Discovery of a Novel Chemotype of Histone Lysine Methyltransferase

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Discovery of a novel chemotype of histone lysine methyltransferase EHMT1/2 (GLP/G9a) inhibitors: rational design, synthesis, biological evaluation and co-crystal structure Ciro Milite, Alessandra Feoli, John R Horton, Donatella Rescigno, Alessandra Cipriano, Vincenzo Pisapia, Monica Viviano, Giacomo Pepe, Giorgio Amendola, Ettore Novellino, Sandro Cosconati, Xiaodong Cheng, Sabrina Castellano, and Gianluca Sbardella J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b02008 • Publication Date (Web): 12 Feb 2019 Downloaded from http://pubs.acs.org on February 13, 2019

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Journal of Medicinal Chemistry

Discovery of a novel chemotype of histone lysine methyltransferase EHMT1/2 (GLP/G9a) inhibitors: rational design, synthesis, biological evaluation and cocrystal structure Ciro Milite,‡,║Alessandra Feoli,‡,║ John R. Horton,§ Donatella Rescigno,‡,∫,† Alessandra Cipriano,‡,∫ Vincenzo Pisapia,‡,∫ Monica Viviano,‡ Giacomo Pepe,‡ Giorgio Amendola,∆ Ettore Novellino,¥ Sandro Cosconati,∆ Xiaodong Cheng,*,§ Sabrina Castellano,*,‡ and Gianluca Sbardella*,‡ ‡Department

of Pharmacy, Epigenetic Med Chem Lab, University of

Salerno, via Giovanni Paolo II 132, I-84084 Fisciano (SA), Italy §Department

of Molecular and Cellular Oncology, The University of

Texas MD Anderson Cancer Center, Houston, Texas 77030, United States

ACS Paragon Plus Environment

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∫PhD

Page 2 of 115

Program in Drug Discovery and Development, University of

Salerno, Via Giovanni Paolo II 132, I-84084 Fisciano (SA), Italy ∆DiSTABiF,

University of Campania “Luigi Vanvitelli”, Via Vivaldi 43, 81100 Caserta, Italy

¥Department

of Pharmacy, University Federico II of Naples, Via D.

Montesano 49, 80131 Naples, Italy Keywords.

G9a/GLP

inhibitors;

EHMT1/2;

co-crystal

structure;

DNMT1; benzodiazepine; scaffold hopping; epigenetics.

Abstract.

Since the discovery of compound BIX01294 over 10 years ago, only a very limited number of non-quinazoline inhibitors of H3K9specific

methyltransferases

G9a

and

GLP

have

been

reported.

Herein we report the identification of a novel chemotype for G9a/GLP inhibitors, based on the under-investigated 2-alkyl-5amino- and 2-aryl-5-amino-substituted 3H-benzo[e][1,4]diazepine scaffold.

Our

research

efforts

yielded

the

identification

of

compound 12a (EML741), which not only maintained the high in vitro and cellular potency of its quinazoline counterpart, but also

displayed

improved

inhibitory

potency

against

DNMT1,

improved selectivity against other methyltransferases, low cell toxicity,

and

improved

apparent

permeability

values

in

both


2

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PAMPA and PAMPA-BBB assays and, therefore, might potentially be a better candidate for animal studies. Finally, the co-crystal structure of GLP in complex with 12a provides the basis for the further development of benzodiazepine-based G9a/GLP inhibitors.

Introduction Lysine methylation on histone tails plays a primary role in the regulation of chromatin structure and gene transcription, both in

physiological

and

conditions.1-5

pathological

Besides

epigenetic regulation of gene expression, lysine methylation is also involved in the regulation of cellular signal transduction pathways. In

fact,

a number of

nonhistone proteins are also

methylated on Lys residues, leading to changes in their function or stability.6,

7

Protein lysine methyltransferases (PKMTs or KMTs) catalyze the addition of a methyl group from S‐adenosyl‐L‐methionine (SAM) to the ε-amino group of the targeted lysine residue in histone and nonhistone substrates, leading to mono-, di-, or trimethylation. 8-11

This process does not modify the charge state of the residue.

Instead,

lysine

methylation

increases

the

bulkiness

and

the

hydrophobicity of the amino group and improves its hydrogen bond formation potential.10 As a result, lysine methylation has dramatic effects on the interactions of the modified protein with other proteins, thus


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its physiological function.10 Depending on the methylation state and

the

position

of

the

modified

residue,

histone

lysine

methylation can lead to either activation or repression of gene expression and transcription.2, histone

H3

hallmark

lysine

of

9

and

27

transcription

11-14

In general, methylation of

(H3K9

and

repression

H3K27)

whereas

is

a

typical

methylation

of

histone H3 lysine 4, 36, and 79 (H3K4, H3K36, and H3K79) is associated with transcription activation.15-18 G9a

(also

known

as

lysine

methyltransferase

1C,

KMT1C,

or

euchromatic histone-lysine N-methyltransferase 2, EHMT2) and GLP (G9a-like protein, also known as KMT1D or EHMT1) are two closely related PKMTs with approximately 80% sequence identity in their respective trithorax

suppressor (SET)

heterodimeric

variegation

domains.19,

complex

methyltransferase

of

in

20

It

of G9a and mouse

3–9

has GLP

embryonic

enhancer-of-zeste

been

shown

is the

main

functional

cells,

depositing

stem

mono- and dimethylated H3K9 (H3K9me1 and H3K9me2).21, H3K9, other histone substrates, such as histone H1,23, 27

that

22 24

the

Besides H3K27,25-

and H3K56,28 have been reported. The two enzymes can also

methylate a variety of nonhistone proteins,29 including the tumor suppressor p53,30 sirtuin 1 (SIRT1),31 the ATPase Associated with various

cellular

Activities

estrogen

receptor



(ER),33

(AAA+) the

helicase

chromodomain

reptin,32 Y-like

the

protein

(CDYL1),34 the myogenic differentiation 1 (MyoD),35 the widely


4

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interspaced zinc finger motifs protein (WIZ),34 as well as G9a itself.36 Furthermore, G9a and/or GLP have been reported to play roles37

in

germ

development,21,

22

cell tumor

development cell

growth

and

meiosis,38

and

metastasis,30,

embryo 39-42

cocaine-induced plasticity,43 cognition and adaptive behavior,12, 44

immune

diseases

response,45, such

as

46

and

provirus

cancer,

silencing.47

inflammatory

Many

human

diseases,

and

neurodegenerative disorders are associated with dysregulation of G9a and/or GLP.12, biological

13, 40, 44, 48, 49

activities

and

In addition to the many common

regardless

of

the

high

sequence

homology, G9a and GLP also possess distinct physiological and pathophysiological functions.50-53


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A

N

N

COOH

NH2

NH N

O

HO

HO

O N

HO

N

N

O

HO

N

N

H 2N

N

N

O

NH2

NH2

NH2

HO 1 G9a/GLP IC50= 1.5/1.6 M

sinefungin G9a/GLP IC50= 30.1/28.4 M

2 (BRD9539) G9a/GLP IC50= 30.1 M/ not reported

B N

N

HN H3CO H3CO

H 3C

N

N CH3 3 (BIX-01294), X=N G9a/GLP IC50= 1.9/0.7 M 10 (HKMTI-1-248), X = CH G9a/GLP IC50= 13 nM/ n.r.

X

N CH3

CH3 N

H3CO O

H3CO

N

O

N

H 2N

N

CH3

N

N

H3CO

N

N

O

N

CH3

N

8 (UNC0642) G9a/GLP IC50=

Discovery of a Novel Chemotype of Histone Lysine Methyltransferase

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