chemical biology & drug design volume 70 issue 1 2007 [doi 10.1111_j.1747-0285.2007.00535.x] jeffrey...

8/12/2019 Chemical Biology & Drug Design Volume 70 Issue 1 2007 [Doi 10.1111_j.1747-0285.2007.00535.x] Jeffrey R. Huth

1/12

Discovery and Design of Novel HSP90 InhibitorsUsing Multiple Fragment-based Design Strategies

Jeffrey R. Huth, Chang Park, Andrew M.Petros, Aaron R. Kunzer, Michael D.Wendt, Xilu Wang, Christopher L. Lynch,Jamey C. Mack, Kerry M. Swift, Russell A.Judge, Jun Chen, Paul L. Richardson, ShaJin, Stephen K. Tahir, Edward D.Matayoshi, Sarah A. Dorwin, Uri S. Ladror,Jean M. Severin, Karl A. Walter, Diane M.Bartley, Stephen W. Fesik, Steven W.Elmore and Philip J. Hajduk*

Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, IL 60064, USA*Corresponding author: Philip J. Hajduk, [email protected]

The molecular chaperone HSP90 has been shownto facilitate cancer cell survival by stabilizing keyproteins responsible for a malignant phenotype.We report here the results of parallel fragment-based drug design approaches in the design ofnovel HSP90 inhibitors. Initial aminopyrimidineleads were elaborated using high-throughputorganic synthesis to yield nanomolar inhibitors ofthe enzyme. Second site leads were also identiedwhich bound to HSP90 in two distinct conforma-

tions, an open and closed form. Intriguingly,linked fragment approaches targeting both ofthese conformations were successful in producingnovel, micromolar inhibitors. Overall, this studyshows that, with only a few fragment hits, mul-tiple lead series can be generated for HSP90 dueto the inherent exibility of the active site. Thus,ample opportunities exist to use these lead seriesin the development of clinically useful HSP90inhibitors for the treatment of cancers.

Key words: Drug Design, Heat Shock Protein, NMR Screening,Structure-Based Drug Design

Received 14 May 2007, revised and accepted for publication 4 June2007

The codependence of many cancer-related proteins on heat shockprotein 90 (HSP90) suggests that inhibitors of this molecular chaper-one would have broad efficacy across many tumor types (1). Indeed,numerous oncoproteins including Cdk4, Akt, BCR-ABL, mutated P53,and v-src are known to be client proteins of HSP90 (for review seeRef. 2). In fact, the chaperone activity of HSP90 is so extensive thathundreds of proteins were found to interact with HSP90 in a com-

prehensive yeast protein interaction study (3). This biologythat inhibitors of HSP90 would be broadly toxic with little sbetween normal and malignant cells. For reasons that are npletely understood, HSP90 inhibitors do exhibit a tolerabmargin. For instance, derivatives of the natural product gmycin, such as 17-allylamino-17-demethoxygeldanamycin show appropriate safety in animal models and in man (2). be due to the dependence of cancer cells on higher levmolecular chaperones and the subsequent concentration oaffinity HSP90 inhibitors in these cells (4). Alternativelyand related inhibitors may selectively target HSP90 complare specific to tumor cells (5).

The structure of HSP90 is complex and affords multiple avdrug targeting. It is composed of three domains that assema dimer (6,7), which then forms a scaffold for co-chaperonOf particular interest has been the N-terminal ATPase where the natural product inhibitors geldanamycin (9) and (10) have been shown to bind. Thus, HSP90 inhibitors cougeted to the active site as well as numerous proteinprotein tion sites known to stabilize the functional complex anprotein binding. In addition to these sites, relevant conformchanges are known to occur upon ATP binding (7,11), openpossibility to target transient conformational states of the pr

A significant limitation in the clinical use of geldanamycinrelated natural product derivatives 17-AAG and 17-DMAassociated liver toxicity and difficult formulation that hinadministration (2). Because the liver toxicity is thought to bto the quinone ring structure rather than direct inhibition HSP90 (2), it has been of high interest to find alternativmolecule inhibitors that are completely distinct from geldaThis approach seems to be successful given that a numberine- and pyrazole-based inhibitors that bind to the geldansite have been developed (12). Here, we report the use oment-based drug design (13,14) in the discovery of aminopderivatives that inhibit HSP90. This study shows that wit

few fragment hits, multiple distinct lead compounds can structed with the aid of structural studies, particularly whformational flexibility exists in the binding site.

Results

Identication of rst site ligands In order to identify ligands that can serve as starting pothe development of HSP90 inhibitors, an NMR-based scre

1

Chem Biol Drug Des 2007; 70: 112Research Article

2007 The Authors Journal compilation 2007 Blackwell Munksgaard

doi: 10.1111/j.1747-0285.2007.00535.x


2/12

N-terminal domain of human HSP90 was conducted using a library of11 520 compounds with an average molecular weight of 225 Da.Chemical shift changes of Leu, Val, and Ile methyl groups in the pres-ence of compound were detected in two-dimensional 1H/13 C hetero-nuclear single quantum correlation (2D HSQC) NMR experiments andused to confirm compound binding (15). As shown in Table 1, tworelated chemotypes were discovered, an aminotriazine (representedby 1 ) and an aminopyrimidine (represented by 2 ) series. The bindingaffinities of these compounds were measured in two ways. NMRtitration studies (16) indicated that compound 2 binds with a K D of20 l M. This is in agreement with the results of a complementaryFluorescence Resonance Energy Transfer (FRET) assay where the K iwas determined to be 18 l M. The binding of the larger aminotriazine(compound 1 ) was found to be 100-fold more potent. By NMR thecompound was in slow exchange, consistent with the K i of 0.32 l Mmeasured in the FRET assay. Both of these leads were attractivestarting points for affinity optimization since the potencies relative tothe molecular weights are high. One method to quantify this relation-ship is calculate a binding efficiency index (BEI; 17), where theBEI ) 1000log(K i)/(MW). As an example, a drug with a molecularweight of 350 Da and a potency of 1 nM can be described with a BEIof 25.7. By comparison, the binding efficiencies of the compoundsidentified in the HSP90 screen are high (21 for compound 1 and 27for compound 2 ; Table 1). In order to improve the binding affinitiesof these lead compounds, a structure-based drug design approach

was taken. The structure of HSP90 when bound to the aminopdine is similar to that reported for the ADP-bound state Figure 1A). The exocyclic amino groups of 2 and the adenine ring ofADP both make hydrogen bounds to the side chain of D79 as wa bound water molecule. A second water molecule also makes structural hydrogen bond to the N3 pyrimidine nitrogen of 2 , analog-ous to the hydrogen bond observed to the N1 of adenine. Unexedly, the naphthyl-substituted aminotriazine was found to induconformational change that resulted in opening up a larger binsite even though the key hydrogen bonds from the triazine rithe water molecules and D79 were unaffected (Figure 1B). Tha clear example of induced-fit binding that was difficult to prfrom the structure of the ADP complex alone (18). Furthermosuggested that additional binding potency could be obtained bymally filling the pocket of this open conformation of the catdomain. The structures in Figure 1 indicated that the aminotriand aminopyrimidine rings bind in a similar way to HSP90 andlikely be interchanged in second-generation compounds. The haffinity of compound 1 and the associated structural results made itclear that aromatic substitution of the heterocycle could increasbinding affinity. The features of both leads were incorporatedhigh-throughput chemical approach in which the trifluorometh 2was replaced by a variety of aromatic groups according to ScheOne hundred and twenty-eight compounds with aryl substituat the four position of the aminopyrimidine were prepa

Table 1: HSP90 inhibitors identified from NMR fragment-based screening

Number Structure NMR K D (l M) FRET K i (l M) MW BEIa

1

N

N

Br

N

NH2

NH2


3/12

Compound 4 (Table 2) was among the most potent compoundsdiscovered with a K i of 170 nM. Minor modification by replacing the4-methyl with a 4-chloro substituent resulted in a threefold increasein potency (K i of compound 5 60 nM). Interestingly, the resultingbinding efficiency for compound 5 (BEI 26) is very close to thatobserved for the initial hit (compound 2 , BEI 27; Table 1), consis-tent with the general observation that binding efficiencies tend toremain constant when optimal substituents are added to a fragment

core (19). Overall, by combining features of two fragment lehigh-throughput organic synthesis (Scheme 1), a highly opotent, low-molecular weight lead was discovered.

Discovery of a second site ligand While elaboration of 2 into nanomolar inhibitors was successthe chemical diversity that could be explored was severely

by the availability of chemical reagents compatible with thistry shown in Scheme 1. Moreover, the aminopyrimidipounds, including 5 , were subsequently reported in a pateapplication (see international patent application WO 2006In order to identify more novel substituents for the adjacenlibrary of 3360 compounds with an average molecular w150 Da was screened by 2D NMR for the ability to bind tin the presence of saturating amounts of compound 2 . The mostpotent hit identified in the screen was compound 3 (containing afuranone moiety), which binds to HSP90 with a K D of 150 l M inthe presence of compound 2 (Table 1). The binding of 3 was foundto be co-operative, as the observed K D is >5000 l M in the absenceof compound 2 . The furanone hit satisfied the initial objectivthe screen as it was novel, had not been investigated in lead optimization, and was known to bind with significant a

Structure-based linking strategy In order to guide the linking of the furanone (3 ) to the aminopy-rimidine (2 ), structures of the ternary complex were solveboth NMR and X-ray crystallography. The crystal s(Figure 2A) showed a p-stacking interaction between the phenof compound 3 and the pyrimidine ring of compound 2 , which isconsistent with the co-operativity observed in NMR experiments. To accommodate the second site ligand, no cational change in HSP90 relative to binding of compound 2 alonewas observed. Unexpectedly, the NOE data from NMR expcollected in parallel structural studies could not be reconcithis X-ray crystallographic structure. In particular, unamNOEs were observed between the furanone moiety of 3 andL103, L107, F138, and V150 of HSP90 (see Figure 2NOEs can only be accommodated if the furanone inducesconformation similar to that observed for compound 1 (Figure 1B).In fact, simple docking of compounds 2 and 3 to the proteinpocket formed by compound 1 (Figure 1B) yielded a ternary coplex that satisfied all of the observed intermolecular NOillustrated in Figure 2B, this alternative conformation pafuranone ring against the hydrophobic side chain of L10undergoes a v1 rotation upon remodeling of the binding poThis binding orientation was also consistent with the cotivity observed in NMR titration experiments because the phenyl ring was calculated to be in van der Waals contacompound 2 (Figure 2B).

As it was clear from the structural studies that compound 2 and3 could bind to both the open and closed conformationsHSP90 N-terminal domain, two separate linking strategies wsued. For the stacked orientation illustrated in Figure 2A, thgroup must bend by 180. Based on this geometry, a methylsulfamide linker was suggested to link the aminopyrimidine

A

B

D79

D79

Figure 1: X-ray crystallographic structures of NMR screening hitsbound to the N-terminal domain of HSP90. Grey surfaces depict thecompound binding site for two of the conformations of HSP90N-terminal domain that were observed. Atom coloring: carbons inmagenta, nitrogens in blue, and fluorines in light blue. Structuralwaters shown as red spheres. Hydrogen bonds indicated with dot-ted lines. (A) Compound 2 bound to a 'closed' conformation. (B)Compound 1 bound to the 'open' conformation where a larger bind-ing pocket is formed, sufficient to accommodate the naphthyl sub-

stituent of compound 1 .

Discovery and Design of Novel HSP90 Inhibitors

Chem Biol Drug Des 2007; 70: 112 3


4/12

m -position of the furanone phenyl ring to generate compound 6 .This design (Scheme 2) yielded a 10-fold improvement in potencyrelative to compound 2 alone, indicating that favorable interactionsbetween HSP90 and the furanone in the linked state occurred.

Indeed, an X-ray crystallographic structure of the linked com(Table 2, compound 6 ) bound to HSP90 confirmed that the two fragments bind to the protein in the same way in the linked aunlinked states (Figure 2C).

NNCl

NH2

ArB

OH

OH

NN

NH2

Ar Pd(PPh 3)4, CsF

70C, DME / MeOH

Scheme 1: Synthesis of 6-methyl-pyrimidin-2-ylamine library (example: compound 4 )

Table 2: HSP90 inhibitors from fragment-based lead optimization

Number Structure FRET K i (l M) MW BEIa

4

NN

NH2

Cl

Cl

0.17 (0.01) 255 26

5 NN

NH2

ClCl

Cl 0.06 (0.01) 274 26

6

N

NNH2

NH

SOOHN

O O

1.9 (0.2) 389 15

7

O N

ON

N

N

NH2

4.0b (1.0) 321 17

a Binding efficiency index (BEI). See legend of Table 1 for a description.bCalculated from a direct binding assay where the fluorescence of 7 was detected (see Methods and Materials).

Huth et al.

4 Chem Biol Drug Des 2007; 70: 112


5/12

The design of a furanone-based inhibitor for the open form ofHSP90 (Figure 2B) was also pursued. Here, the NMR structuralstudies indicated that a 90 twist was required to position the fura-none near L107 in the open conformation. Twelve compounds weresynthesized based on this structural information. An acetylene linkerfrom the aminopyrimidine to the o -position of the phenyl ring of3-(benzylideneamino)oxazolidin-2-one, an analog of compound 3 ,satisfied this geometry (Scheme 3), and resulted in a five-foldincrease in potency (Table 2, compound 7 ). X-ray crystallographic

studies confirmed that this inhibitor does indeed bind to tconformation of HSP90 as designed (Figure 2D).

Discussion

Flexibility of HSP90 The compounds reported here probe various conformationHSP90 N-terminal domain that may be targeted by the

BA

L107L103 F138V150

C D

Figure 2: Binding of fragment hits compared to binding of linked compounds. Atom coloring for the fragment hitsnitrogens in blue, oxygen in red, and fluorines in light blue. Atom coloring for the linked compounds is the same exorange. (A) X-ray crystallographic structure of a ternary complex with compounds 2 and 3 . (B) NOE-based model of the same ternary cowith compounds 2 and 3 showing an alternate binding mode observed in solution. The side chains of L107, L103, F138, anin red to indicate key NOEs from HSP90 to 3 that were used to construct the model. (C) X-ray crystallographic structure of a bwith compound 6 overlaid with the ternary complex shown in A. Note the near perfect alignment of the linked and unliX-ray crystallographic structure of a binary complex with compound 7 overlaid with the model of the ternary complex shown in B. the oxazolidinone accesses the back of the pocket in an 'open' conformation of HSP90 as suggested by the NOE-base




6/12

agents. While two states of the domain are depicted in Figure 1,a much broader range of conformations are sampled by the pro-tein. For instance, X-ray crystallographic studies of numerous com-plexes identified at least six distinct conformations for aminoacids A101A123 (C. Park, unpublished observations). Furthermore,most of the complexes studied required co-crystallization with the

compound of interest, and multiple crystal forms were obse(R. Judge, unpublished observations). Finally, using NMR spscopy, the backbone assignments could not be made for amacids 105121 because of extensive line broadening, whicconsistent with protein mobility (J. Huth, unpublished obstions). Taken together, these data indicate a high level of pro

O

O

O

OO

O

O Na

NNCl

NH2

NN

NH2

NC

NN

NH2

NH2 SCl

O

ONO 2

NN

NH2

NH

S

O

OO2N

NN

NH2

NH

S

O

ONH2

O

O

O Na

NN

NH2

NH

S

O

ONH

OO

NaH, EtOH

E+Z Z

Zn(CN) 24Pd(PPh3)

DMF, 80C(77%)

H2 (60 psi)10% Pd/C

EtOAc / AcOH(97%)

AcOH

NEt 3

DCM(20%)

Dioxane / DMF(95%)

SnCl 2. H2O

NMP / EtOH(49%)

AcOH(40%)

6 (E only)

Scheme 2: Synthesis of N -(2-amino-6-methyl-pyrimidin-4-ylmethyl)-3-{[2-oxo-dihydro-furan-(3E)-ylidenemethyl]-amino}-(compound 6 )

Huth et al.



7/12

flexibility that may explain how two different binding modes ofcompound 3 could be captured using both NMR and X-ray crystal-lography.

Implications for fragment-based drug design Particularly for fragment-based drug design, structural information isan important component for guiding the chemistry of elaborationand linking (20). However, the X-ray crystallographic structure of thepyrimidine (compound 2 ) alone did not reveal the binding site thatcould be induced and productively exploited by compound 1 , northe subsequent optimization leading to compounds 4 and 5 . Inaddition, the two observed binding modes for the ternary complexwith 2 and 3 (Figure 2A,B) were completely unanticipated and, toour knowledge, unprecedented. The fact that both binding modescould be productively exploited to develop parallel lead series thatbind with higher affinity to the protein as designed is ample evi-dence that both modes can in fact be accessed by the ternary com-plex. While the structural surprises highlight the remarkableinsights that structural data can provide on ligand binding, they alsocaution against the rigid interpretation of any single piece of struc-tural data. It was by taking advantage of multiple structures (andeven multiple structural techniques) that the two avenues foroptimization were identified.

The drug discovery case study described here adds to the list ofexamples where fragment-based drug design was used to iden-tify novel lead compounds (20,21). However, this case study also

illustrates the inherent challenge in linking two fragmenin such a way that the individual binding energies are ized. We have previously reported that the observed baffinity for a linked compound is, on average, one log uthan the sum of the pK D values (negative base-10 logarithmthe K D) for the two compounds (22). Using this rule of the expected affinity that could be obtained upon link 2(pK D 4.7) and 3 (pK D 3.8) would be approximately 30 M(pK D 7.5). The observed K i values of 1.9 and 4 l M (Figure 3)would suggest that the linkers used to form 6 and 7 from 2and 3 are not yet optimal. This is likely due, at least into the limited number of linkers that were evaluated fobinding mode (one for the stacked arrangement and 12 other). It is also interesting to consider the binding efindices of the linked compounds. The values obtained fpounds 6 and 7 (BEI values of 15 and 17, respectively) arenificantly lower than the binding efficiency of 27 for t(Figure 3), suggesting that the furanone and oxazolgroups, as linked, contribute very inefficiently to the energy (19).

Concluding Remarks

We have described how multiple fragment-based drug desigegies capitalized on the same set of NMR screening hits tothree distinct HSP90 lead inhibitors (Figure 3). In one apfragment elaboration strategy, guided by X-ray crystall

N

N

NH2

Cl

O

N

N

NH2

O

NO

O

NH2

N

N

NH2

NNO

O

Pd(PhCN) 2Cl2CuI[HP(tBu) 3]BF4DIPEA

Dioxane, 50C(91%)

AcOH

EtOH, 75C(21%)

7

Scheme 3: Synthesis of 3-{[1-[2-(2-amino-6-methyl-pyrimidin-4-ylethynyl)-phenyl]-meth-(E)-ylidene]-amino}-oxazol 7 )




8/12

structures of HSP90 ligand complexes and high-throughput organicsynthesis, resulted in the rapid discovery of a 60 nM inhibitor(Figure 3A). In a parallel approach, SAR by NMR and X-ray crystal-lography were effective in the discovery of two additional series(Figure 3B). Surprisingly, the two structural methods identified dis-tinct binding modes for fragments in a ternary complex. In onecase, binding to the open conformation of the N-terminal domainwas observed. For the other, we observed binding to a closed con-formation. With further optimization, one or more of these seriescould result in HSP90 inhibitors with clinical utility for the treat-ment of tumors that rely on this chaperone for proliferation andsurvival.

Methods and Materials

Protein preparation The gene for amino acids D9-E236 of human HSP90 (Geaccession number NM_005348) was cloned into pET28a resin the fusion of (MGSSHHHHHHSSGLVPRGSHM) to the -end ofthe gene. For NMR screening and titration studies HSP90 expressed in Escherichia coli BL21(DE3) cells and labeled with13

C at the methyl groups of valine and leucine and the d-methylof isoleucine by including [3-C-13]-a-ketobutyrate and [3,3-C-13]-a-ketoisovalerate in the medium (15). For NMR structural stthis clone of HSP90 was expressed in E. coli BL21(DE3) with15 N-ammonium chloride and 13 C-glucose as the sole nitrogen andcarbon sources. The protein was purified by nickel affinity matography and dialyzed into 30 mM sodium phosphate buffer,pH 7.2, 50 mM NaCl, 5 mM DTT, and 2 mM MgCl2 for all NMRstudies. For X-ray crystallography, HSP90 (6His-(Thr)-(9236)]) was expressed in E. coli , and purified by nickel affinitychromatography. Subsequently, the poly-HIS tag was cleavedbiotinylated thrombin, the thrombin removed with straptavidarose, and the poly-HIS with a second nickel affinity purificThe cleaved HSP90 was then purified by gel filtration chromraphy using an S100 column in 25 mM HEPES buffer, pH 7.5,100 mM NaCl, and 1 mM sodium azide, and concentrated to50 mg/mL.

NMR-based screening Nuclear magnetic resonance-based screening (23) of HSP90conducted by acquiring 1H13 C HSQC spectra of 2550 l M proteinsamples. About 11 500 compounds of diverse structure withaverage molecular weight of 225 Da were solvated in 2H-DMSOto 1 M. Mixtures of 10 compounds were then randomly prepain 96-well plates (primary library). A second library of miwhere each mixture was prepared by combining three mixtur10 was prepared in 96-well plates. The final concentrationeach compound in the mixture was 33.3 mM. For screening, 6 l Lof each 30-compound mixture was mixed with 0.5 mL of a 2 l Mprotein NMR sample resulting in 400 l M of each compound and1.2% DMSO. Subsequently, mixture hits were deconvolutemixtures of 10 using the primary library. A second roundeconvolutions, where individual compounds were tested400 l M, resulted in identification of the fragment hits. Specwere acquired in 1530 min with 38 complex points on a B(Bruker Biospin, Billerica, MA, USA) 500 MHz spectrequipped with a cryoprobe. For titration studies, HSQC spec25 l M protein samples were acquired with 20, 40, 60, 120, 2or 600 l M compound 2 ; 20, 50, or 100 l M compound 1 , whichwas found to be in slow exchange on the NMR time-scale.the second site screen, 3360 compounds with an average mollar weight of 150 Da were screened at 5 mM in mixtures of fivecompounds against 50 l M HSP90 in the presence of 200 l Mcompound 2 (final concentration of DMSO 3%). Mixtures thatcaused additional chemical shift changes in addition to thcaused by compound 2 were deconvoluted by testing individualcompounds at 5 mM in the presence of 200 l M compound 2 . Thebinding affinity of compound 3 was measured by NMR titration

Fragment elaboration approachA

B Linked fragment approaches

Closed state Open state

N

NBr

N

NH2

NH2 N

N

CF 3

NH2

NN

NH2

Cl

Cl

Cl

O

O

NH N

N

CF 3

NH2

NN

NH2

NH

SOOHN

O O

O N

ON

N

N

NH2

1

BEI = 21

2

BEI = 27

5

BEI = 26

3

2

BEI = 27

6Ki= 1.9 MBEI = 15

Ki= 4 M

Ki= 18 M

Ki= 0.06 M

Ki= 0.32 M

KD= 150 M

BEI = 17

7

Open state

Figure 3: Hit-to-lead approaches using NMR screening hits.Structural studies were used to guide (A) the high-throughputorganic synthesis that incorporated a fragment elaboration approachand (B) the linked fragment approaches as described in the text.Atoms are colored in blue and green to highlight components fromeach lead, and in red to identify linking groups (panel B only).Atoms in the NMR hits that are colored in black were not used inthe final compounds. Binding efficiency indices were calculated asdescribed in the legend for Table 1.

Huth et al.



9/12

studies using 50, 150, 400, 1500, or 5000 l M compound 3 in thepresence and absence of 200 l M compound 2 . Dissociation con-stants were obtained by monitoring the chemical shift changes asa function of ligand concentration using a single binding sitemodel. A least-squares grid search was performed by varying thevalues of K D and the chemical shift of the fully saturated protein.Errors in the dissociation constants were obtained using a MonteCarlo simulation of the data assuming a Gaussian distribution for

errors in chemical shifts with a standard deviation of 0.01 p.p.m.100 Monte Carlo simulations were performed for each dissoci-ation constant, and the reported errors are the standard devia-tions of the simulated values.

Fluorescence Resonance Energy Transfer assay Compounds were serially diluted into assay buffer containing50 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.05%Triton-X-100, 0.04 l M Hsp90, 0.08 l M geldanamycin-biotin, 0.08 l Mstreptavidin-APC (Perkin-Elmer, Catalog number R130-100, Perkin-Elmer, Waltham, MA, USA), and 0.001 l M Eu-W1024-labeled anti-6XHis antibody (Perkin-Elmer, Catalog number AD0110) for 3 h atroom temperature. In the (geldanamycin-biotin/streptavidin-APC/HSP90/Eu-labeled anti-6XHis antibody) complex, fluorescence energytransfer occurs from europium on the anti-6XHis antibody to APCon the streptavidin. Following excitation of Eu at 340 nM, the fluor-escence transfer was detected as the ratio of fluorescence intensityat 655 nm (emission of APC) to that at 615 nm (emission of Eu)using the Envision plate reader (Perkin-Elmer). A decrease in fluor-escence energy transfer occurred upon displacement of geldanamy-cin-biotin with added compound. The resulting decrease influorescence energy transfer was used to calculate compound IC50values with the GRAPHPAD software package. The average and stand-ard deviations of replicate values are reported.

Fluorescence spectroscopy for compound 7 binding An assay for the inhibition of HSP90 by compound 7 was per-formed using the intrinsic fluorescence of the compound in themanner like that used to monitor warfarin binding to a singlebinding site on albumin (24). Compound 7 has broad, weak fluor-escence emission. Using an excitation wavelength of 325 nm, thefluorescence spectral maximum of the compound shifts uponbinding from 415 to 395 nm, and intensifies approximately 3.5-fold. Three titrations were compared, using 2, 5, and 20 l Mcompound and titrating the protein to 90 l M. After subtractingbackground fluorescence arising from the protein, the K D wascalculated to be 5.1, 3.0, or 2.8 l M, respectively, using theintensity at 390 nm. The independence of the K D on compoundconcentration supports the assumption that 7 binds to a singlesite on HSP90.

Model of ternary complex based on NMR structural studies All NMR experiments were carried out at 30 C on either a Bruker(Bruker Biospin) DRX500 or DRX600 spectrometer, both with a cryo-probe accessory. The side chain resonances of L103, L107, and Val

150 of HSP90 were assigned using data from a 13 C-edited, 12 C-fil-tered NOESY spectrum and a 13 C-edited NOESY spectrum (80 monds mixing time for both) recorded on a complex of Val(d1) methyl-protonated 15 N-, 13 C-, and 2H-labeled HSP90(9-236) (2with unlabeled ADP and 5 mM MgCl2 , in conjunction with the crystal structure of Mg-ADP-bound HSP90 [pdb 1BYQ (18)]the side chain resonances of F138 were assigned from NOrecorded on a uniformly 13 C- and 15 N-labeled sample of HSP0 i

conjunction with the crystal structure. NMR samples of thcomplex of HSP90 with compounds 2 and 3 were 1 mM in protein,1.6 mM in compound 2 , and 2.4 mM in compound 3 . Protein-ligandNOEs were extracted from 13 C-edited, 12 C-filtered NOESY spectr(80 mseconds mixing time) recorded on either uniformly 13 C- and15 N-labeled protein, or Val, Leu, Ile (d1) methyl-protonated 15 N-,13 C-, and 2H-labeled protein.

For the model of the ternary complex, compound 2 was first dockedinto the binding site of HSP90 (open conformation) basecrystal structures of compound 1 and that of compound 2 alone(Figure 1). This was performed with the program INSIGHT II (Accelrys,San Diego, CA, USA). Compound 3 was then manually docked intthe HSP90, ATP-binding site based on eight observed intermNOEs. Only the open conformation of the binding site watent with the observed intermolecular NOEs.

X-ray crystallography X-ray diffraction data were collected at the APS 17 BMbeamline of Argonne National Laboratory. Co-crystallizaused to obtain crystal structures for each of the compoundpound stock solutions were made by dissolving compdimethyl sulfoxide (DMSO). Compound and protein wplexed in a 3:1 molar ratio, giving a final DMSO concent2.42.7% (v/v) and incubated overnight at 4 C prior to crystalliza-tion. Crystallization was performed using the vapor d(hanging drop) technique. The drops were made using l L ofprotein solution combined with 2 l L of reservoir solution and supended over a 1 mL reservoir. Crystals grew within 4 4 C. For compound 1 , crystals grew from 30 mg/mL protein a reservoir solution of 22% (w/v) PEG 8000, 0.1 M sodium cacody-late pH 6.5, 0.2 M ammonium sulfate [adapted from publiconditions (9)]. Crystals were cryo-protected using reservtion with 25% (v/v) glycerol. The crystal diffracted twith space group P43212 (a b 118.8, c 180.8, a b c 90 ). Compounds 2 and 3 were obtained as a ternary complex. Protein was incubated with compound 2 overnight with acompound to protein molar ratio of 4:1. Compound 3 was thenadded at a compound to protein molar ratio of 6:1 and infor 6 h. Crystallization was performed using protein at 35with a reservoir of 2230% (w/v) PEG 4000, 0.1 M TrisHCl pH8.5, 0.2 M MgCl2 [adapted from published conditions (9)]. Thetals were cryo-protected by increasing the amount of PEG35% (w/v) and adding 10% (v/v) glycerol. In this instancompounds were present in the cryo-protectant soluti0.5 mM. The crystal diffracted to 1.7 with space group P1212(a 64.16, b 88.76, c 98.45, a b c 90). For com-pound 6 , crystals grew from 40 mg/mL protein with a ressolution of 2028% (w/v) PEG 3350, 0.1 M Bis-Tris-Propane pH




10/12

6.5, 0.2 M sodium chloride. The crystals were cryo-protected usingreservoir solution with 15% (v/v) glycerol. The crystals diffractedto 1.9 with space group C2 (a 113.35, b 89.14, c 64.46,a 90 , b 121.45, c 90 ). For compound 7 , crystals grewfrom 40 mg/mL protein with a reservoir solution of 818% (w/v)polyethyleneglycol monomethyl ether (PMME) 2000, 0.1 M sodiumcacodylate pH 6.5, 0.2 M MgCl2 [adapted from published condi-tions (26)]. Crystals were cryo-protected by increasing the PMME

concentration to 35% (w/v). The crystal diffracted to 1.8 withspace group I222 (a 66.79, b 91.38, c 98.34, a b c 90 ).

Chemistry

Geldanamycin-biotinGeldanamycin-biotin was synthesized with minor modificationsfrom the method described previously (27). The product was veri-fied pure (>95%) by reverse-phase HPLC and identified by elec-trospray ionization mass spectrometry ( MESI-MS), m/z 1097.7[(M + H)+].

4-(2,4-Dichloro-phenyl)-6-methyl-pyrimidin-2-ylamine (compound 4)To a mixture of 2-amino-4-chloro-6-methylpyrimidine (143 mg,1.0 mmol) and Pd(PPh3)4 (58 mg, 0.05 mmol) in DME (4 mL) andMeOH (2 mL) was added 2,4-dichlorophenylboronic acid (228 mg,1.2 mmol) immediately followed by CsF (456 mg, 3.0 mmol). Themixture was purged with argon, sealed and heated up to 70 Cfor 12 h. The mixture was diluted with EtOAc (200 mL) andwashed with water then brine. After drying over Na2SO4, evapor-ation of the solvent gave crude product, which was dissolved inDMSO and MeOH (1:1, 3 mL) and purified by preparative HPLC ona Waters Nova-Pak (Waters, Milford, MA, USA) HR C18 6 l m60 Prep-Pak cartridge column (40 100 mm) to give 4 as themono TFA salt (328 mg, 89%). 1H-NMR (300 MHz, DMSO-d6) dp.p.m. 7.75 (t, 1H), 7.56 (m, 2H), 6.77 (s, 1H), 2.33 (s, 3H). MS(ESI) m/e 252/254, 254/256. Analytical calculated forC11 H9Cl2N3C2HF3O2: C 42.41%, H 2.74%, N 11.41%; found: C42.66%, H 2.44%, N 11.36%.

4-(2,4-Dichloro-phenyl)-6-chloro-pyrimidin-2-ylamine (compound 5)To a mixture of 2-amino-4,6-dichloropyrimidine (163 mg, 1.0 mmol)and Pd(PPh3)4 (58 mg, 0.05 mmol) in DME (20 mL) and MeOH(10 mL) was added 2,4-dichlorophenylboronic acid (228 mg,1.2 mmol) immediately followed by CsF (456 mg, 3.0 mmol). Themixture was purged with argon, sealed and heated up to reflux for4 h. The reaction mixture was evaporated under vacuum, dissolvedin DCM (200 mL), washed with water, brine, and dried on Na2SO4 .Solvent volume was reduced on vacuum, and the residue was puri-fied on silica gel (EtOAc:hexanes, 1:4 to 1:2) to give 5 (135 mg,49%). 1H-NMR (300 MHz, CDCl3) d p.p.m. 7.55 (d, 1H), 7.51 (d, 1H),7.36 (dd, 1H), 7.01 (s, 1H), 5.34 (s, 2H). MS (ESI) m/e 272/274,274/276.

N -(2-Amino-6-methyl-pyrimidin-4-ylmethyl)-3-{[2-oxo-dihydro-furan-(3E)-ylidenemethyl]-amino}-benzenesulfonamide (compound 6)A mixture of c-butyrolactone (12.00 g, 139.4 mmol) and ethyl fmate (10.33 g, 139.4 mmol) was added dropwise to a solutiosodium hydride (60%, 5.85 g, 146.4 mmol) and ethanol (1 m1,4-dioxane (360 mL) and DMF (45 mL). After the addition wplete, the solution was stirred at room temperature overni

Diethyl ether (300 mL) was then added, and the resulting prtate was filtered on vacuum, washed with diethyl ether, and dfurther on vacuum to give a mixture of E- and Z-isomers osodium enolate product: sodium; [2-oxo-dihydro-furan-3-ylmethanolate (18.06 g, 95%). Separately, 2-amino-4-chloro-6-mpyrimidine (12.00 g, 83.6 mmol), Zn(CN)2 (5.40 g, 46.0 mmol), andPd(PPh3)4 (4.83 g, 4.2 mmol) were heated at 80 C in DMF (220 mL)for 72 h. The solution was cooled, added to water (700 mL)extracted with EtOAc (3 300 mL). The combined extracts weredried with brine, then Na2SO4 . Solvent was removed under reducedpressure, and two recrystallizations of the crude material (Etgave 2-amino-4-cyano-6-methylpyrimidine (8.63 g, 77%). 2-Acyano-6-methylpyrimidine (8.62 g, 64.3 mmol) was dissolved Ac (150 mL) and AcOH (150 mL). And, 10% Pd/C (1.72 g, 1was added, and the solution was subjected to an H2 atmosphere(60 psi). The mixture was stirred at room temperature for 2 hvents were removed on vacuum, and the crude material washed with 70% EtOAc (hexanes) to give 2-amino-4-aminom6-methylpyrimidine as the mono AcOH salt (12.51 g, 92-Amino-4-aminomethyl-6-methylpyrimidine mono AcOH (212.0 mmol) and NEt3 (2429 mg, 24.0 mmol) were dissolved in DC(60 mL). 3-Nitrobenzenesulfonyl chloride (2659 mg, 12.0 mmadded, and the mixture was stirred at room temperature 30 min. The solution was added to water (100 mL), adjustedpH of 8, and extracted with EtOAc (3 50 mL). The combinedextracts were dried with brine, then Na2SO4 and concentrated.The material was purified on silica gel (5% MeOH/EtOAgive N -(2-amino-6-methyl-pyrimidin-4-ylmethyl)-3-nitro-benzefonamide (765 mg, 20%). N -(2-Amino-6-methyl-pyrimidin-4-ylmethy3-nitro-benzenesulfonamide (760 mg, 2.4 mmol) was dissolvNMP (4 mL). EtOH (0.2 mL) and SnCl2H2O were subsequentlyadded, and the mixture was heated to 70 C overnight. The solu-tion was cooled, added to water (12 mL), adjusted to a pH and extracted with EtOAc (3 8 mL). The combined extracts weredried with brine, then Na2SO4 and concentrated. The material waspurified on silica gel (5% MeOH/EtOAc) to give 3-aminoN -(2-amino-6-methyl-pyrimidin-4-ylmethyl)-benzenesulfonamide (49%). To 3-amino-N -(2-amino-6-methyl-pyrimidin-4-ylmethyl)-bzenesulfonamide (100 mg, 0.3 mmol) and the previously synth[2-oxo-dihydro-furan-3-ylidene]-methanolate (46 mg, 0.3 mmadded AcOH (3 mL). The solution was stirred at room tempeovernight. The solution was added to water (10 mL), adjustedpH of 7, and extracted with EtOAc (3 5 mL). The combinedextracts were dried with Na2SO4 . The solution volume was reducedon vacuum to precipitate pure 6 exclusively as the E-isomer (53 mg,40%). 1H-NMR (300 MHz, DMSO-d6) d p.p.m. 9.31 (d, 1H)(t, 1H), 7.69 (dt, 1H), 7.57 (t, 1H), 7.517.38 (m, 2H), 7.32 (dt, (s, 2H), 6.38 (s, 1H), 4.32 (t, 2H), 3.80 (d, 2H), 2.89 (td, 2H(s, 3H). Analytical calculated for C17 H19 N5O4S0.5H2O: C 51.25%, H

Huth et al.



11/12

5.06%, N 17.58%; found: C 51.46%, H 5.11%, N 17.37%. Structurewas confirmed by X-ray crystallography (Figure 2C).

3-{[1-[2-(2-Amino-6-methyl-pyrimidin-4-ylethynyl)-phenyl]-meth-(E)-ylidene]-amino}-oxazolidin-2-one (compound 7)2-Amino-4-chloro-6-methylpyrimidine (459 mg, 3.2 mmol), 2-ethynyl-

benzaldehyde (500 mg, 3.8 mmol), and N ,N -diisopropylethylamine(496 mg, 3.8 mmol) were dissolved in 1,4-dioxane (5 mL). Dichloro-bis(benzonitrile)palladium(II) (37 mg, 0.10 mmol), copper(I) iodide(12 mg, 0.06 mmol), and tri-t -butylphosphonium tetrafluoroborate(61 mg, 0.21 mmol) were added, and the solution was heated at50 C overnight. The mixture was cooled, concentrated, and purifiedon silica gel (50100% EtOAc/hexanes) to give 2-(2-amino-6-methyl-pyrimidin-4-ylethynyl)-benzaldehyde (693 mg, 91%). 2-(2-Amino-6-methyl-pyrimidin-4-ylethynyl)-benzaldehyde (50 mg, 0.21 mmol) and3-amino-oxazolidin-2-one (21 mg, 0.21 mmol) were dissolved in eth-anol (1.5 mL) and acetic acid (one drop) was added. The solutionwas heated at 75 C for 105 min. The mixture was cooled, concen-trated, and purified on silica gel (5% MeOH/EtOAc) to give 7(14 mg, 21%). 1H-NMR (300 MHz, DMSO-d6) d p.p.m. 8.08 (s, 1H),7.58 (dd, 1H), 7.65 (dd, 1H), 7.597.47 (m, 2H), 6.77 (s, 1H), 6.71(s, 2H), 4.53 (t, 2H), 4.03 (t, 2H), 2.26 (s, 3H). MS (ESI) m/e 300/322. Structure was confirmed by X-ray crystallography (Figure 2D).

Acknowledgment

The authors would like to thank Dr Jonathan Greer for criticallyreading the manuscript.

Note added in proof

The PDB accession codes for the X-ray crystallography structuresshown in Figures 1 and 2 are: 2QF6, 2QF0, 2QG0, 2QG2.

References

1. Solit D.B., Rosen N. (2006) Hsp90: a novel target for cancertherapy. Curr Top Med Chem;6:12051214.

2. Cullinan S.B., Whitesell L. (2006) Heat shock protein 90: aunique chemotherapeutic target. Semin Oncol;33:457465.

3. Zhao R., Davey M., Hsu Y.C., Kaplanek P., Tong A., Parsons A.B.,Krogan N., Cagney G., Mai D., Greenblatt J., Boone C., Emili A.,Houry W.A. (2005) Navigating the chaperone network: an inte-grative map of physical and genetic interactions mediated bythe hsp90 chaperone. Cell;120:715727.

4. Gooljarsingh L.T., Fernandes C., Yan K., Zhang H., Grooms M.,Johanson K., Sinnamon R.H., Kirkpatrick R.B., Kerrigan J., LewisT., Arnone M., King A.J., Lai Z., Copeland R.A., Tummino P.J.(2006) A biochemical rationale for the anticancer effects ofHsp90 inhibitors: slow, tight binding inhibition by geldanamycinand its analogues. Proc Natl Acad Sci U S A;103:76257630.

5. Kamal A., Thao L., Sensintaffar J., Zhang L., Boehm M.FBurrows F.J. (2003) A high-affinity conformation of Hsptumour selectivity on Hsp90 inhibitors. Nature;425:4074

6. Chadli A., Bouhouche I., Sullivan W., Stensgard B., MN., Catelli M.G., Toft D.O. (2000) Dimerization and Ndomain proximity underlie the function of the molecularone heat shock protein 90. Proc Natl Acad Sci U1252412529.

7. Ali M.M., Roe S.M., Vaughan C.K., Meyer P., PanaretouP.W., Prodromou C., Pearl L.H. (2006) Crystal structuHsp90-nucleotide-p23/Sba1 closed chaperone compleure;440:10131017.

8. Meyer P., Prodromou C., Hu B., Vaughan C., Roe S.M., B., Piper P.W., Pearl L.H. (2003) Structural and functionsis of the middle segment of hsp90: implications for ATlysis and client protein and cochaperone interactionsCell;11:647658.

9. Stebbins C.E., Russo A.A., Schneider C., Rosen N., HPavletich N.P. (1997) Crystal structure of an Hsp90-geldcomplex: targeting of a protein chaperone by an anagent. Cell;89:239250.

10. Roe S.M., Prodromou C., O'Brien R., Ladbury J.E., PPearl L.H. (1999) Structural basis for inhibition of thmolecular chaperone by the antitumor antibiotics radicigeldanamycin. J Med Chem;42:260266.

11. Huai Q., Wang H., Liu Y., Kim H.Y., Toft D., Ke H. (200of the N-terminal and middle domains of E. coli Hsp90 andconformation changes upon ADP binding. Structure;13:57

12. McDonald E., Workman P., Jones K. (2006) InhibitoHSP90 molecular chaperone: attacking the master regucancer. Curr Top Med Chem;6:10911107.

13. Shuker S.B., Hajduk P.J., Meadows R.P., Fesik S.W. (1covering high-affinity ligands for proteins SAR by Nence;274:15311534.

14. Hajduk P.J., Meadows R.P., Fesik S.W. (1997) Drug decovering high-affinity ligands for proteins. Science;278:4

15. Hajduk P.J., Augeri D.J., Mack J., Mendoza R., Yang JS.F., Fesik S.W. (2000) NMR-based screening of proteinsing C-13-labeled methyl groups. J Am Chem S78987904.

16. Hajduk P.J., Sheppard G., Nettesheim D.G., OlejnicShuker S.B., Meadows R.P., Steinman D.H. et al. (1997ery of potent nonpeptide inhibitors of stromelysin usingNMR. J Am Chem Soc;119:58185827.

17. Cele A.Z., Metz J.T. (2005) Ligand efficiency indices posts for drug discovery. Drug Discov Today;10:464469

18. Prodromou C., Roe S.M., O'Brien R., Ladbury J.E., PPearl L.H. (1997) Identification and structural characterthe ATP/ADP-binding site in the Hsp90 molecular chCell;90:6575.

19. Hajduk P.J. (2006) Fragment-based drug design: how bbig? J Med Chem;49:69726976.

20. Hajduk P.J., Greer J. (2007) A decade of fragment-badesign: strategic advances and lessons learned. Nat ReDiscov;6:211219.

21. Erlanson D.A., McDowell R.S., O'Brien T. (2004) Fragdrug discovery. J Med Chem;47:34633482.




12/12

22. Hajduk P.J., Huth J.R., Sun C. (2006) SAR by NMR: an analysisof potency gains realized through fragment-linking and frag-ment-elaboration strategies for lead generation. In: Jahnke W.,Erlanson D.A., editors. Fragment-based Approaches in Drug Dis-covery. Weinheim: Wiley-VCH;p. 181192.

23. Huth J.R., Sun C., Sauer D.R., Hajduk P.J. (2005) Utilization ofNMR-derived fragment leads in drug design. Methods Enzy-mol;394:549571.

24. Maes V., Engelborghs Y., Hoebeke J., Maras Y., Vercruysse A.(1982) Fluorimetric analysis of the binding of warfarin to humanserum albumin. Equilibrium and kinetic study. Mol Pharma-col;21:100107.

25. Goto N.K., Gardner K.H., Mueller G.A., Willis R.C., K(1999) A robust and cost-effective method for the productiVal, Leu, Ile (delta 1) methyl-protonated 15N-, 13C-, 2H-lproteins. J Biomol NMR;13:369374.

26. Wright L. et al. (2004) Structure-activity relationships in pbased inhibitor binding to HSP90 isoforms. Chem Biol;11:77

27. Zhou V., Han S., Brinker A., Klock H., Caldwell J., Gu X.A time-resolved fluorescence resonance energy transfer-b

HTS assay and a surface plasmon resonance-based bindassay for heat shock protein 90 inhibitors. Anal Bchem;331:349357.

Huth et al.


chemical biology & drug design volume 70 issue 1 2007 [doi 10.1111_j.1747-0285.2007.00535.x] jeffrey...

Documents