DNMT-focused library Medicinal and Computational Chemistry Dept., ChemDiv, Inc., 6605 Nancy Ridge Drive, San Diego, CA

92121 USA, Service: +1 877 ChemDiv, Tel: +1 858-794-4860, Fax: +1 858-794-4931, Email:

ChemDiv@chemdiv.com

DNA methylation is an essential intracellular event critically involved in a wide range of

endogenous processes that play significant role in the foundation of genetic phenomena and diseases.

Such epigenetic modifications play a key role in the patho-physiology of many tumors and the current use

of agents targeting epigenetic changes has become a topic of intense interest in cancer research.

Particularly, DNA methyltransferase (DNMT) is a crucial enzyme for cytosine methylation in

DNA [1]. In mammals, DNMTs can be broadly divided into two functional families (DNMT1 and

DNMT3). All mammalian DNA methyltransferases are encoded by their own single gene, and consisted

of catalytic and regulatory regions (except DNMT2). Via interactions between functional domains in the

regulatory or catalytic regions and other adaptors or cofactors, DNA methyltransferases can be localized

at selective areas (specific DNA/nucleotide sequence) and linked to specific chromosome status

(euchromatin/heterochromatin, various histone modification status). With assistance from UHRF1 and

DNMT3L or other factors in DNMT1 and DNMT3a/ DNMT3b, mammalian DNA methyltransferases can

be recruited, and then specifically bind to hemimethylated and unmethylated double-stranded DNA

sequence to maintain and de novo setup patterns for DNA methylation. Complicated enzymatic steps

catalyzed by DNA methyltransferases include methyl group transferred from cofactor Ado-Met to C5

position of the flipped-out cytosine in targeted DNA duplex. In the light of the fact that different DNA

methyltransferases are divergent in both structures and functions, and use unique reprogrammed or

distorted routines in development of diseases, design of new drugs targeting specific mammalian DNA

methyltransferases or their adaptors in the control of key steps in either maintenance or de novo DNA

methylation processes will contribute to individually treating diseases related to DNA methyltransferases.

Various functional protein domains in mammalian DNA MTases are presented in Fig 1. As shown

in the figure below, except Dnmt2, all mammalian DNA MTases are divided into catalytic and regulatory

regions. From N-terminal to C-terminal, domains in the regulatory region of Dnmt1 are: PBD domain

[PCNA (proliferating cell nuclear antigen) binding domain], NLS (nuclear localization sequence) domain,

RFT (replication-foci targeting) domain, CXXC (cysteine-rich, cysteine-x-x-cysteine type) domain, and

randomly arranged BAH (bromo/Brahma-adjacent homology) domain. The ATRX (alpha-thalassemia

and mental retardation on X chromosome)-homology domain is located in the C-terminal part of the

regulatory region of Dnmt3a, Dnmt3b, and Dnmt3L. Another essential functional domain—PWWP

  1

(proline-tryptophan-tryptophan-proline motif) domain locates at C-terminal to the ATRX-homology

domain of the regulatory region in Dnmt3a and Dnmt3b.

Fig. 1. Functional domains in mammalian DNA MTases.

Figure 2 provides the overview of epigenetic modulation of gene expression by posttranslational

DNA methylation. Transcriptionally inactive chromatin is characterized by the presence of methylated

cytosines within CpG dinucleotides (CH3), which is sustained by DNA methyltransferases.

Fig. 2. Modulation of gene expression by DNMTs.

The underlying mechanism of cytosine C5 methylation (1–4) and its possible side-effect reactions

(5–8) are depicted in Fig. 3. The sulfhydryl group at the side chain of the key cysteine residue, in the

active-site loop of DNA MTase, could nucleophilically attack to the C6 position of cytosine followed by

transient protonation of the N3 nitrogen atom via the glutamate residue in conserved motif VI of DNA

MTase (1–2). Consequently, aromaticity of cytosine is broken so as to strengthen activity of nucleophilic

attack of the C5 position in cytosine to the methyl group in the sulfonium centre of Ado-Met (2).

Following the methyl group transferred to the C5 position of cytosine, the N3 position in cytosine is re-

deprotonated (b). To release enzyme moiety covalently bound in the C6 position of cytosine, the C5

  2

position could be deprotonated with assistance of unknown residues in the enzyme (3). Following the β-

elimination reaction involved in C5 and C6 positions in cytosine, C5-methyl cytosine could finally be

formed (4). Under certain conditions (e.g. Ado-Met inadequate), water can penetrate into the active site of

DNA MTase towards the relatively localized π electron in the activated cytosine (5). Then the C4 position

in cytosine is hydroxylated so that deamination reaction is triggered (6). The amino group in the C4

position of cytosine then is replaced to form a carbonyl oxygen atom (7). The conversion of cytosine to

uracil is accomplished (7–8).

Fig. 3. The chemical pathway of cytosine C5 methylation by DNMTs (1–4) and its possible side-effect

reactions (5–8)

Fig. 4 shows the interactions between hemimethylated DNA duplex and SRA domain of UHRF1.

In Fig. 4(A) - the interface toward DNA in the SRA [SET (Su(var), E(z), Trithorax) and RING module

associated] domain of UHRF1 [ubiquitin-like, containing PHD (plant homology domain) and RING

(really interesting new gene) finger domains 1] is imagined as a grasping hand, with its finger and thumb

projecting into the major and minor groove of DNA duplex, respectively. Its palm holding a deep pocket

for binding C5-methyl cytosine. In Fig. 4(B) - the guanidinium side chain of Arg residue in the finger can

form hydrogen bonds with O6 and N7 positions in the intrahelical orphaned guanine ring in order to

simulate the Watson-Crick hydrogen bonds, which is important for binding of the SRA domain and target

DNA duplex. The main-chain carbonyl group of another amino acid residue—Asn, located near the Arg

residue in the finger, can effectively form essential hydrogen bonds with the cytosine ring in the   3

unmethylated strand. The collaborative organization of the Arg and Asn residues in the finger is

prerequisite for specificity of the SRA domain binding to hemimethylated CG site. In Fig. 4(C) - in the

palm, amide groups in the main chain of the Gly and Ala residues, and carbonyl groups in the side chain

of the Asp residue and the main chain of the Thr residue, are toward to O2 and N4 positions of C5-methyl

cytosine to form hydrogen bonds so as to discriminate cytosine from thymine base. Moreover, the van de

Waals interaction is formed by interaction of C5 methyl group of the target methylated cytosine and the

Cα and Cβ atoms of the Ser residue of the deep pocket in the palm. In addition, π stacking force given by

the two benzene rings of two Tyr residues in the palm located in the two sides of the C5-methyl cytosine

ring.

Fig. 4. The biochemical outcome of DNMT action

DNMT inhibitors and DNMT-mediated cell signaling

DNMT inhibitors represent a promising class of epigenetic modulators. More than 70 small

molecule DNMT inhibitors have been disclosed to date. DNA methyltransferases represent promising

targets for the development of unique anticancer drugs. Recent researches have revealed promising anti-

tumorigenic activity for DNMT inhibitors in vitro and in vivo against a variety of hematologic and solid

tumors. These epigenetic modulators cause cell cycle and growth arrest, differentiation and apoptosis.

Rationale for combining these agents with cytotoxic therapy or radiation is straightforward since the use

of DNMT inhibitor offers greatly improved access for cytotoxic agents or radiation for targeting DNA-

protein complex. The positive results obtained with these combined approaches in preclinical cancer

models demonstrate the potential impact DNMT inhibitors may have in treatments of different cancer

types. Therefore, as the emerging interest in use of DNMT inhibitors as a potential chemo- or radiation

sensitizers is constantly increasing, further clinical investigations are inevitable in order to finalize and   4

confirm the consistency of current observations. However, all DNMT inhibitors currently in clinical use

are nonselective cytosine analogs with significant cytotoxic side-effects.

Errors in cell signaling are deeply implicated in the development as well as in the progression of

cancer. Aberrant DNMT activity has been involved in these processes (Fig. 5). PTEN/PI3K/Akt pathway

physiologically plays a key role in the control of many processes essential for the cellular life. PTEN

(phosphatase and tensin homolog deleted on chromosome ten) is a tumor suppressor gene and its

functional loss has been documented in bladder cancer, glioblastoma, melanoma and cancers of the

prostate, breast, lung and thyroid [2]. PTEN negatively controls the PI3K/Akt pathway and its epigenetic

loss, frequent in cancer cells, leads to the aberrant pathway activation. DNMT inhibitors restore the PTEN

expression by epigenetic mechanisms. This tumor suppressor gene controls PI3K by preventing the

activation of PDK-1 and Akt.

Fig. 5. DNMT inhibitors and PTEN/PI3K/Akt pathway.

The functional loss of PTEN is higher than that attributable to LOH of chromosome 10q and post-

translational mechanisms, including hypermethylation, explain the other part of this phenomenon [3].

Evidence indicates that 5-Aza is a chemosensitizer in prostate cancer [4] and its property seems mediated

by PTEN. After infection with a recombinant adenovirus containing wild-type PTEN, bladder tumor cells

acquire greater chemosensitivity to the cytotoxic effect of doxorubicin [5]. The chemosensitivity induced

by PTEN is partially mediated by PI3K and Akt/PKB [6]. Other evidence, in a different experimental

model, suggests that the transfection of TC-32 Ewing sarcoma cells with Akt/PKB inhibits doxorubicin-

induced apoptosis suggesting that PTEN increases doxorubicin cytotoxicity through the PI3K signaling

pathway. Additional indication of chemosensitizing properties of PTEN derived from data obtained in

endometrial cancer cells [7]. In this system, PTEN significantly enhanced chemosensitivity to

doxorubicin. This effect was associated with the levels of phospho-Akt/PKB and phospho-Bad (Ser-136),

  5

which were reduced in the PTEN expressing clones. Results from other studies performed on brain

tumors clearly show that decreasing activity of the PI3K/Akt pathway in tumor cells with mutant PTEN

may contribute to the increased sensitivity to chemotherapy [8]. Down-regulating the Akt pathway by

inducing PTEN also increases the sensitivity of glioblastoma cells to temozolomide.

Despite the promising anticancer activity in haematological malignancies [9], early clinical trials

showed that DNMT inhibitors have low anticancer activity and significant toxicity as single agent in solid

tumors. Recent studies, however, suggest that low concentrations of DNMT inhibitors such as decitabine

and its analogs (Table 1) may act synergistically when combined with chemotherapy and contribute to

overcoming intrinsic or acquired chemoresistance [10]. These properties are considered clinically

significant as the resistance of tumor cells to cytotoxic agents remains the major obstacle in

chemotherapeutic-based treatments. The mechanisms underlying chemoresistance remain in some

measure elusive even though multifactorial mechanisms, including epigenetic modifications may drive

this mechanism [11]. Therefore, any effort to overcome multi-drug resistance represents the primary goal

in cancer research. Based on the chemical mechanisms, DNMT inhibitors act through different

mechanisms. Among the different mechanisms postulated, alterations in differentiation, changes in

apoptosis, and induction of a beneficial immune response are considered of main importance [12]. Finally,

the induction of DNA damage due to the formation of irreversible covalent enzyme-DNA adducts has

also been taken into consideration.

Table 1. DNMT inhibitors which have been used as reference compounds.

Name/Phase Structure/Orginator Mechanism of action

Azacitidine/ Launched in 2004

Pfizer

Apoptosis Inducers, multicancer therapy

This drug is a ribonucleoside analogue and it binds to RNA and DNA. This molecule interrupts mRNA translation and when incorporated into DNA inhibits methylation by trapping DNMTs. At relatively higher concentrations this drug results in the formation of high levels of enzyme-DNA adducts. Azacitidine injectable suspension is a DNA methyltransferase inhibitor launched in the U.S. in 2004 by licensee Pharmion for the treatment of myelodysplasia (MDS). In 2007, the FDA approved a formulation for i.v. injection that is administered once-daily for seven days, every four weeks. In 2005, Pharmion withdrew its marketing authorization application (MAA) for the product in Europe following a request for additional data from the EMEA and in 2006, the application was resubmitted. In 2008, a positive opinion was received in the E.U. for the treatment of MDS. Final approval was obtained in 2008. Approval was also obtained in the E.U. in 2008 for the treatment of acute myeloid leukemia and for the treatment of chronic myelomonocytic leukemia. First launches of the product took place in Germany in 2008 and 2009. In 2009, Nippon Shinyaku filed for approval in Japan for the

  6

treatment of myelodysplastic syndrome. Approval was obtained in 2010.

Decitabine/ Launched – 2006

Pharmachemie

Multicancer Therapy Hematologic Agent

(Miscellaneous) Hematopoiesis Disorders

Therapy

This drug is a deoxyribonucleoside analogue. For this reason, this molecule does not bind to RNA but only to DNA. When incorporated into DNA inhibits methylation by trapping DNMTs resulting in the reduced methylation of cytosines in DNA synthesized after drug treatment. When used at relatively high concentrations this drug results in the formation of high levels of enzyme-DNA adducts. Decitabine was discovered by Pharmachemie and licensed to SuperGen in 1999. The compound has been shown to have a broad spectrum of activity in several hematological malignancies as well as solid tumors. Recent research demonstrates tumor suppressor genes and DNA repair genes are hypermethylated and silenced in a majority of cancers, typically at an early stage of the disease. Hypermethylation may prove useful for early cancer detection and screening of cancer patients. Therapy that removes the methyl groups by hypomethylation may activate natural defense mechanisms against cancer.

Zebularine/ Preclinical

University North Carolina,

Chapel Hill

This drug is a deoxyribonucleoside analogue. For this reason, this molecule does not bind to RNA but only to DNA. When incorporated into DNA inhibits methylation by trapping DNMTs resulting in the reduced methylation of cytosines in DNA synthesized after drug treatment. When used at relatively high concentrations this drug results in the formation of high levels of enzyme-DNA adducts. The compound also inhibits Cytidine Deaminase activity.

(-)-EGCG / Phase II/III

Multicancer Therapy

EGCG, the major polyphenol from green tea, can inhibit DNMT activity and reactivate methylation-silenced genes in cancer cells. With nuclear extracts as the enzyme source and polydeoxyinosine-deoxycytosine as the substrate, EGCG dose-dependently inhibited DNMT activity, showing competitive inhibition with a K(i) of 6.89 �M. Molecular modeling studies suggest that EGCG can form hydrogen bonds with Pro(1223), Glu(1265), Cys(1225), Ser(1229), and Arg(1309) in the catalytic pocket of DNMT. In addition, it was also shown that other tea polyphenols, including curcumin, catechins, epicatechins and bioflavonoids (quercetin, fisetin, and myricetin) as well as genistein from soybean, can inhibit SssI DNMT- and DNMT1-mediated DNA methylation in a concentration-dependent manner.

MG98/ Discontinued

Structure has not been disclosed yet

MethylGene

Multicancer Therapy

This antisense oligonucleotide targets the 3 UTR of DNMT1 causing a methylation decrease in cell lines and animal models. Second-generation phosphorothioate antisense oligodeoxynucleotide targeting DNA methyltransferase-1 (DNMT1). MG-98 had been in clinical development by MethylGene for the treatment of metastatic renal cell cancer and several other cancer indications. In 2007, the company discontinued

  7

development of the compound, reporting that it would focus its resources on more commercially viable product candidates. MG-98 was being assayed in combination with interferon alpha. MG-98 inhibits the synthesis of DNA methyltransferase-1 (DNMT-1) by targeting its mRNA and therefore reactivating silenced tumor suppressor genes. In 2000, MethylGene and MGI Pharma established a research and development agreement covering North American rights relating to MG-98. MethylGene will retain all rights outside of North America.

RG108 / Biological

Testing

Institute Biochem. Biophysics PAS

This small molecule agent is not straightly incorporated into DNA but it binds to the catalytic site of DNMTs causing inhibition of DNA methylation.

Procainamide / Launched

Bristol-Myers Squibb Pfizer

This molecule reduces DNMT1’s affinity for both DNA and S-adenosyl-methionine causing a decrease in DNA methylation.

Disulfiram / Launched

Pfizer

Anticataract Agent

Cancer Therapy Treatment of Alcohol and

Cocaine Dependency

Multitargeted drug, including Aldehyde Dehydrogenase, DNMT1 and TRPA1. Disulfiram was first launched by Wyeth Pharmaceuticals (now Pfizer) under the brand name Antabuse(R) in 1951 for the oral treatment of alcoholism. At present, the National Institute on Drug Abuse (NIDA) is conducting phase II clinical trials with the compound to evaluate its potential for the treatment of cocaine dependency. The University of California, Irvine is conducting early clinical studies of the compound in combination with arsenic trioxide in patients with metastatic melanoma and at least one prior systemic therapy. Johns Hopkins University is also investigating the compound in phase II clinical trials for the treatment of recurrent prostate cancer.

Compounds in Biological

Testing

-

  8

Pfizer

Oncolytic Drugs

RG108 / Biological

Testing

Institute Biochem. Biophysics PAS Oncolytic Drugs

RG108, a DNMT1 inhibitor, represents promising candidate for cancer drug development. The structural design of the chemically modified inhibitor was aided by molecular modeling, which suggested the possibility for extensive chemical modifications at the 5-position of the tryptophan moiety in RG108. The inhibitory activity of the corresponding derivative was confirmed in a cell-free biochemical assay, where bio-RG108 showed an undiminished inhibition of DNA methyltransferase activity (IC50 = 40 nM).

Compounds in Biological

Testing

-

  9

Cancer Research

Technology Oncolytic Drugs

Compounds in Biological

Testing

MethylGene

Oncolytic Drugs

DNMT1 and DNMT3b2 inhibitors

Compounds in Biological

Testing

-

  10

Nippon Kayaku Oncolytic Drugs

Thymolphthalein / Biological

Testing

University Medicine Dentistry New Jersey

Oncolytic Drug

IC50 = 9 μM in PRSET-7 cell line (HKMTS assay)

XB-05 / Preclinical

University of Louisville Oncolytic Drug

XB-05 - a novel non-nucleoside inhibitor of DNMT1 with potent activity in colon, breast and prostate cancer cells.

Biological Testing

Deutsches Krebsforschungszentrum

(DKFZ) Univ degli studi di Napoli

This compound is able to induce a recognizable demethylation of chromosomal satellite repeats in HL60 human myeloid leukemia cells and thus represents a lead compound for the development of a novel class of non-nucleoside DNMT1 inhibitors.

  11

Federico II Universita degli Studi di

Salerno (Originator) Oncolytic Drug

BIX-01294 / Biological

Testing

Alexis Biochemicals

Emory University M.D. Anderson Cancer

Center Sigma-Aldrich

University of Illinois at Chicago

Oncolytic Drug

Histone-lysine N-methyltransferase GLP/Eu-HMTase1, inhibition: IC50=27 nM (Chemiluminescent assay); Euchromatic histone-lysine N-methyltransferase 2a [G9a], inhibition: IC50=0.106 µM (Fluorescent assay).

SGI-1027 / Biological

Testing SuperGen

Oncolytic Drug

This compound inhibits the activity of DNMT1, DNMT3A, and DNMT3B as well M. SssI with comparable IC50=6-13 μM/L) by competing with S-adenosylmethionine in the methylation reaction. Treatment of different cancer cell lines with SGI-1027 resulted in selective degradation of DNMT1 with minimal or no effects on DNMT3A and DNMT3B.

KED-4-69 Georgetown University Prostate Cancer Therapy

DNA methyltransferase inhibitor that inhibited prostate PC-3 cell proliferation (GI50=1.5 μM) and exhibited strong cytotoxic activity against a panel of 60 human cancer cell lines (GI50=1.50-56.1, LC50 >100 μM), with no toxicity in mice up to 550 mg/kg. p.o. In mice bearing PC-3 tumor xenografts, it reduced tumor volume (40%) at 10 mg/kg/day i.p. over 28 days. Potentially useful for the treatment of prostate cancer.

Biological Testing

Nagoya City University (NCU)

Oncolytic Drug

This compound belongs to a parent series of related maleimide derivatives. It was found to be more potent DNMT1 inhibitor than RG108, a DNMT1 inhibitor described above.

A huge range of experimental evidences suggests that DNMT inhibitors may serve as efficient

chemo- and radiosensitizers in solid tumors. However, the observation reported need more support in

order to indicate intimate molecular mechanisms of chemo- and radiosensitization. Additionally,

  12

  13

theunderstanding of different mechanisms as well as the long term safety of DNMT inhibitors on normal

cells alone or in combination with standard treatments remains very limited and requires further research

efforts. Although clinical and preclinical data indicate a substantial toxicity of these inhibitors, other

evidence seems to suggest that these drugs may have potential in reducing toxicity under specific

conditions. Therefore, the future goal is to indentify compounds able to enhance the therapeutic index and

protect the non malignant tissues from side effects. Finally, other preclinical data suggest that the

sensitizing effects of DNMT inhibitors seem to depend on the epigenetic modulation of a wide array of

genes. A non negligible part of this effect may be related to the off-target mechanisms. Future research

will focus on establishing clinically relevant combinations of DNMT inhibitors and conventional cancer

therapies. In particular, a longstanding interest exists in the development of molecules that can modify

cellular responses to radiation and chemotherapy. The future perspectives lie in identifying more similar

compounds and elucidating their mechanisms of action in order to develop more effective cancer

therapies and treatments.

CADD for novel small molecule DNMT inhibitors

There are several CADD approaches which have been effectively applied to design novel small-

molecule DNMT inhibitors; these include: structure similarity-based approach and isosteric/bioisosteric

morphing, 3D-molecular docking and 3D-pharmacophore modeling as well as advanced neural-net

techniques with related SAR analysis. For instance, Medina-Franco and colleagues [13] have conducted a

virtual screen of a large database of natural products with a validated homology model of the catalytic

domain of DNMT1. The virtual screening focused on a lead-like subset of the natural products docked

with DNMT1, using three docking programs, following a multistep docking approach. Prior to docking,

the lead-like subset was characterized in terms of chemical space coverage and scaffold content.

Consensus hits with high predicted docking affinity for DNMT1 by all three docking programs were

identified. One hit showed DNMT1 inhibitory activity in a previous study. The virtual screening hits were

located within the biological-relevant chemical space of drugs, and represent potential unique DNMT

inhibitors of natural origin. Validation of these virtual screening hits is warranted.

Kuck et al [14] have recently performed a virtual screening of more than 65K lead-like compounds

selected from the National Cancer Institute collection using a multistep docking approach with a

previously validated homology model of the catalytic domain of human DNMT1. Experimental

evaluation of top-ranked molecules led to the discovery of novel small molecule DNMT1 inhibitors. VS

hits were further evaluated for DNMT3B inhibition revealing several compounds with selectivity towards

DNMT1. These are the first small molecules reported with biochemical selectivity towards an individual

DNMT enzyme capable of binding in the same pocket as the native substrate cytosine, and are promising

candidates for further rational optimization and development as anticancer drugs. The availability of

  14

enzyme-selective inhibitors will also be of great significance for understanding the role of individual

DNMT enzymes in epigenetic regulation.

In [15] the modulating effects of several tea catechins and bioflavonoids on DNA methylation

catalyzed by prokaryotic SssI DNA methyltransferase and human DNMT1 were studied. It was found that

each of the tea polyphenols [catechin, epicatechin, and (-)-epigallocatechin-3-O-gallate (EGCG)] and

bioflavonoids (quercetin, fisetin, and myricetin) inhibited SssI DNMT- and DNMT1-mediated DNA

methylation in a concentration-dependent manner. The IC50 values for catechin, epicatechin, and various

flavonoids ranged from 1.0 to 8.4 μM, but EGCG was a more potent inhibitor, with IC50 values ranging

from 0.21 to 0.47 μM. When epicatechin was used as a model inhibitor, kinetic analyses showed that this

catechol-containing dietary polyphenol inhibited enzymatic DNA methylation in vitro largely by

increasing the formation of S-adenosyl-L-homocysteine (a potent noncompetitive inhibitor of DNMTs)

during the catechol-O-methyltransferase-mediated O-methylation of this dietary catechol. In comparison,

the strong inhibitory effect of EGCG on DNMT-mediated DNA methylation was independent of its own

methylation and was largely due to its direct inhibition of the DNMTs. This inhibition is strongly

enhanced by Mg2+. Computational modeling studies showed that the gallic acid moiety of EGCG plays a

crucial role in its high-affinity, direct inhibitory interaction with the catalytic site of the human DNMT1,

and its binding with the enzyme is stabilized by Mg2+. The modeling data on the precise molecular mode

of EGCG's inhibitory interaction with human DNMT1 agrees perfectly with the experimental finding.

Concept and Applications

DNMT-targeted library design at CDL involves:

• A combined profiling methodology that provides a consensus score and decision based on various

advanced computational tools:

1. Bioisosteric morphing, structure diversity & similarity concept, topological pharmacophore and

funneling procedures in designing novel potential DNMT ligands with high IP value. We apply CDL’s

proprietary ChemosoftTM software and commercially available solutions from Accelrys, MOE, Daylight

and other platforms.

2. Neural Network tools for target-library profiling, in particular Self-organizing Kohonen Maps,

performed in SmartMining Software.

3. 3D-molecular docking approach to focused library design.

4. Computational-based `in silico` ADME/Tox assessment for novel compounds includes prediction of

human CYP P450-mediated metabolism and toxicity as well as many pharmacokinetic parameters, such

as Brain-Blood Barrier (BBB) permeability, Human Intestinal Absorption (HIA), Plasma Protein binding

(PPB), Plasma half-life time (T1/2), Volume of distribution in human plasma (Vd), etc.

The fundamentals for these applications are described in a series of our recent articles on the design of

exploratory small molecule chemistry for bioscreening [for related data visit ChemDiv. Inc. online

source: www.chemdiv.com].

• Synthesis, biological evaluation and SAR study for the selected structures:

1. High-throughput synthesis with multiple parallel library validation. Synthetic protocols, building

blocks and chemical strategies are available.

2. Library activity validation via bioscreening; SAR is implemented in the next library generation.

We practice a multi-step approach for building DNMT-focused library:

Virtual screening

(1) The small-molecular ligands for all DNMT classes (see Table 1) are compiled into a unique

knowledge base (reference ligand space) and annotated according to the particular DNMT subtype. The

knowledge base has been thoroughly analyzed (a) using Tanimoto similarity measure (Table 2) towards

divers compounds from ChemDiv collection as well as (b) specific bioisosteric rules and topological

pharmacophores (Fig. 6) used in the subsequent morphing procedures.

Table 2. Structure similarity between reference compounds and structures from DNMT-targeted library

DNMT inhibitors Similarity coeff. ChemDiv compound

Azacitidine

0.73 N

N

O

O

O H

N2

H

O HOH

 1075-0005

  15

N NH

OHOO

OCl

0.59 NHNH

O

O

OH

Cl

 1682-7513

O

OH

C H 3

OOO

OH

C3H

0.52

O

O

CH3

CH3

 0402-0007

N

NH

N H

ONH

N N

C H 3

N H 2

0.51 N

NHN

NHN HO

C3H

 C301-7344

O

OH OH

SOH

O N

N

N

N

NH2

0.50 N

N+ N

O

N

O-

NH2

OH

OH

OH

 0723-2064

  16

N

NH

N H

ONH

N N

C H 3

N H 2

0.49 NH

NH

OO

CH3

D421-0689

N NH

OHOO

OCl

0.48 N

NH

NH

O

O

Cl

 8207-0142

 

  17

N

O

O

N

MeO

X

NN

O OO

ArArlinker (6C)

linker (6C)

Hb-acceptors Hb-acceptors

Ar

Ar

Ar

Ar

HBA

E518-1248

DNMT inhibitor

  18

N

N N

N

NN

N

N

N

N

O

O

N

N

Ar

Ar Het

HetAr

Ar

HBA

HBA

HBD

HBD E760-0018DNMT inhibitor

Fig. 6. Specific topological pharmacophores and bioisosteric modifications (examples)

3D-molecular Docking

For the DNMT-targeted library design we have been used a molecular docking approach.

Currently, several crystallographic complexes of DNMT with SAM, SAH as well as DNA are available in

PDB databank - 3AV6, 3AV5, 3PTA (Fig. 7). The obtained data has been used for the active site

construction, 3D-modeling and virtual scoring generation.

 

(A)

(B)

  19

(C)

Fig. 7. Crystallographic data used for 3D-molecular docking construction: (A) DNMT1 with SAM; (B)

DNMT1 with SAH; (C) mouse DNMT1 bound to DNA containing unmethylated CpG sites [16].

For example, Song and colleagues have recently solved structures of mouse and human DNMT1

composed of CXXC, tandem bromo-adjacent homology (BAH1/2), and methyltransferase domains bound

to DNA-containing unmethylated CpG sites (Fig. 7A). The CXXC specifically binds to unmethylated

  20

CpG dinucleotide and positions the CXXC-BAH1 linker between the DNA and the active site of

DNMT1, preventing de novo methylation. In addition, a loop projecting from BAH2 interacts with the

target recognition domain (TRD) of the methyltransferase, stabilizing the TRD in a retracted position and

preventing it from inserting into the DNA major groove. These studies identify an autoinhibitory

mechanism, in which unmethylated CpG dinucleotides are occluded from the active site to ensure that

only hemimethylated CpG dinucleotides undergo methylation. Figure 8 shows the 3D-alignment

(superposition) of two protein data: 3AV6 and 3PTA. As clearly shown in the fig below, these DNMT1

samples have a good 3D-similarity, except the activation DNA-loop for 3PTA (active conformation).The

active SAM-binding sites are quite identical in structure and position; the SAM/SAH positions and

related binding points are also very close.

 

  21

Fig. 8. The superposition of 3AV6 and 3PTA

Based on the obtained data we have constructed and thoroughly validated the DNMT1 3D-

molecular docking model using reference compounds listed in Table 1. Then, the model has been used for

the prioritization of ChemDiv chemotypes to be included in the final DNMT-targeted library. Several

representative examples of ChemDiv compounds in the active SAM-binding site are shown in Fig. 9.

(A) E518-1248

  22

(B) E760-0018

Fig. 9. Representative compounds (yellow) from DNMT-focused library in the SAM-specific binding

site; Reference DNMT-active compound (orange)

Key statistical parameters for compounds in DNMT-targeted library are shown below.

 

 

   23

 

 

 

 

   24

Several representative examples of compounds from DNMT-focused library are shown in fig. 10.

  25

N

O

O

O

N

N

O O

N

O

O

O

N

N

NN

O

O

ON

ON

O

OO

O

OO

O

SN

N O

OO

O O

Cl

SN

O

ONN

N

O

O

O

N

OO

S

NN

N

N

O

O

F F

FS

NO

O

ON

O

NN

NN

N

NS

O

F

F F

N

N N

O

N

O

O

N

N

S

N

Cl

O

N

N

N

O

OO

S

N

N S

NO

OO

O

OO

N

O

OO

NO

NO

N

NN

O

N

S N

NN

O

OO

N N

N

O

O

N SN

ONO

NO

OO N

N

NN N

N

O

OBr

Fig. 10. Compounds from the DNMT-targeted library (representative selection from more than 28K structures with high virtual score)

As a result of the performed CADD, we have selected approx. 29K small-molecule compounds which are the main content of the final DNMT-targeted library. We also provide rapid and efficient tools for follow-up chemistry on discovered hits. Targeted library is updated quarterly based on a “cache”

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principle. Older scaffolds/compounds are replaced by templates resulting from our in-house development (unique chemistry, literature data, CADD) while the overall size of the library remains the same (ca. 28-35K compounds). As a result, the library is renewed each year, proprietary compounds comprising 50-75% of the entire set. Clients are invited to participate in the template selection process prior to launch of our synthetic effort.

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