Homocysteine-targeting compounds as a new treatment strategy for diabetic wounds via inhibition of the histone methyltransferase SET7/9

In hypoxia and hyperglycemia, SET7/9 plays an important role in controlling HIF-1α methylation and regulating the transcription of HIF-1α target genes, which are responsible for angiogenesis and wound healing. Here, we report the Ir(III) complex Set7_1a bearing acetonitrile (ACN) ligands as a SET7/9 methyltransferase inhibitor and HIF-1α stabilizer. Interestingly, Set7_1a could engage SET7/9 and strongly inhibit SET7/9 activity, especially after preincubation with homocysteine (Hcy), which is elevated in diabetes. We hypothesize that Set7_1a exchanges ACN subunits for Hcy to disrupt the interaction between SET7/9 and SAM/SAH, which are structurally related to Hcy. Inhibition of SET7/9 methyltransferase activity by Set7_1a led to reduced HIF-1α methylation at the lysine 32 residue, causing increased HIF-1α level and recruitment of HIF-1α target genes that promote angiogenesis, such as VEGF, GLUT1, and EPO, in hypoxia and hyperglycemia. Significantly, Set7_1a improved wound healing in a type 2 diabetic mouse model by activating HIF-1α signaling and downstream proangiogenic factors. To our knowledge, this is the first Hcy-targeting iridium compound shown to be a SET7/9 antagonist that can accelerate diabetic wound healing. More importantly, this study opens a therapeutic avenue for the treatment of diabetic wounds by the inhibition of SET7/9 lysine methyltransferase activity.


Materials and cell lines
Dulbecco's Modified Eagle's Medium (DMEM) and Fetal bovine serum (FBS) were available from Gibco BRL. Human embryonic kidney HEK293 cells (CRL-1573) and Human Umbilical Vein Endothelial Cells (HUVEC) (CRL-1730) were obtained from ATCC and maintained in Dulbecco's modified Eagle's medium (DMEM, Gibco, USA) containing 10% fetal bovine serum (FBS) at 37 °C and in the presence of 5% CO2. Luciferase reporter assay system was purchased from Promega (Madison, WI, USA). β-actin antibody (4967L) was purchased from Cell Signaling Technology. EPO antibody (ab129452) was purchased from Abcam. All other reagents and chemicals were obtained from commercial sources and used as received. All animal experiments were approved by the Animal Ethical and Welfare Committee of University of Macau (No. ICMS-AEC-2014-06). All procedures involved in the animal experiments were carried out in accordance with the approved guidelines and regulations.

Mass spectrometry analysis
Hcy was incubated with complex Set7_1a in Tris-HCl buffer pH = 7.5,10 mM KCl) 30 min at room temperature, then was directly injected into an MSQ Plus Single Quad Detector (Thermo Scientific MSQ Plus single quadrupole mass spectrometer, Thermo Scientific, USA) at a flow rate of 5 μL min −1 . Data were analyzed by the software Data Analysis.

Amino acids selectivity assay
The selectivity of complex Set7_1a (10 μM) was tested over possible biologically relevant common amino acids (100 μM), including Cys, Hcy, Glu, Arg, Asp, Tyr, Val, Trp, Pro, Ala, Met, Lys, Gly, His, Phe, Leu, Asn, Ile, Ser, Thr and Gln. A stock solution of complex Set7_1a (0.5 mM) was prepared in dimethyl sulfoxide (DMSO) for further use. The complex was diluted in Tris-HCl buffer pH = 7.5,10 mM KCl) to a final concentration of 20 μM for 1.5 mL. After that, various amino acids were added into each eppendorf tube with a final concentration of 100 μM. The emission spectra were recorded in a cuvette at 25 °C on a PTI QM-1 spectrofluorometer (Photo Technology International, Birmingham, NJ). The intensity of the emission peak of complex Set7_1a in the presence or absence of various amino acids at 597 nm were recorded to obtain the bar chart.

Cell viability assay
The cytotoxicity of the compounds towards normal human cells will be measured by an MTT assay. Hyperglycemia induced human umbilical vein endothelial cells HUVEC will be seeded at a density of 5,000 cells per well in 96-well plates and incubated for 12 h under hypoxia.
Compounds dissolved in DMSO will be added to cells followed by incubation for 48 h. Then 10 μL of 5 mg/mL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reagent will be added to each well. After 4 h incubation in the dark, 100 μL of DMSO will be added to each well, and the intensity of absorbance will be determined by a SpectraMax M5 microplate reader at a wavelength of 570 nm.

Western blotting
The transfected cells from BiFC assay will be harvested to obtain whole-cell extracts by the addition of one volume of 250 mM Tris-HCl (pH 6.8), 20% glycerol, 2% sodium dodecyl sulfate (SDS), 5% 2-mercaptoethanol, and 0.2% bromophenol blue to cells in one volume of PBS followed by boiling for 5 min. Samples will resolved on sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS/PAGE) gels and transferred to polyvinylidene fluoride (PVDF) membranes. Blots will be probed with antibodies to SET7/9, HIF-1α, GAPDH (cell signaling). After incubation with secondary antibodies (Santa Cruz Biotechnology), blots will be developed with ECL reagent (Thermo Fisher Scientific).

Cellular thermal shift assay
Cellular thermal shift assay was performed to monitor the target engagement of complex Set7_1a in hyperglycemia-induced HUVEC cell lysates. Briefly, 2 × 10 6 hyperglycemiainduced HUVEC cells cultured under hypoxia will be lysed and collected. 300 mg of cell lysates will be diluted and divided into aliquots (200 μL) using PBS. Each lysate will be treated with the most promising compound for 30 min at room temperature and will be then divided into 6 aliquots of 50 µL and placed into separate PCR tubes. The PCR tubes will be heated individually at different temperatures ranging from 25 °C to 60 °C for 5 min (Applied Biosystems 7500, Life Technologies). The heated lysates will be then centrifuged for 5 min at 13 000 g and the supernatants will be subjected to SDS-PAGE followed by immunoblotting with SET7, HIF-1α protein antibodies (CST, 1: 1000 dilution).

SET7/9 protein expression and purification
The purification of recombinant proteins His-SET7/9 was carried out as described previously 8 .

Fluorescence-based protein thermal shift assay
The protein thermal shift assay with the purified protein was performed as described previously 9 .
Briefly, purified human recombinant SET7/9 protein was appropriately diluted in ddH2O. All assay experiments used 2 μg protein per well and final concentration 10 × Sypro Orange and 10 μM of complex Set7_1a up to a total volume of 20 μL. The PCR plates were sealed with optical seal, shaken, and centrifuged after protein and compounds were added. Thermal scanning (35 to 62 °C at 1 °C/min) was performed using a real-time PCR setup (Mx3005P Q-PCR system) and fluorescence intensity was measured after every 1 min.

Isothermal titration calorimetry
ITC experiments were carried in a MicroCal PEAQ-ITC Isothermal Titration Calorimeter (Malvern Panalytical), as previously described with minor modification 10 . Briefly, the complex Set7_1a and recombinant SET7/9 protein were dialyzed into the ITC buffer (20 mM Bis-Tris, 150 mM NaCl, 2 mM DTT, 5% DMSO) overnight. The SET7/9 protein (500 μM) was titrated against 50 μM of complex Set7_1a, consisted of 19 injections of 2 μL complex Set7_1a solution at a rate of 2 sec/μL at 150 s time intervals. An initial injection of ligand (0.4 μL) was made and discarded during data analysis. The experiment was carried out at 25 °C while stirring at 750 rpm. The generated data was analyzed using the Setup MicroCal PEAQ-ITC Analysis Software provided by the manufacturer.

Biolayer interferometry
The binding affinities of inhibitors to recombinant SET7/9 were measured by biolayer interferometry on an OctetRed 96 (Fortebio). Ni-NTA biosensors were loaded with His-tagged SET7/9 in BLI kinetics buffer (PBS buffer containing 0.02% Tween 20 and 0.1% BSA), washed in the same buffer and transferred to wells containing complex Set7_1a or HIF-1α peptide at indicated concentrations in the same buffer. The Ni-NTA biosensor tips coated with His-tagged protein complex were dipped in increasing concentrations of complex Set7_1a for 300 s and subsequently dissociated in the wells containing buffer for another 300 s. Negative control performed with BLI kinetics buffer against Ni-NTA biosensors was subtracted from the sample response against SET7/9-loaded Ni-NTA biosensors. The equilibrium dissociation constant (Kd) value for a 1:1 interaction was calculated from the steady state fit. The Kd and associated standard errors were calculated using Octet analysis software.

Dual luciferase reporter assay
To evaluate the effect of the compounds on HIF-1α-directed transcription, HRE activity will be determined using a dual luciferase assay. Briefly, HEK-293 cells will be transiently transfected with pRL-TK and the HIF-1α HRE-luciferase reporter plasmids for 36 h. Hyperglycemiainduced cells will be treated with compounds (10 μM) under hypoxia for 8 h before measurement. Luciferase activity will be measured using a spectrophotometer (Spectra-max M5, Molecular Devices, USA) and will be integrated over a 10 second period. The results will be standardization with the activity of Renilla luciferase.

SET7/9 knockdown assay
Hyperglycemia-induced HUVEC cells were seeded in plates at about 80% confluence in DMEM for 12 h. LipofectamineTM 3000 reagent and siRNAs were gently mixed in FBS-free DMEM medium. After 15 min incubation at room temperature, 500 μL of siRNA-lipid complex were directly added to cells in 1.5 mL DMEM culture medium. Then, HUVEC cells were incubated at 37 o C in a CO2 incubator for 48 h before use.

Dot blot assay for HIF-1α methylation detection
The samples were incubated at 30 o C for 60 min in a reaction buffer containing 50 mM Tris-HCl (pH 8.5), 5 mM MgCl2, 4 mM DTT, active SET7/9 protein and 1 µM SAM. HIF-1α K32 peptide or K32R peptide (1 µg) were used as substrates. The total volume of the reaction mixture was adjusted to 10 μL. The samples were subjected to dot blot and conducted to detect methylation with an anti-methyllysine antibody.

ELISA assay for VEGF detection
The levels of the VEGF released into the cell culture medium will be determined using a commercial ELISA kit. Briefly, 50 µL of cell culture medium samples, assay buffer and diluted detect antibody will be pipetted into the VEGF antibody coated microplate 96 wells. The plate will be covered with an adhesive strip and incubated at room temperature for 2 h. After washing with wash buffer six times, 100 µL of diluted streptavidin-HRP solution will be added in each well followed by incubation for 45 min. After washing six times, 100 µL of substrate solution will be added to the wells followed by incubation for 5-30 min. Color development will be stopped by adding 100 µL of stop solution and the intensity of the color will be measured by a spectrophotometer (Spectra-max M5, Molecular Devices, USA). The endogenous VEGF levels may also be detected via immunoblotting with anti-VEGF antibody.

Measurement of proteasome activity
The commercial Proteasome Activity Fluorometric Assay Kit (BioVision Incorporated, CA, USA) was used to detect the proteasome activity after complex Set7_1a treatment.

Measurement of oxygen consumption
The effect of complex Set7_1a towards oxygen consumption was determined using commercial oxygen consumption rate assay kit (Cayman Chemical, MI, USA).

HIF-1α methylation assay
HUVEC cells grown for 48 h in normal (5.5 mM) or high glucose concentrations (30 mM) will be incubated for an additional 24 h in hypoxia (1% O2) in the presence of the most promising compound or DMSO. Proteins will be extracted and analyzed by Western blotting. An antimethyl HIF-1α antibody will be applied to examine whether HIF-1α methylation occurs after treatment with the active compounds in hyperglycemia-induced cells.

Zebrafish experiments
Transgenic Tg(fli-1:EGFP) zebrafish will be kept separately with a 14 h light/10 h dark cycle under standard conditions. Zebrafish embryos will be generated by natural pair-wise mating (3-12 months old) and will be raised at 28.5 °C in embryo water. 24 hpf zebrafish embryos will be collected, distributed into a 12-well microplate with 6 fish in each well and co-treated with 300 nM VRI (VEGFR tyrosine kinase inhibitor II) and the most promising compounds for 24 h.
Embryos receiving embryo water with 0.1% DMSO served as a vehicle control and will be equivalent to no treatment. All of these experiments will be repeated three times, with 8 embryos per group.

Histological analysis and microvessel density assay
After fixation in 4% paraformaldehyde, the skin samples were embedded in paraffin and sectioned (5 μm). For histological evaluation, sections were deparaffinized and rehydrated, followed by hematoxylin and eosin (H&E) or Masson's trichrome staining. For immunohistochemical staining of CD31, the wound tissue sections were deparaffinized and stained with CD31 antibody (ABclonal, Cambridge, MA, USA). The slides were examined under ×400 magnification to identify the area with the highest vascular density, and five randomly high-power field areas of the highest microvessel density were selected for each section. The average was calculated as the microvessel density of this sample.

Skin wound model
Dorsum was clipped in anesthetized mice, and then two full-thickness wounds were made with a 6-mm biopsy punch on each side of midline. A circular reference was placed alongside to permit correction for the distance between the camera and the animals. Wound areas were measured every other day by taking digital photographs and quantitated using ImageJ (National Institutes of Health). The wound closure rates were calculated as the following formulation: (wound area on day 0 -wound area on day X)/wound area on day 0 × 100%.  Fig. 1. Chemical structure of sinefungin (Sine).

Quantitative RT-PCR
Supplementary Fig. 2. Dose-dependent effect of positive control sinefungin with SET7/9 in vitro was estimated using an FP assay. Sinefungin displays a SAM site-binding probe in SET7/9 with an IC50 of about 6.17 μM. Error bars represent the standard deviations of the results obtained from three independent experiments. Supplementary Fig. 3. Bacterially expressed recombinant human wild type SET7/9 protein (Red arrow) was resolved on 10% SDS-PAGE and visualized by G250 staining. BSA: Bovine serum albumin.