DNMT-Focused Library

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: [email protected]

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 posttranslational 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 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, Azacitidine/ every four weeks. In 2005, Pharmion withdrew its Launched in 2004 marketing authorization application (MAA) for the product in Europe following a request for additional data from the Pfizer EMEA and in 2006, the application was resubmitted. In

2008, a positive opinion was received in the E.U. for the Apoptosis Inducers, treatment of MDS. Final approval was obtained in 2008. multicancer therapy 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. 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 Decitabine/ licensed to SuperGen in 1999. The compound has been Launched – 2006 Pharmachemie shown to have a broad spectrum of activity in several hematological malignancies as well as solid tumors. Recent Multicancer Therapy research demonstrates tumor suppressor genes and DNA Hematologic Agent repair genes are hypermethylated and silenced in a (Miscellaneous) majority of cancers, typically at an early stage of the Hematopoiesis Disorders disease. Hypermethylation may prove useful for early Therapy cancer detection and screening of cancer patients. Therapy that removes the methyl groups by hypomethylation may activate natural defense mechanisms against cancer. 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 Zebularine/ of cytosines in DNA synthesized after drug treatment. Preclinical When used at relatively high concentrations this drug results in the formation of high levels of enzyme-DNA University North Carolina, adducts. The compound also inhibits Cytidine Deaminase Chapel Hill activity. 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 (-)-EGCG / Phase hydrogen bonds with Pro(1223), Glu(1265), Cys(1225), II/III 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 Multicancer Therapy well as genistein from soybean, can inhibit SssI DNMT- and DNMT1-mediated DNA methylation in a concentration-dependent manner. This antisense oligonucleotide targets the 3 UTR of Structure has not been DNMT1 causing a methylation decrease in cell lines and disclosed yet animal models. Second-generation phosphorothioate MG98/ antisense oligodeoxynucleotide targeting DNA Discontinued MethylGene methyltransferase-1 (DNMT1). MG-98 had been in clinical development by MethylGene for the treatment of Multicancer Therapy 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 / This small molecule agent is not straightly incorporated Biological into DNA but it binds to the catalytic site of DNMTs Testing causing inhibition of DNA methylation.

Institute Biochem. Biophysics PAS

This molecule reduces DNMT1’s affinity for both DNA Procainamide / and S-adenosyl-methionine causing a decrease in DNA Launched methylation. Bristol-Myers Squibb Pfizer 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 Disulfiram / conducting phase II clinical trials with the compound to Launched evaluate its potential for the treatment of cocaine Pfizer dependency. The University of California, Irvine is

conducting early clinical studies of the compound in Anticataract Agent Cancer Therapy combination with arsenic trioxide in patients with Treatment of Alcohol and metastatic melanoma and at least one prior systemic Cocaine Dependency 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

Pfizer Oncolytic Drugs RG108, a DNMT1 inhibitor, represents promising candidate for cancer drug development. The structural design of the chemically modified inhibitor was aided by RG108 / molecular modeling, which suggested the possibility for Biological extensive chemical modifications at the 5-position of the Testing tryptophan moiety in RG108. The inhibitory activity of the

corresponding derivative was confirmed in a cell-free Institute Biochem. Biophysics PAS biochemical assay, where bio-RG108 showed an Oncolytic Drugs undiminished inhibition of DNA methyltransferase activity (IC50 = 40 nM).

Compounds in Biological - Testing

Cancer Research Technology Oncolytic Drugs

Compounds in Biological DNMT1 and DNMT3b2 inhibitors Testing

MethylGene

Oncolytic Drugs

Compounds in Biological - Testing

Nippon Kayaku Oncolytic Drugs

Thymolphthalein / Biological IC50 = 9 μM in PRSET-7 cell line (HKMTS assay) Testing

University Medicine Dentistry New Jersey Oncolytic Drug

XB-05 / XB-05 - a novel non-nucleoside inhibitor of DNMT1 with Preclinical potent activity in colon, breast and prostate cancer cells. University of Louisville Oncolytic Drug

This compound is able to induce a recognizable demethylation of chromosomal satellite repeats in HL60 Biological human myeloid leukemia cells and thus represents a lead Testing Deutsches compound for the development of a novel class of non- Krebsforschungszentrum nucleoside DNMT1 inhibitors. (DKFZ) Univ degli studi di Napoli 11 Federico II Universita degli Studi di Salerno (Originator) Oncolytic Drug

Histone-lysine N-methyltransferase GLP/Eu-HMTase1, BIX-01294 / inhibition: IC =27 nM (Chemiluminescent assay); Biological Alexis Biochemicals 50 Euchromatic histone-lysine N-methyltransferase 2a [G9a], Testing Emory University M.D. Anderson Cancer inhibition: IC50=0.106 µM (Fluorescent assay). Center Sigma-Aldrich University of Illinois at Chicago Oncolytic Drug This compound inhibits the activity of DNMT1, DNMT3A, and DNMT3B as well M. SssI with comparable SGI-1027 / IC50=6-13 μM/L) by competing with S- Biological adenosylmethionine in the methylation reaction. Treatment Testing of different cancer cell lines with SGI-1027 resulted in SuperGen selective degradation of DNMT1 with minimal or no Oncolytic Drug effects on DNMT3A and DNMT3B. 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 KED-4-69 lines (GI50=1.50-56.1, LC50 >100 μM), with no toxicity in Georgetown University mice up to 550 mg/kg. p.o. In mice bearing PC-3 tumor Prostate Cancer Therapy xenografts, it reduced tumor volume (40%) at 10 mg/kg/day i.p. over 28 days. Potentially useful for the treatment of prostate cancer.

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

described above. Nagoya City University (NCU) Oncolytic Drug

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 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 13 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 14 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

H N 2

O N 0.73

O O H

H O Azacitidine O H

1075-0005

15 O O OH O Cl N H N N H 0.59 H N OH O O Cl

1682-7513

CH O 3

H O OH

H C C H 3 O O 3 0.52 O O

O CH 3

0402-0007

H C C H 3 H N 3 O H N N N N N H O N H N H N N H H N 2 0.51

C301-7344

NH 2 - O NH + N 2 N

N N N N OH O N N 0.50 O O H O S

OH H O OH H O

0723-2064

16 O O H N C H O 3 N N H N N H N H 2 H N 0.49 NH CH 3

D421-0689

O O O OH N

N N H 0.48 H N O

O Cl NH

8207-0142

linker (6C) X

linker (6C) Ar N Ar N O N Ar O O O MeO N O E518-1248 Hb-acceptors Hb-acceptors

DNMT inhibitor Ar

HBA

17 HBA N N O Ar Het N N N HBA Ar N O Het Ar N HBD N N N DNMT inhibitor HBD E760-0018

N Ar N

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)

(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.

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

(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.

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

25 O O O O N O O O N N O N O O

O O O O O O O N O N O N N O O N N N S O N O O N Cl N N O N

N S O O O O N N N S O O O N O F N N O S O F N O F O N N O S N S N N N O N O N Cl O N N N N N F N N F F O N S O N N N S O N N O O O N O O O O N O O O O O N N N O O SN N O N O N O N N O O N O O NN O N O N N N N S N O Br O

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”

26 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.

27 References

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