Heat Shock Protein 90/70 (HSP90/70) Targeted Libraryreported HSP90 and HSP70 inhibitors, isosteric modifications, topological analogues compounds successfully passed through the developed

Heat Shock Protein 90/70 (HSP90/70)

Targeted Library

June, 2012

YAI, ChemDiv, Inc.

The Core Strategy

Analyze the structures of reported HSP90/70 inhibitors (more than 1K

molecules);

► Analyze the structures of HSP90 and HSP70 proteins and conformational

shifts within the active binding site observed upon ligand binding;

► Binding modes and allosteric binding sites identification/analysis;

► Include selective HSP90 and HSP70 binders;

► 3D-model construction/validation;

► Select small-molecule compounds from ChemDiv store (more than 1.5 mil

molecules) with potential HSP90/70 activity following several structure

determinant, including similarity, privileged scaffolds and “med-chem” filters;

► Molecular Docking of selected compounds / “Pro-Drug” approach

Representative examples of reported HSP90 inhibitors

O

O

O O

N

OO

O

O

O

N

O

ONa

+

Novobiocin Hsp90 C-terminal inhibitor NB disrupts Bcr-Abl /Hsp90and Bcr-Abl /Hsp70 interactions Launched (anticancer) Pfizer

N

O

O

N

O

O

O O

O

N

O

Tanespimycin (17-AAG)ATP-site ligand, HSP90 Phase III (anticancer) Pfizer

N

NN

N

N

Cl

N

O

CNF-2024 Phase II (anticancer)ATP-site ligand, HSP90Conforma Therapeutics

N

N

OFF

F

O N

N

O

PF-04928473 Phase I (anticancer)ATP-site ligand, HSP90 Pfizer

N N

N O

O

O

N GanetespibPhase II/III (anticancer)ATP-site ligand, HSP90 Synta PharmaceuticalsN

N

N

S

N

N

N

O

O

I

PU-H71 Phase I (anticancer) ATP-site ligand, HSP90Memorial Sloan-Kettering Cancer Center Sloan-Kettering Institute

N

N

N

O

O O AT-13387AU Phase II (anticancer)ATP-site ligand, HSP90 Astex Pharmaceuticals

O

N

O

O

O

O

O

N

O

O

O KW-2478Phase I/II (anticancer) Kyowa Hakko Kirin

O

N

N

O

OO

O N

NVP-AUY922Phase II (anticancer) Novartis

NN

O

F

N O

N N

NVP-HSP990Phase I (anticancer) Novartis

N

O

N

O

N

N

N

O

XL-888 Phase I (anticancer)selective HSP90 inhibitor Exelixis

N

NN

N

N O

N

Biological Testingselective HSP90 inhibitor Sanofi

ClCl

ON

N

F

N

N

N

N

O

F F

N Biological Testingselective HSP90 inhibitor Pfizer

O

O

N

O O

KNK-437 PreclinicalInhibits the synthesis of HSP90 Kaneka

N

N

O

O

O

N

O

N O

O

O

O

O

N+

O

O

O

DMT series Biological TestingUniversity of Pittsburgh

NCl

Cl

NN

O

N

O

N

N

O

O

N

VER-155008 Preclinical VernalisIC50 = 0.5 mcM

NN

O

O

ON

N

O

O

O

N+O O

Mal-2-213 Biological Testing ATP-site ligandColumbia University IC50 ~30 nM

N

NN

N

O

S

N

N

O

N

N

O

YK-5 Biological Testing Allosteric site inhibitorMemorial Sloan-Kettering Cancer Center

S

N

N

S

N+

O

MKT-077 Phase II (anticancer) allosteric inhibitorDana-Farber Cancer Institute

O

OH

OH

OH

OH

OH

OH

O MyricetinAllosteric site inhibitor

Representative examples of reported HSP70 inhibitors

The final library content (more than 15K compounds)

compounds with „moderate-to-high‟ structural similarity (Tanimoto coeff. >0.5) to the

reported HSP90 and HSP70 inhibitors, isosteric modifications, topological analogues

compounds successfully passed through the developed 3D-models

O

OH

NH

O

OH

similarity = 0.52

O

NH

O

ID: K808-1985

Compound developed by Kyowa Hakko KirinHSP90 inhibitor under early biological evaluation Kitamura, Y. et al. US 7781485 Aug 24, 2010

N

N

N

OH

ID: G856-2554

N

N

O

O

similarity: 0.50

Compound developed by Emory UniversityHSP90 inhibitor under early biological evaluation Min, J. et al. US 022070, 2012

IC50=0.770 µM (Displacement of geldanamycin)

H417 human small-cell lung carcinoma cells

N NH

OOH

Me

MeO

HIGH SCRORE

O

NH

O

O

OH

OMe

K219_0094

MEDIUM SCRORE

G857_1210

EXAMPLES:

EXAMPLES:

non-trivial a-helix and b-sheet mimetics (HSP90/70-‟client protein‟ interaction mimetics)

compounds with a high diversity in structure (minor subset), including nature-like

compounds, e.g. spiro-compounds (Escape from flatland)

N

N

O

O

EtO

ID: K780-1065

N

N

OO Me

Me

Me

Me

ID: P681-0294

NS

NN

O

ClID: 5408-0495

N

NH

S

O

O Me

Me Me

ID: 3046-8072

N

O

O

NH

Me Me

Me

ID: C700-2278

NN

NN

O

O

O

O

F

ID: C796-0949

N

NN

NH

O

OO

Me

Cl

Me

MeMe

MeID: E223-2328

N

N

NH

O

O

ID: K788-9376

NO

O

OH

MeO

ID: K780-0501

EXAMPLES:

EXAMPLES:

Molecular weight H_acceptor

H_donor B_rotN

Key Properties

LogP logSw

PSA

Num. of unique heterocycles: 176;

Num. of unique screens: 3,663;

Diversity: 0,801 (cosine coefficient);

Num. of comb.templates: 340

Statistic

Hsp90 exists as two isoforms: Hsp90α and Hsp90β

► Hsp90 consists of a highly conserved N-terminal domain, a charged linker, and a highly

conserved C-terminal region, in general, the charged linker is not important for Hsp90 function,

and the inhibitors of Hsp90 mainly bind to the N-and C-terminal regions. The N-terminal

domain binds the natural products geldanamycin (GDA), radicicol and their derivatives, which

modulate at least two different conformational states. Novobiocin binds to the C-terminal

nucleotide binding site and inhibits Hsp90 function

► The first Hsp90 inhibitor drug is geldanamycin, a natural product isolated from

Streptomyces hygroscopicus. The geldanamycin analogue 17AAG (17-allylamino-17-

demethoxy-geldanamycin) possesses all the Hsp90-related characteristics of geldanamycin

but with lower toxicity. Another natural Hsp90 inhibitor is radicicol.

Geldanamycin 17AAG Radicicol

3D-molecular Docking

Besides the natural compounds, some synthesized inhibitors have also shown promising

effects on Hsp90, for example, purine-based inhibitors the Pyrazole-isoxazole analogues,

Novobiocin and coumarin scaffold analogues, such as 4TCNA

► Benzamide tetrahydro-4H-carbazol-4-one analogs (BT), AT13387 derivatives (AT) and

Dihydroxylphenyl amides (DA) have been developed as further potent Hsp90 inhibitors

3D-QSAR, CoMFA, CoMSIA, ANN and 3D-pharmacophore as well as 3D-docking methods

were employed to investigate several HSP90 purine-based inhibitors, which provided useful

models for designing the Hsp90 targeted compounds

► the common pharmacophoric features, i.e., one H-bond donor, one H-bond acceptor, one

hydrophobic (aromatic), and two hydrophobic (aliphatic) features, which might be of import

to develop inhibitors as anti-cancer agents

Novobiocin

BT AT

DA

to date, there are no 3D-QSAR studies of many ligands reported except very few classes.

Moreover, no comprehensive feature for the ligand-receptor interactions, such as the

hydrophobic contact between the key amino acid residues has been demonstrated

Purine-based HSP90 pharmacophore

developed by Chen includes

hydrophobic (blue), hydrogen bond

acceptor (green) and hydrogen bond

donor (purple). The distances of

features were labeled by blue lines

Chen CY. Insights into designing the dual-targeted HER2/HSP90 inhibitors. J. Mol. Graph. Model. 2010, 29(1), 21-31

► More than 50 crystallographic complexes of small-molecule compounds including several

BBs with HSP90 have been described to date. A majority of these complexes were

investigated during the study

► MolSoft SoftwareTM was used to analyzed these complexes with regard to their structural

homology and the ligand binding modes

► the ATP-binding site was thoroughly reconstructed based on the 3D-alignment of many

protein-ligand complexes

reported HSP90 ligands were flexibly redocked to the binding site of Hsp90 protein to

internaly evaluate the model. The optimum docking conformations as the most probable

binding conformation corresponding with a low energy score were analyzed

Hsp90a N-terminal domain bound to ATP (3T0Z)

Homo sapiens

The 3D-alignment of two crystallographic

data obtained for ATP and geldanamycin

D93, T184 and G97 are the most crucial conservative (rigid) points in the first

common binding mode

► AAs which coordinate the cofactor (Mg2+) are the second target

► there are several other AAs which can also provide significant contacts with

ligands within the ATP-binding site, including N106 (adjusted), K112, K58 (adjusted),

F138 and G137, N106 (adjusted), L107 (adjusted), N51, G97

Radicicol analogue bound to the ATP-

binding site of HSP90 (orange), VHD in the

site (yellow). Crystallographic data,

superposition

VHD

Woodhead AJ, et al. Discovery of (2,4-dihydroxy-5-isopropylphenyl)-[5-(4-methylpiperazin-1-ylmethyl)-1,3-dihydroisoindol-2-

yl]methanone (AT13387), a novel inhibitor of the molecular chaperone Hsp90 by fragment based drug design. J Med Chem. 2010

Aug 26;53(16):5956-69.

VHD is one compound among a wide

fragment-based library screened recently

against HSP90. For many fragments the

related crystallographic data was

obtained. We also used these data to

develop our 3D-docking model

ATP bound to the binding site of HSP90

(orange), direct ATP analogues in the site

(yellow). Crystallographic data, superposition

From the

fragment-based

library

Crystal Structure of HSP90

with N-Aryl-benzimidazolone

Key contacts: G97, T184, D93

K58-sylfonyl oxygen (weak)

Another binding mode was

revealed for several tricyclic HSP90

inhibitors

H-bonding: Y139, L103

Hydrophobic and stacking contacts:

Phe138, Y139, Leu103, Ala111, Leu107,

Phe170, Gly106, Phe22, W162

Combined features form the third

binding mode for HSP90 ligands

Vallée F. et al. J Med Chem. Tricyclic series of heat shock protein 90 (Hsp90) inhibitors part I: discovery of tricyclic imidazo[4,5-

c]pyridines as potent inhibitors of the Hsp90 molecular chaperone. 2011; 54(20): 7206-19

N

N

NN N

NO

N(conf):24

minE:-99.97

maxE:-71.49

Energy (calc):-82.78

RMSD: 6.401e-007

H

N

N

N

N

O

N(conf):15

minE:-85.68

maxE:-54.12


RMSD: 6.915e-007

N

NN

O

N(conf):17

minE:-77.63

maxE:-41.11


RMSD: 6.718e-007

N

N N

N

O

O

N

N(conf):21

minE:-111.7

maxE:-67.89


RMSD:6.193e-007

S

O

ON

N

ON

O

O

Cl

N(conf):11

minE:-86.47

maxE:-32.4

Energy (calc):-69.8

RMSD: 6.119e-007

0.1 sens

N

N N

N

O

N

N(conf):11

minE:-100.1

maxE:-22.38


RMSD: 6.924e-007

0.1 sens

H

N

N N

H N

O

N

N(conf):24

minE:-77.57

maxE:-58.96


RMSD:6.581e-007

N(conf):24

minE:-108.2

maxE:-70.52


RMSD:6.589e-007

N

N

N

NO

N N

3D-Molecular Docking Hsp70► Hsp70 chaperones are key to cellular protein homeostasis

► Hsp70 proteins are allosteric machines and offer, besides classical active-site targets,

also opportunities to target the mechanism of allostery

► Potent anticancer compound MKT-077 has recently been described as an "allosteric

drug“

► Using NMR spectroscopy the compound's binding site on human Hsc70 (hHsc70 has

85% identity with human Hsp70) was identified. Fractional shifts upon addition of MKT-

077 in the 15N,2H-labeled hHsc70 include T222, A223, D225, H227, L228. For each

resonance, the shift at a molar ratio of 0.5 was defined as 50% completed

► We have reconstructed the allosteric binding site using this data and docked MKT-

077

Rousaki A elt al. Allosteric drugs: the interaction of antitumor compound MKT-077 with human Hsp70 chaperones. J Mol Biol. 2011, 411(3):614-32

MKT-077

N(conf):23

minE:-58.95

maxE:-46.94


RMSD:5.131e-007

S

N

N

S

N+

O E231

R261

V59

P91

K257

P63

Y41

2.43.2

2.8

ATP-binding site for

competitive inhibitors

Selective Binding to HSP90

► XL888 is a potent and selective ATP-competitive inhibitor of HSP90 developed by Exelixis

► XL888 binds to the target in a manner that is structurally distinct from other HSP90

inhibitors currently in the clinic

► XL888 selectively inhibits members of the HSP90 family and it is inactive against HSP70,

the structurally related Bergerat-fold ATPase Topoisomerase II, and a diverse panel of

serine/threonine and tyrosine kinases

► Crystallographic analysis of XL888 bound to HSP90 has showed that XL888 extends

across the width of the ATP-binding domain. A flexible region of ~30 amino acids referred to

as the “lid” (magenta ribbon) adopts a different conformation when bound to XL888 compared

with ADP or Geldanamycin

N

O

N

O

N

N

N

O

XL888A co-crystal structure of XL888 bound to the N-terminal

domain of HSP90a: comparison to ADP and Geldanamycin.

Lyman SK, et al. High-content, high-throughput analysis of cell cycle perturbations induced by the HSP90 inhibitor XL888. PLoS One. 2011;6(3):e17692;

Paraiso KH, et al. The HSP90 Inhibitor XL888 Overcomes BRAF Inhibitor Resistance Mediated through Diverse Mechanisms. Clin Cancer Res. 2012

;18(9):2502-14.

NH

N

N

NH

O

O

NH2

Compound 1

Vallée F, et al. Tricyclic series of heat shock protein 90 (Hsp90) inhibitors part I: discovery of tricyclic

imidazo[4,5-c]pyridines as potent inhibitors of the Hsp90 molecular chaperone. J Med Chem.

2011;54(20):7206-19.

yellow – ATP; orange – compound 1 Terminal region adjustment and alternative

binding cavity formation for penetration

Insight into the selectivity, XL888 binding mode

yellow – docking results

orange – crystallographic data

Selective HSP90 inhibitors docked into the reconstructed binding site

XL888

N

N

OF F

F

N

O

ON

SNX-2112

Huang KH, et al. Discovery of

novel aminoquinazolin-7-yl 6,7-

dihydro-indol-4-ones as potent,

selective inhibitors of heat shock

protein 90. Bioorg Med Chem

Lett. 2012;22(7):2550-4

NVP-AUY922 O

N

O

O

NO

N

O

(Novartis)

Eccles SA, et al. NVP-AUY922: a novel

heat shock protein 90 inhibitor active

against xenograft tumor growth,

angiogenesis, and metastasis. Cancer

Res. 2008 ;68(8):2850-60

(Pfizer)

ClCl

ON

N

F

N

N

N

N

O

F F

N

(Pfizer)

Zehnder L, et al. Optimization of potent, selective,

and orally bioavailable pyrrolodinopyrimidine-

containing inhibitors of heat shock protein 90.

Identification of development candidate 2-amino-4-

{4-chloro-2-[2-(4-fluoro-1H-pyrazol-1-yl)ethoxy]-6-

methylphenyl}-N-(2,2-difluoropropyl)-5,7-dihydro-

6H-pyrrolo[3,4-d]pyrimidine-6-carboxamide. J Med

Chem. 2011,12;54(9):3368-85

Overlapping of several inhibitors

Yellow - XL888

Orange – compound 1

Green - SNX_2112

Blue - NVP_AUY922

Core residues (H-bonding): T184,

D93, S52, K58;

Planar Stacking: F138

Additional H-bonding: Y139, W162,

F22, Q23, L103

Representative examples of compounds from HSP90/70 library

N NH

OOH

Me

OMe

G857_1210

Possible O-dealkylation by CYP3A4

G97

T184

D93N51

H2O

HIGH SCRORE

Ep = -60 kcal/mol

O

NH

O

O

OH

OCH

3

K219_0094

presumably> Pgp-target> poor BBB permeability

T184

K58 H20H20

MEDIUM SCRORE

Ep = -64 kcal/mol

N

O

O

O

Me

Me

Me

N

O

OH

OH

Me

D398_0454 D398_0454_1

D93

S52

H2O

N52O-dealkylatyon

CYP3A4

Pro-Drug Strategy

MEDIUM SCRORE

Ep = -47 kcal/mol

N

N

SNH

OO

O Me

Me

Cl

NH

NH

SNH

OO

O

Cl

N-dealkylation

CYP3A4

D93

S52

N51/S52

H2O

G801_0201 G801_0201_1

Pro-Drug Strategy

MEDIUM SCRORE

Ep = -74 kcal/mol

N

N

NH

N

OO

CH3

OH

OH

N

N

NH

N

OO

CH3

metabolic sites

hydroxylation

CYP3A4

possibledeacetylasesite S062-1044 S062-1044-1

D93

N51

T184

G97

K58

H2O

Pro-Drug Strategy

HIGH SCRORE

Ep = -74 kcal/mol

Documents

Heat Shock Protein 90/70 (HSP90/70) Targeted Libraryreported HSP90 and HSP70 inhibitors, isosteric modifications, topological analogues compounds successfully passed through the developed