domain bound by a DFG-in inhibitor, PPY-A, weshowed the ethyl group of the side chain of the mutated gate-keeper Ile315 partially occupied the selectivity pocket (14). This iso-leucine side chain conformation however does not appearcompatible with inhibitors such as AP24163 or nilotinib that bind inthe selectivity pocket. Alternative side chain conformations ofIle315 are feasible, e.g. where the ethyl group would point towardsthe vinyl linker of the purine template of AP24163. Modeling stud-ies suggest that with this alternative conformation of Ile315 side

chain and a slight adjustment of the bound inhibitor, AP24163 isable to bind to the ATP site in a DFG-out binding mode withoutserious steric clashes, consistent with the moderate Abl T315Iactivity of AP24163.

Comparison between AP24283 and AP24163Abl Co-structuresAlthough designed to target different conformations of Abl kinase,namely DFG-in and DFG-out, AP24283 and AP24163 share a com-mon purine template and a vinyl linkage. As a result the bindingpositions of these groups in both inhibitors are found to be nearlyidentical (Figure 3C). Additionally, the smaller cyclopropyl substitu-ent in AP24163 binds in a similar pocket as the DMPO-phenyl ofAP24283, although the hydrophobic interactions involved are lessextensive. Both inhibitors form four hydrogen bonds with the pro-tein, two of which are made to the hinge and the remaining two tothe side chain of Glu286 and to the backbone of Asp381 of theDFG-motif. While the hydrogen bond made to Asp381 is similar, thehydrogen bond formed to Glu286 of helix aC is accompanied by aslight adjustment of Glu286 and the helix aC in Abl:AP24283 (Fig-ure 3C). This perturbation is mainly caused by the difference ingeometry between the hydrogen bond donor-acceptor pair in theamide of AP24163 and the indazole group of AP24283. Neitherstructure has ordered water molecules at the ligand binding siteindicating that potential structural water molecules at the ATP bind-ing site have been replaced by the individual bound ligands (datanot shown).

The activation loop adopts very different conformations (DFG-in andDFG-out) and interacts with the bound inhibitors differently. Forexample, Phe382 of the DFG-motif is involved in multiple vdw inter-actions with AP24163 but forms no such interactions with AP24283from which it is located more distantly. While the P-loop in bothco-structures adopts a twisted conformation folding over the boundinhibitor, there exist slight differences in the overall main chaintrace and large differences in the inhibitor interactions of Tyr253from the P-loop (Figure 3C). In Abl:AP24283, Tyr253 is brought tothe vicinity of the bound inhibitor and makes extensive vdw con-tacts with the DMPO-phenyl group as well as the purine template.On the other hand, Tyr253 in Abl:AP24163 is positioned furtheraway from the bound inhibitor and plays only an indirect role bystabilizing the side chain conformation of Phe382.

Dual Src-Abl inhibitory activities of AP24283and AP24163In addition to potently inhibiting Abl kinase, both AP24283 andAP24163 are potent Src kinase inhibitors despite their differentbinding modes (Src IC50 of <0.5 nM for AP24283 and 7.6 nM forAP24163, respectively) (Table 1). Given the high degree of sequencesimilarity between Abl and Src kinase domains, this is perhaps notsurprising. While dasatinib, together with many other DFG-in Ablinhibitors, demonstrates potent Src inhibition, nilotinib has onlyweak Src activity (IC50 of >5 lM) (21). The reason why the DFG-outinhibitor AP24163 exhibits Src inhibitory activity, whereas nilotinibdoes not, remains unclear. Comparison of the interactions of theseinhibitors with the hinge region may provide some rationale. Among



the four inhibitors discussed in detail here, i.e. two DFG-in inhibi-tors (AP24283 and dasatinib) and two DFG-out (AP24163 and niloti-nib), nilotinib is the inhibitor that has least interaction with thehinge region, forming a single hydrogen bond through a monocyclictemplate, and furthermore possesses no Src activity. The remainingthree inhibitors each make two coordinated hydrogen bonds to thehinge region, and form more vdw contacts with conserved residuesin the adenine binding site including with Ala269, and Leu370 fromthe N- and C-lobes respectively and with Leu248 from the P-loop.The presence of these coordinated hinge hydrogen bonds not onlyserves to increase the binding affinities of the inhibitors, but alsoallows the purine and thiazole (of dasatinib) templates to bind moredeeply in the adenine binding pocket. Such increased interactionsappear to make AP24163 molecular recognition more reliant on fea-tures that are conserved between Src and Abl kinases, such asoptimized hinge binding, and conversely less dependent on kinasespecific features, such as the P-loop interactions observed only inAbl kinase.

The importance of forming two hinge hydrogen bonds to retain Srcactivity is further supported by structure-activity relationship withinthe purine arenyl DFG-out compounds. AP24348 is an analog ofAP24163 which lacks the cyclopropylamine group and hence canmake only one hydrogen bond to the hinge. The Src kinase IC50 ofAP24348 is 84 nM, approximately 10-fold worse than the Src IC50 ofAP24163, 7.6 nM (Table 1). The Abl kinase activity of AP24348 ismaintained, however, Abl IC50 = 48 nM, compared to that ofAP24163 Abl IC50 = 25 nM. Although AP24348 has reduced hingeinteractions compared to AP24163 in both Src and Abl, in the caseof Abl this loss may be offset by extra interactions, both directlyand indirectly, to the flexible P-loop. Similar compensatory interac-tions are unlikely to occur in Src, which has an extended P-loopstructure (24,33,34), leading to a marked loss in potency against Src.

Conclusions

Here, we have presented Abl co-structures of two inhibitors,AP24283 and AP24163, which share a common, novel N9-arenylpurine template and which bind in DFG-in or DFG-out binding mode,depending on the template substituents. These structures sharecharacteristic hinge binding features, in combination with respectivefeatures characteristic of a DFG-in or -out targeted inhibitor. Nota-bly, both inhibitors lack formal hydrogen bonds to the Thr315 gate-keeper residue, present in most Abl and Src inhibitors, insteadforming a pseudo-hydrogen bond between the gatekeeper residueand the vinyl linkage present in both compounds. Nevertheless,despite the absence of this conserved hydrogen bond both com-pounds exhibit potent Abl inhibitory activity. The crystallographicco-structures presented here reveal additional binding interactionsthat compensate for the loss of the gatekeeper hydrogen bond. Forthe DFG-in Abl inhibitor, AP24283, these additional bindinginteractions include a pair of coordinated hydrogen bonds from theindazole ring of the inhibitor to the protein, together with additionalinteractions between the P-loop and inhibitor, notably from the sidechain of Tyr 253. In the case of the DFG-out Abl inhibitor, AP24163,the additional interactions include correlated hydrogen bonds to thehinge region of Abl, and interaction with the side chain of the DFG-

motif Phe382, rather than with Tyr 253. Comparison of our DFG-inand DFG-out Abl co-structures with other Abl:inhibitor co-structures,such as those of dasatinib and nilotinib respectively, also revealsdiffering inhibitor molecular recognition within DFG-in and DFG-outclasses. These differences are evident in the P-loop conformationsof the different structures, and particularly in the differing interac-tions of Tyr 253 with specific inhibitors. These structural compari-sons highlight the plasticity of the kinase binding site, andfurthermore the ability of kinases to optimize their binding interac-tions to inhibitors with diverse chemotypes.

Our DFG-out inhibitor, AP24163, exhibits modest activity against theAbl T315I mutant, demonstrating the potential for appropriatelydesigned DFG-out inhibitors to overcome this key mutation. In part,the Abl T315I activity of AP2413 can be attributed to the incorpora-tion of a less sterically demanding vinyl linkage proximal to theincreased bulk introduced by incorporation of an isoleucine sidechain at the gatekeeper position. Abl T315I activity is typically notpresent in optimized DFG-in Abl inhibitors that bind to the selectiv-ity pocket, including AP24283 (20,21), indicating that both an appro-priate linkage and a privileged DFG-out binding element arenecessary to obtain Abl T315I activity. Furthermore, in contrast tothe DFG-out inhibitors imatinib and nilotinib, AP24163 possessespotent Src kinase activity. AP24163 is expected to bind Src in asimilar mode to that observed in Abl, forming two hydrogen bondsto the hinge region in both proteins. Formation of these correlatedhydrogen bonds allows a deeper binding of the template to theadenine site. Such enhanced hinge interaction appears to makeAP24163 less dependent on P-loop interactions than imatinib or nil-otinib for potency, and in consequence, AP24163 is able to retainpotency against kinases with an extended P-loop conformation, asobserved in Src.

It is noteworthy that kinase inhibitors devoid of functionality capa-ble of making hydrogen bonds to the gatekeeper residue are likelyto lose selectivity without incorporation of additional specificityenhancing interactions. For example, AP24163 exhibits a lower IC50

in the Ba ⁄ F3 parental cell proliferation assay compared with niloti-nib (Table 1), even though autophosphorylation data in Abl T315Itransformed Ba ⁄ F3 cells clearly demonstrated inhibition of the AblT315I kinase target (data not shown). Due to its modest activityagainst the Abl T315I mutant, AP24163 is unlikely to be furtheradvanced, hence the critical need for additional inhibitors withimproved potency against Abl T315I remains.

The work presented here offers insights which may prove useful fornew design approaches for inhibitors of Abl kinase mutants, includ-ing the T315I mutant, as well as for dual Src-Abl inhibitors, includ-ing in both cases DFG-out designs.

Experimental Procedures

Cloning, protein expression and purificationThe kinase domain of murine c-Abl spanning from 229 to 515 wasco-expressed in E. coli with Yop H protein tyrosine phosphatase, asdescribed previously (14). Abl and Yop H plasmids were co-trans-formed into BL21DE3 cells. The protein expression was carried out at

Zhou et al.


18 �C. The kinase domain of Abl was co-purified with eitherAP24283 or AP24163 since the addition of either compound wasfound to stabilize the protein in the purification process. A three tofive fold molar excess of compound over the estimated amount ofAbl protein was added to the thawed cell pellets before cell lysis.The purification consists of nickel affinity column (Qiagen, Valencia,CA, USA) followed by TEV treatment to remove the NH2-terminalhexa-histidine-tag, anion exchange column (MonoQ 5 ⁄ 5; GE Health-care, Piscataway, NJ, USA) and size exclusion column (Superdex 75HiLoad 16 ⁄ 60; GE Healthcare). The Abl protein in complex with eithercompound was stored in a crystallization buffer composed of 25 mM

Tris (pH 7.3), 75 mM NaCl, 10% glycerol and 5 mM dithiothreitol(DTT), and concentrated to 25–30 mg ⁄ mL before crystallization trials.

CrystallographyCo-crystals of Abl kinase domain with either AP24283 or AP24163were grown by the hanging drop vapor diffusion method at 4 �Cusing a drop size of 1.2 lL (0.6 + 0.6 lL) from a condition contain-ing 0.1 M Tris–HCl (pH 8.5), 30% w ⁄ v polyethylene glycol 4000, and0.2 M sodium acetate. The crystals were cryoprotected in motherliquor supplemented with 30% v ⁄ v glycerol, and flash-frozen inliquid nitrogen. X-ray diffraction data were collected at beamline19 BM (Advanced Photon Service, Argonne, IL, USA), and indexedand scaled using HKL2000 package (35).

The structures were determined by molecular replacement byAMoRe (36) using one monomer from the Abl:dasatinib complex(Protein Data Bank code 2GQG) as the search model for Abl:AP24283and the Abl:imatinib (Protein Data Bank code 1IEP) for Abl:AP24163,respectively. Both complex crystals belong to space group P21, butwith different unit cell dimensions (Table 2). Two molecules occupyin the asymmetric unit in both Abl:AP24283 and Abl:AP24163. Theelectron density for AP24163 as well as those Abl residues sur-rounding the inhibitor is well resolved (Figure 1C), as is the electrondensity for Abl:AP24283, leaving no ambiguities for the bindingmode of the inhibitors. The structures were refined with CNX (Accel-rys Inc., San Diego, CA, USA) combined with manual rebuilding inQuanta (Accelrys). After several cycles of refinement and modelbuilding, each compound was built into the density. Further refine-ment in CNX and Quanta was carried out until convergence wasreached. The data collection and refinement statistics are summa-rized in Table 2. The final model for Abl:AP24283 consists of resi-dues from 229 to 514 except 275–279 (which are disordered) formolecule A, and from 229 to 501 except 274–276 for molecule B,with 515 of molecule A and 502–515 of molecule B missing in thedensity. For Abl:AP24163, both molecules possess residues from 229to 511 plus an additional Gly at its NH2-terminus (numbered 228)due to the cloning strategy, with residues 512–515 being disordered.In Abl:AP24163, an alternative conformation was observed forAsn231 in molecule A located in a loop at the NH2-terminus.

Compound synthesis and activity assayThe synthesis of AP24283, AP24163 and their analogs have beenreported elsewhere (27,28). The in vitro kinase assay and cell prolif-eration assay using Ba ⁄ F3 cells were performed according to proto-cols described previously (27).

Accession numbersAtomic coordinates have been deposited with the Protein Data Bankand are available under the following accession codes: 3KF4 forAbl:AP24283 and 3KFA for Abl:AP24163.

Acknowledgments

The authors would like to thank Shuangying Liu and R. MathewThomas from the ARIAD Chemistry department for their assistancein preparing AP24283 and AP24163 and their analogs, as well asJeff Keats and Qihong Xu of the ARIAD Biology department for pro-viding enzyme and cellular assay data. In addition we acknowledgeFeng Li and Allyn Martin for their technical support, Manfred Weig-ele, David Berstein and Victor Rivera of ARIAD for their commentsand enthusiastic support of this work.

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Documents

Structural Analysis of DFG-in and DFG-out Dual Src-Abl Inhibitors Sharing a Common Vinyl Purine Template