(human lung cancer cell line harboring wild-type p53), an IC 50 of 20 μM for U373MG cell line (human glioblastoma cell line harboring inactive mutant p53) [18] and an IC 50 of 30 μM for Hut78 cell line (cutaneous T-cell lymphoma). [19] [20] Despite the fact there are two DNMT inhibitors approved for clinical use, both azacitidine and decitabine have low specificity, poor bioavailability, and instability in physiological conditions and toxicity.
Therefore, it has been the interest of our [21] [22] [23] [24] [25] [26] [27] [28] and several other research groups [29] [30] [31] [32] [33] [34] [35] [36] to identify DNMT inhibitors with novel chemical scaffolds for further development. Inhibition of DNMTs remains a major topic of research not only because of its potential therapeutic benefits but also to understand the essential mechanisms of epigenetic events in cells. There are currently more than 256 compounds in annotated public chemical databases [37] with measured activity vs. DNMTs. Figure S1 in the Supporting Information shows the most frequent scaffolds of active molecules. Figure 1 shows the chemical structures of representative DNMT inhibitors or compounds with DNA demethylation activity from different sources, including drugs for other indications, screening compounds from synthetic origin, and natural products. [38] [39] [40] Also, there are several compounds from dietary origin. [41] [42] [43] Of note, strong evidence indicates that environmental factors and nutrients play a major role in establishing epigenetic mechanisms, including irregular DNA methylation patterns. Thus, a regular uptake of DNA demethylating agents (which are not necessarily very potent DNMT inhibitors) is hypothesized to have a chemopreventive effect. [44] The DNMT inhibitors have been identified from different approaches or their combination [44] [45] such as virtual screening, [21, 30] high-throughput screening, lead optimization, [34, 46] and structure-guided design [36] to name a few. Amongst the most promising inhibitors are molecules with "long scaffolds" such as the quinolone-based SGI-1027 ( Figure 1 ) and analogs. With the aid of molecular modeling, it has hypothesized that such compounds with long scaffolds occupy the catalytic site and SAM's cavity. [47] It has also been proposed that analogs of SGI-1027 exert their mechanism through interaction with DNA. [48] As part of an ongoing effort to identify novel DNMT1 inhibitors from different sources and further increase the availability of novel scaffolds, herein, we report five new inhibitors of DNMT1 with distinct chemical scaffolds. Two compounds, glyburide, and panobinostat are approved drugs with potential drug-repurposing applications. In particular, panobinostat is a pan histone deacetylase inhibitor (HDAC), another major epigenetic target, and could be used as a dual epigenetic agent.
We aimed to test compounds from different sources: approved drugs, synthetic compounds from a DNMT focused library, and one compound from dietary origin. We experimentally tested ten molecules with diverse chemical scaffolds and chemical structures different from reported DNMT inhibitors ( Figure   2 ). Thus, we screened in biochemical assays as inhibitors of DNMTs two approved drugs with "long" chemical scaffolds (Figure 2A ): the sulfonylurea glybenclamide, approved for the treatment of diabetes (vide infra), and panobinostat, a non-selective and potent zinc-dependent HDAC inhibitor (the most potent deacetylase inhibitor on the market), approved in 2018 by the FDA for the treatment of multiple myeloma. Of note, currently epigenetic multitargeting is focused on the inhibition of zinc-dependent HDACs as one of action mechanisms. [14] Due to the known activity of polyphenols and previous evidence of theaflavin activity with DNMT3A, [49] we hypothesized that theaflavin, a dietary component ( Figure 2B ) present in the back tea, is an inhibitor of DNMT1 and DNMT3B. We also theorized that the naphthalene sulfonamide 7936171 ( Figure 2C ) with a long scaffold is an inhibitor of DNMT1 and DNMT3B. [50] Finally, we tested six molecules from a DNMT focused library of synthetic molecules ( Figure 2D ), which are becoming attractive to experimentally screen molecules. [6] To select the ten compounds in this work, commercial availability and reasonable price (the latter an important factor considering the current economic situation imposed by the COVID-19 pandemic) were additional criteria. First, the ten compounds were tested at a single concentration (100 M) in duplicate in a biochemical DNMT assay. SAH was used as a reference. The five most active molecules at one-single concentration were further tested in a dose-response manner to obtain the half-maximal inhibitory concentration (IC 50 ).
Predicted binding modes for the five compounds were studied using molecular docking, implementing a novel re-scoring algorithm recently developed. [51] 
All compounds were purchased from chemical vendors. Glyburide, panobinostat, and theaflavin were purchased from TargetMol. Molecules 7936171 and 6631802 were purchased from Chembridge Corporation. Molecules CSC027480404, CSC026286840, CSC027694519, and CSC027083851 were acquired from ChemSpace. The compound purity confirmed by the chemical vendors was equal or higher than 90 % (Table S1 in the Supporting information).
The inhibition of the enzymatic activity of DNMT1, DNMT3B, and DNMT3B/3L was tested using the HotSpot SM platform for methyltransferase assays available at Reaction Biology Corporation. [52] HotSpot SM is a low volume radioisotope based assay that uses tritium-labeled AdoMet ( 3 H-SAM) as a methyl donor.
The test compounds diluted in dimethyl sulfoxide were added using acoustic technology (Echo550, Labcyte) into enzyme/substrate mixture in the nano-liter range. The corresponding reactions were commenced by the addition of 3 H-SAM, and incubated at 30°C. Total final methylations on the substrate (Poly dI-dC in DNMT1 assay, and Lambda DNA in DNMT3B; DNMT3B/3L assay) were identified by a filter binding method implemented in Reaction Biology. Data analysis was conducted with Graphed Prism software (La Jolla, CA) for curve fits. Reactions were carried out at 1 μM of SAM. In all assays, SAH was used as a standard positive control. The ten compounds were tested first with DNMT1 and DNMT3B at one 100 M concentration in duplicate. The five most active compounds were tested in 10-dose IC 50 (effective concentration to inhibit DNMT1 activity by 50%) with a three-fold serial dilution starting at 100
M. Theaflavin was also tested with DNMT3B and DNMT3B/L in 10-dose IC 50 with three-fold serial dilution starting at 100 M. The authors have previously contracted the screening services of Reaction Biology Corporation to identify a novel inhibitor of DNMT1. [23] 
The five compounds tested in a dose-response manner, and that showed activity with DNMT1 (vide infra)
were docked with this enzyme using the program Molecular Operating Environment (MOE), version 2018.08. [53] The chemical structures of the five compounds were built with MOE. The docking was carried out with the crystal structure of the catalytic domain of DNMT1 obtained from the Protein Data Bank [54] PDB ID: 4WXX. [55] This crystal structure is in complex with SAH and has a resolution of 2.62 Å. The structure of the protein was prepared with the "QuickPrep" tool of MOE v.2018 using the parameters established by default, which help to remove the molecules of structural water and add hydrogens atoms to the protein. In this process, the co-crystallized SAH was removed for the binding site to realize a direct docking. The docking was conducted using default parameters in MOE. Before docking the five newly tested compounds, the docking protocol was validated by re-docking the SAH obtaining a root-meansquare deviation of 1.3 Angstroms (and a docking score of -8.96 kcal/mol).
Docking poses obtained with MOE were re-scored with the Extended Connectivity Interactions Features (ECIF) method recently developed. Briefly, ECIF is a recently reported set of descriptors to represent protein-ligand complexes. These descriptors are defined as a set of protein−ligand atom-type pair counts relying on a detailed description of the connectivity of the atoms involved. It has been shown that machine-learning scoring functions built on ECIF and Gradient Boosting Trees, consistently outperform the performance of scoring functions reported so far regarding the obtention of binding scores in a linear correlation with experimental data, particularly when a distance cutoff criterion of 6 Angstroms is used to derive the descriptors, and purely ligand-based descriptors are added (ECIF6::LD-GBT). [51] From the poses obtained from MOE, the best-ranked one for each compound was prepared using X-Tool [56] and standardized using Standardizer, JChem 20.11.0, 2020, ChemAxon to perceive aromaticity in an interpretable way for the model. All poses were re-scored using the ECIF6::LD-GBT model. [51] Table 1 summarizes the relative enzymatic activities of DNMT1 and DNMT3B in the presence of 100 M compound. Compounds that had more than 20% inhibition were regarded as inhibitors and were moved forward to dose-response evaluations. We used a similar criterion in a previous identification of novel chemical scaffolds. [21] Seven molecules showed detectable inhibition with DNMT1, of which the five most active were theaflavin (65 % inhibition), CSC027694519 (65 %), panobinostat (63 %), 7936171 (62 %), glyburide (60 %). The least active were CSC027480404 (29 %) and CSC026286840 (27 % inhibition).
Theaflavin and 7936171, showed detectable inhibition with DNMT3B (33 and 22 % inhibition, respectively). All other eight molecules were inactive with DNMT3B. The five compounds with the highest percentage of inhibition at 100 M with DNMT1 were tested in dose-response manner. Theaflavin that showed the best activity at a single dose with DNMT1 and DNMT3B was also tested drug-response manner with DNMT3B and DNMT3B/3L. Table 2 summarizes the results. 
The docking was done in MOE v.2018 for the 10 compounds (shown in Figure 2 The docking poses of the five compounds were re-scored with ECIF, as described in the Methods section. Table 3 values, the most active, glyburide, is predicted correctly by the ECIF re-scoring method. Also, the rescoring of MOE poses ranks glyburide as the one with the highest affinity overall, while MOE score predicts a higher affinity for compounds 6631802 and CSC026286840. Figure 4 shows the association between the experimental pIC 50 of the five compounds evaluated in biochemical inhibition assays of DNMT1 with the MOE's docking scores ( Figure 4A ) and re-scoring with ECIF6::LD-GBT ( Figure 4B ).
Clearly, the ECIF re-scoring scheme improved the correlation with the experimental pIC 50 values. 
The initial goal of this study was to identify novel small molecule DNMT1 and DNMT3B inhibitors. In this work, we deprioritize testing with DNMT3A because it has been reported that this enzyme can act as both an oncogene and as a tumor suppressor gene, at least in lung cancer (it remains to test if this paradox of DNMT3A applies to other cancers). [8] Seven molecules with detectable inhibition of DNMT1
and two with DNMT3B were initially identified at single dose concentrations (Table 1 ). An approved drug for the treatment of type 2 diabetes (glyburide) and an approved anticancer drug known as HDAC inhibitor (panobinostat) were among the active compounds with DNMT1. Other active compounds against DNMT1 were a dietary component (theaflavin), a naphthalene sulfonamide (7936171) previously reported to be an inhibitor of dengue virus methyltransferase, [50] and three small molecules from a DNMT focused library (CSC027480404, CSC026286840, and CSC027694519) .
Overall, the tested compounds showed less activity with DNMT3B (Table 1) . Only two compounds, the dietary component theaflavin, and the naphthalene sulfonamide, showed inhibition higher than 20 % with DNMT3B at high concentration. Moreover, theaflavin showed the highest percentage of inhibition (33 %), which was lower than the highest percentage of inhibition showed for DNMT1 (65 %). None of the two compounds had an IC 50 lower than 100 M with DNMT3B.
Dose-response assays for the most active compounds at a single dose ( have not been fully established and the results show a large variation between assay conditions. This point has been largely discussed in the literature for DNMT inhibitors. [47, 57] In this work, we used SAH as a positive control, since SAH is a very well-known inhibitor of DNMT. [58] Even though the active compounds' potency values are not high, all compounds have chemical scaffolds different from the scaffolds reported for known DNMT inhibitors.
Glyburide ( Figure 2 ) is a sulfonylurea. In 1984 it received approval by the United States FDA for the treatment of patients with diabetes mellitus type II. To the best of our knowledge, there are no reports of this compound as an inhibitor of DNMT1. However, several studies support that sulfonylureas are potential anticancer drugs candidates for several cell lines such as colon cancer, human ovarian cancer, kidney cancer, melanoma, and lung cancer. [59] [60] Therefore, we propose that glyburide could be further investigated and developed as an epi-drug with potential anticancer activity.
Panobinostat is an orally available pan-deacetylase inhibitor with broad antitumor activity. [61] It has shown inhibitory activity vs. different types of cancer, for example, malignant glioma cells with an IC 50 range of 0.3 μM to 0.23 μM, [62] carcinoma cells (IC 50 between 0.4 nM -1.3 nM), [63] and in human erythroleukemic cell lines (IC 50 between 40 nM to 51 nM). [64] One of the principal benefits of the panobinostat is that being an HDAC inhibitor will avoid the histone acetylation, which helps to maintain closed the chromatin and decrease the gene transcription involved with proliferation, differentiation, and progress of the cancer cells. Zopf et al. reported that panobinostat reduced DNMT1 (and DNMT3a) activities and expression in liver cancer cell lines. [65] In the study of Zopf et al., it was concluded that inhibitors of HDACs can indirectly control DNA methylation. In this work, it was confirmed that panobinostat also inhibits the enzymatic activity of DNMT1 directly. Of note, there is a recent interest in developing dual inhibitors of DNMTs and HDACs, and results in this direction look promising. [17] Indeed, Yuan et al. reported hydroxamic acid derivatives of a small molecule previously identified as a week inhibitor of DNMT1 from virtual screening of a large chemical library. [21] In the work of Yuan et al., the hydroxamic acid derivatives of NSC 319745 showed inhibition of DNMT1, HDAC1, and HDAC6, plus cytotoxicity activity against human cancer cells. [46] In a second example in the clinic, the combined use of decitabine (a DNMT inhibitor) and belinostat (an HDAC inhibitor) increases the chemotherapy efficiency. [66] Besides, Min et al. showed that an increase in transcription of DNMT1 is one of the mechanisms of resistance of anti-cancer drugs targeting HDACs, such as vorinostat. Consequently, it has been shown that co-targeting DNMT1 improved the antitumor efficacy of vorinostat and other HDAC inhibitors. [67] These contributions, including the findings of this work, are in line with the epigenetic multitargeting. The advantages of this approach have been demonstrated in both in vitro and in vivo disease models by the co-administration of an epigenetic agent with another drug. [68] The naphthalene sulfonamide 7936171 (also with ID ZINC 01078518) is a known inhibitor of the viral NS5 RNA methyltransferase with an IC 50 of 64.2 M and an EC 50 of 12 M. [50] The viral RNA domain involves the use of SAM as the methyl donor and generates SAH as the final product. Compound 7936171 has a "long" and different scaffold from the known DNMT1 inhibitors published so far (Table   S1 ).
Theaflavin is a natural product polyphenol found in green and black tea and coffee (vide supra) with previously measured enzymatic inhibitory activity of DNMT3A. [49] However, there were no reports on its inhibitory potential of DNMT1 and DNMT3B. In this work, it was measured for the first time its activity with both enzymes. Despite the fact its IC 50 is high (85.3 M, under the assay conditions of this work), this dietary component could contribute to the modulation of DNMT1. Interestingly, it has been proposed that the modulation of normal levels of DNMT could be conveniently achieved through the dietary uptake of food chemicals (or other "safe" natural products). A prominent example of this hypothesis has been suggested for the polyphenol compound from green tea, EGCG (Figure 1 ), which has been proposed to inhibit DNMT1 and reactivate methylation-silenced genes in cancer. [44] The other two small molecules, 7936171 and CSC027694519, had no previous reports of inhibition of DNMTs. As discussed in the next section, both molecules have dug-like characteristics and have novel scaffolds that can be further optimized for DNMT1 inhibition.
Herein we employed in silico methods to rationalize at the molecular level the experimental results of the most active molecules. Of the different mechanisms described to inactivate DNMT activity [69] we hypothesized that the herein identified inhibitors are SAM competitors. This hypothesis is based on the "long" scaffolds of the active compounds such as glyburide, panobinostat, 7936171, and Figure S2 ). Similarly, comparing the predicted binding mode of 7936171 with the co-crystallized position of SAH ( Figure S2 ) suggest that the naphthalene ring of 7936171 could be replaced with polar heteroaromatic rings. Of course, the synthesis and testing of the analogs should be made, which is one of the major perspectives of this work. It remains to confirm the hypothesis of the putative binding site of these molecules that can be further tested experimentally with binding completion assays once more potent compounds are identified.
DNMTs are a fundamental class of epigenetic regulatory enzymes. In this work, we tested ten compounds with novel scaffolds as inhibitors of DNMT1 and DNMT3B in biochemical assays. Seven compounds showed at least 20% inhibition of DNMT1 at 100 M. Five molecules showed activity in dose-response inhibition assays with DNMT1 with IC 50 between 55.8 and 85.3 M. Although the molecules' overall potency is not high under this work's assay conditions, four compounds have novel chemical scaffolds, not previously described as inhibitors of DNMT1 in biochemical assays. Notably, glyburide, an approved drug for diabetes type II treatment, showed the best potency of the compounds tested in this study and can be further investigated in its role in epigenetic mechanisms. Also, glyburide could be pursued for drug repurposing applications. It was also concluded that panobinostat is an inhibitor of the enzymatic activity of DNMT1. Remarkably, panobinostat, an epigenetic drug for treating different types of cancer, has shown inhibition of DNMT1 in liver cancer cell lines. [65] Therefore, it is concluded that panobinostat, a known HDAC inhibitor, can be further investigated as a multitarget epigenetic agent. In this work, we also concluded that theaflavin, a natural dietary product, inhibits DNMT1 and does not show significant inhibition of DNMT3B. Therefore, this work also complements theaflavin's DNMT activity profile that had reported activity with DNMT3A. [49] It was also concluded that compounds 7936171 and CSC027694519 and the two compounds that showed 30 % inhibition of Therefore, docking with MOE for compound selection and re-scoring with ECIF for prioritizing experimental tests can guide the optimization programs of the DNMT1 inhibitors identified in this work, which is one of the main perspectives of this work. Another perspective is testing the DNA demethylation activity of the most active compounds. This can be achieved directly with 7936171 and CSC027694519
or after the compounds are optimized.
Chemical structures of the ten most frequent (Bemis-Murcko) scaffolds of the active DNMT inhibitors available in ChEMBL 27 ( Figure S1 ); Chemical vendors of the ten compounds tested and their purity as supplied by the vendor ( Table S1) ; Comparison of the co-crystal position of SAH in the catalytic site of DNMT1 (PDB ID: 4WXX) with the predicted binding mode of A) CSC027694519 and B) 7936171 ( Figure S2 ).