A Photo‐responsive Small‐Molecule Approach for the Opto‐epigenetic Modulation of DNA Methylation

Abstract Controlling the functional dynamics of DNA within living cells is essential in biomedical research. Epigenetic modifications such as DNA methylation play a key role in this endeavour. DNA methylation can be controlled by genetic means. Yet there are few chemical tools available for the spatial and temporal modulation of this modification. Herein, we present a small‐molecule approach to modulate DNA methylation with light. The strategy uses a photo‐tuneable version of a clinically used drug (5‐aza‐2′‐deoxycytidine) to alter the catalytic activity of DNA methyltransferases, the enzymes that methylate DNA. After uptake by cells, the photo‐regulated molecule can be light‐controlled to reduce genome‐wide DNA methylation levels in proliferating cells. The chemical tool complements genetic, biochemical, and pharmacological approaches to study the role of DNA methylation in biology and medicine.

Abstract: Controlling the functional dynamics of DNAwithin living cells is essential in biomedical research. Epigenetic modifications such as DNAmethylation play akey role in this endeavour.D NA methylation can be controlled by genetic means.Y et there are few chemical tools available for the spatial and temporal modulation of this modification. Herein, we present as mall-molecule approach to modulate DNAm ethylation with light. The strategy uses aphoto-tuneable version of ac linically used drug (5-aza-2'-deoxycytidine) to alter the catalytic activity of DNAmethyltransferases,the enzymes that methylate DNA. After uptake by cells,t he photo-regulated molecule can be light-controlled to reduce genome-wide DNA methylation levels in proliferating cells.T he chemical tool complements genetic,b iochemical, and pharmacological approaches to study the role of DNAm ethylation in biology and medicine.
The methylation of DNAatposition 5ofcytosine residues is chemically av ery simple but biologically one of the most important modifications of DNA. It influences many biological processes in humans such as the regulation of cell function, cellular reprogramming,a nd organismal development. [1][2][3][4][5][6][7] Biological effects of higher methylation levels at promoters are mediated by lowering the transcription of genes either by blocking the binding of transcription factors or by recruiting unique methyl-recognizing proteins that lower gene expression. Altered levels of methylation are also associated with several diseases [8][9][10][11] including cancer. [8,[12][13][14][15][16] Driven by the growing importance of DNAmethylation in biomedical research, there is as trong interest to experimentally lower or increase methylation levels [17][18][19][20][21][22][23] to study,f or example,t he role of epigenetic reprogramming in tissue development or regenerative medicine. [24,25] Optical control is of particular relevance given the high spatial and temporal resolution of light. Often, the approach is implemented with photosensitive small molecules of tuneable bioactivity. [26][27][28][29][30][31] These can be used without the need for genetic engineering of cells leading to powerful applications within cell biology. [32] Yet, despite the importance of DNAm ethylation in biology, no light-tuneable small-molecule tool has been developed to manipulate methylation levels in cells.
Herein, we present ap hoto-mediated small-molecule strategy that modulates methylation in light-exposed cells. At the approachsc entre is an inhibitor that interferes with DNAmethyltransferases (DNMTs), the enzymes responsible for DNAm ethylation [33] including the maintenance DNA methyltransferase 1( DNMT1). [34] Thei nhibitorsb ioactivity becomes tuneable with light by the chemical derivatization with ap hotocage.A ss chematically illustrated in Figure 1a, the attached photocage renders the inhibitor biologically inactive.However, light exposure cleaves off the photocage to restore the original inhibitory effect (Figure 1a). Thep hotocaged molecule is hence expected to maintain methylation levels in the dark, while light should decrease methylation levels following replication of cells [35] (Figure 1a).
Our approach was implemented with DNMT inhibitor 5aza-2'-deoxycytidine (dAC, decitabine) [35,36] (Figure 1b). The cytidine analogue is aclinically used drug for myelodysplastic syndromes [37] and is being tested against leukemia and solid tumors [18,38] and as sensitizer for immunotherapies. [39,40] 5-aza-2'-deoxycytidine is the best choice for the photocaging approach given its high inhibitory effect on DNMTs [41] even though it is also known to undergo slow hydrolysis at the 5aza-base ring. [42] To exert its inhibitory effect after cellular uptake,d AC is phosphorylated by deoxycytidine kinase in ar ate-limiting step. [43] Subsequent phosphorylations to triphosphate lead to the polymerase-mediated incorporation into DNA [43] in which the 5-aza-base ring forms ac ovalent adduct with DNMT.T his adduct prevents methylation of DNAi nr eplicating cells but also targets DNMT for proteosomal degradation. [44] Given the tight fit inside the active site of deoxycytidine kinase (Supporting Information, Figure S1), we surmised that photocaging dACw ould block the rate-limiting step of phosphorylation and hence abolish inhibition of DNMT.
To optically control the activity of dAC, we attached aphotocage to each possible coupling site within the nucleoside,the exocyclicNH 2 group of the base and the 3' and 5' OH groups of the deoxyribose (Figure 1b). [27,31] All three positions were modified as the resulting steric blockade was expected to hinder binding of dACi nto the active site of deoxycytidine kinase ( Figure S1). Fort he chemical derivatization, diethylaminocoumarinyl-4-methyl (DEACM) (Figure 1b)w as used given its high extinction coefficient (e = 16 000 m À1 cm À1 )a nd long absorption wavelength (l = 385 nm) that ensure biocompatibility by avoiding mutagenic irradiation at high intensity in the UV spectral region.
Three DEACM derivatives of dAC 1a, 2,a nd 3 (Figure 1b)w ere synthesized. In 1a,t he photocage is attached through acarbamate bond to NH 2 ,while the linkage in 2 and 3 is mediated through acarbonate to 5' and 3' OH, respectively ( Figure 1b). Thesynthetic routes to 1a, 2,and 3 are described in the Supporting Methods.
Additional photocaged compounds were made to demonstrate that the synthetic route is generic. Fore xample, synthesis of 1b and 1c carrying an itrophenyl group on the exocyclica mine ( Figure 1b)s howed that ac hromophore other than DEACM can be attached to dAC. 1b and 1c also served as reference compounds for the spectroscopy analysis (see below). Similarly,p reparation of nitrophenyl-modified azacytidine 1d (Figure 1b)s howed that the clinically used ribonucleotide version of dACcan be equipped with aphotocage (see Supporting Methods for synthetic routes of 1b-d).
DEACM-dACderivatives 1a, 2,and 3 were examined to probe whether the spectroscopic properties are influenced by the chromophoresattachment site.All compounds exhibited strong absorption at ab iocompatible wavelength of l = 365 nm (Figure 2a,T able 1) with e values close to that of unconjugated DEACM (e = 7000 m À1 cm À1 ,F igure S2) [45] implying minimal influence from the coupling to dAC. The data for compounds 1b-d showed similar results (Table 1, Figure S2).
Uncaging efficiency,b yc ontrast, was influenced by the site of dACa tw hich the chromophore was attached. The analysis (Figure 2b)ofcompound 1a revealed afast uncaging rate of k = 1.03 10 À3 s À1 equivalent to a5 0% recovery of dACwithin ahalf-life of t 1/2 = 11 min (Figure 2c)while 2 was slower ( Figure 2c and Figure S3), which is possibly due to aquenching interaction between the photocage and proximal triazine nucleobase.I ns upport, 3 with DEACM at more distant 3' OH to triazine had afast photolysis with t 1/2 = 8min (Figure 2b,c and Figure S3). Thel ikely mechanism for uncaging is shown in Figure S4.  Successful uncoupling of the photocage from the nucleobase was also found for control nucleotides 1b-d. The spectroscopic and photolytic properties were in line with literature values for nitrophenyl (Table 1a nd Figure S3). Nevertheless,t he uncaging rates of 1b-d are too low for subsequent cell work. By comparison, compound 3 has ahigh absorption wavelength and the fastest photolysis.
Analysis of 3 determined its stability in the absence of light. Unmodified dACi sk nown to have as lightly reduced stability owing to hydrolysis at the 5-aza-base ring leading to ahalf-life of 2200 min at 25 8 8C. [42] By comparison, 3 had ahalflife of 690 min 25 8 8C, which reflects partial hydrolysis of the ring and the carbonate linkage to the photocage ( Figure S5). This half-life is almost 86-fold longer than the half-life for photo-induced uncaging of 3 and 7-fold longer than the subsequent incubation duration to cells.This means that after 1h of light-induced deprotection, only 3% or less of compound 3 are still in the caged form. Dark instability is hence not compromising photo-uncaging.R eflecting its adequate stability and fast deprotection rate under illumination, compound 3 was used for subsequent biological investigations.
To test whether methylation levels in cells can be controlled with light, 3 was added to hypermethylated human cancer cell lines SaOS2 and T24. [46] Additional exposure of cells to light was expected to induce passive demethylation owing to photo-uncaging of 3 and the resulting non-methylation during DNAr eplication in dividing cells (Figure 3b). Lack of illumination was anticipated to maintain methylation ( Figure 3a). Consequently,c ells were incubated with 0.1 mm 3 and either illuminated for 1h at 365 nm and 25 8 8Co rk ept in the dark at 25 8 8C. Treatment of cells with unmodified dACs erved as ap ositive control for demethylation (Figure 3c). After incubation with the small molecules, the medium was changed, cells were grown at 37 8 8Cf or 24 h, genomic DNAwas isolated and enzymatically digested, and the nucleotide content was analysed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Figure 3d,e summarize the cellular levels of methylated C as percentage of the total cytosine pool for SaSO2 and T24 cells.E xposure to 3 without illumination maintained ah igh level of methylated DNA (Figure 3d,e, 3), thereby confirming that photocaged dACw as biologically inactive at the tested conditions.H owever,i ncubation with 3 and simultaneous exposure to light caused ad rastic reduction in methylated DNA ( Figure 3d,e, 3-light) to al evel almost identical to uncaged dAC ( Figure 3d,e,d AC). Control experiments in which cells were solely exposed to light in the absence of 3 did not affect methylation (Figure 3d,e,0,and Figure S6; 0 mm 3). Thed ata demonstrate that our strategy of light-induced demethylation is successful;byphotolysis of 3,dACsbiological inhibition was reactivated to block DNAm ethyl transferases within cells.O ur approach was also confirmed by demethylation at ac oncentration of 0.5 mm 3 ( Figure S6). At 1.5 mm or higher, the compound leads to demethylation without light exposure,p ossibly because 3 is hydrolytically inactivated by enzymes.
Molecular analysis confirmed the proposed mechanism for 3sattainment of lower methylation levels in light-exposed cells.First, an enzymatic assay established that the photocage in 3 interferes with deoxycytidine kinase activity.T he kinase usually phosphorylates the 5' OH of uncaged dAC [43] after the compound is taken up by cells.H owever,t he photocage attached to the 3' OH of 3 prevents the compounds phosphorylation ( Figure S7), most likely owing to sterically

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Communications hindering access of 3 to the enzymesa ctive site ( Figure S1). In addition, western blot analysis confirmed that uncaged 3 lowers methylation by decreasing levels of the DNAm ethyltransferase 1( Figure S8). Thea mount of DNMT1 was reduced when cells were exposed to 0.1 mm 3 and light to liberate dAC. Thei nhibitorsm ode of action is thought to involve its incorporation into DNAtoform acovalent adduct with DNMT1, [43] which prevents methylation of DNAi n replicating cells but also targets DNMT for proteosomal degradation. [44] This report has pioneered al ight-gated small-molecule approach to regulate DNAm ethylation levels within cells. Thereby,our study breaks new ground in two areas.F irst, the photocaging of the DNAm ethyl transferase inhibitor achieves optically triggered DNAd emethylation. Previously, there has not been any chemical tool available for lightinduced lowering of cellular methylation levels.Using genetically encoded epigenetic editing has previously yielded sitespecific DNAd emethylation [17] and methylation [23] through TALE-TET1 and Cas9-DNMT fusion proteins,r espectively. Light-mediated regulation of site-specific DNAm ethylation was attained with optogenetic protein pairs fused to DNMT and alocus-targeting protein, [47] similar to optically triggered demethylation with TET1. [48] Photoactivation of am utant dehydrogenase led to ad ecrease in 5-hydroxymethylcytosine. [49] Thebiological tools to target DNAmethylation have been reviewed elsewhere. [22] In awider context, the non-DNA epigenetic mark of histone methylation was modulated by optically controlled histone methyltransferases and histone deacetylases, [50] and by aphotoswitchable inhibitor of adeacetylase. [51] Second, our study is the first to prepare photocaged dAC thereby providing rich chemical insight on an epigenetically important drug molecule as well as expanding the repertoire of caged nucleosides. [27,31,52,53] By generating atotal of six dAC and ribonucleotide versions,w eh ave uncovered information on efficient synthesis and on how the photocagesattachment site influences photolysis yield. Among the photocages tested, DEACM was found to be the best in terms of high wavelength absorption and photolytic efficiency, while carbonate or carbamate-tethered nitrobenzyls 1b-1d were not suitable, similar to previously tested ether-based linkages.Inp ractical terms,t his insight could improve the future synthesis of photocaged versions of clinically tested dAC-related drugs such as SGI-110. [54] Finally,d AC and related drugs could be modified with photoswitches that regulate bioactivity through photo-isomerable conformation changes rather than photolysis. [26][27][28][29] Theo ptically addressable DNMT inhibitor may be developed into ap otentially valuable research tool for studying epigenetic mechanisms in health and disease. Areas of interest include regenerative medicine, [55] developmental biology, [4] development and progression of cancer, [56] and the development of therapeutic routes [18,38,[57][58][59] to treat surface-accessible tissues. [60] Before realizing the potential, the photocaged nucleosidesbioavailability has to be successfully tested and its stability may have to be improved, for example,b yr eplacing the carbonate tether with self-immolating linkages. [61][62][63] In the case of thicker tissues or organs, high-wavelength photocages active in the optical window need to be devised. In conclusion, our photocaged DNMT inhibitor opens up exciting new avenues in basic and clinical research for epigenetics and also the synthesis of photocontrolled molecules.