UNC0638: Selective chemical probe for G9a/GLP methyltransferases

To cite this probe: Vedadi et al, Nat Chem Biol. 7:566-74 (2011).

This probe is available from:
Calbiochem, Cayman, Sigma and Tocris
UNC0638 released on June 1, 2010


Click on the 'Properties' tab above for more details

Biology of the G9a/GLP methyltransferases

G9a (EHMT2) and GLP (EHMT1) catalyze the mono and dimethylation of lysine 9 of histone 3 (H3K9) and other non-histone substrates such as p53 and WIZ.

Cellular Activity

Significant reduction in H3K9 dimethylation at 100nM in MDA-MB231 cells as measured by fluorescence immunostaining without significant cellular toxicity.

Click on the 'Cell-based Assay Data' tab above for more details

Collaborators & Acknowledgements

The work at SGC is supported by Ontario Research Fund Grant RE-03-003, and the work at The University of North Carolina CICBDD is supported by NIH grant Number RC1GM090732.

  1. Mark Minden (OCI)
  2. Rob Bristow (OCI)
  3. Jay Bradner (Dana Farber/Broad Inst)
  4. Alexander Tarakhovsky(Rockefeller)
  5. Martin Hirst (SFU)
  1. Rod Bremner (TWRI)
  2. James Ellis, (Sick Kids, Toronto)
  3. Ben Garcia (Princeton)
  4. David Margolis (UNC)

Selectivity Within Target Family

Protein IC50/nM (Activity) Tm shift °C 1
G9a (EHMT2) <15 (hill slope 1.3) 4
GLP (EHMT1) 19±1 (hill hlope 0.8) 8
SETD7 >10,000 nt
SETD8 >10,000 nd
PRMT3 >10,000 nd
SUV39H2 >10,000 nt
DOT1L nt nd
PRDM1 nt nd
PRDM10 nt nd
PRDM12 nt nd
SMYD3 nt nd
JMJD2E 4660 (AlphaScreen) nt
HTATIP nt nd

(nt=not tested, nd=not detected, 1 singlicate determination @ 100 µM)

Selectivity Beyond Target Family

>30% Inhib @ 1 µM

Receptor %Inhib
Adrenergic alpha1A 90
Adrenergic alpha1B 69
Muscarinic M2 64
Click on the 'Selectivity Profile' tab above for more details

Properties of Probe UNC0638

UNC0638 released on June 1, 2010
Mol/SD file: UNC0638.sdf

2-cyclohexyl-N-(1-isopropylpiperidin-4-yl)-6-methoxy-7-(3-(pyrrolidin-1-yl)propoxy) quinazolin-4-amine

InChI: 1S/C30H47N5O2/c1-22(2)35-17-12-24(13-18-35)31-30-25-20-27(36-3)28(37-19-9-16-34-14-7-8-15-34) 21-26(25)32-29(33-30)23-10-5-4-6-11-23/h20-24H,4-19H2,1-3H3,(H,31,32,33)
Physical and Chemical Properties
Molecular Weight 509.7
Formula C30H47N5O2
cLogP 5.78
cLogS -4.64
Polar Surface Area 62.0 A2
No. of chiral centres 0
No. of rotatable bonds 10
No. of hydrogen bond acceptors 6
No. of hydrogen bond donors 1
Storage Stable as solid in the dark at -20°C.
Dissolution Soluble in DMSO at least up to 10mM.

Selectivity Profile for Probe UNC0638

UNC0638 released on June 1, 2010

Selectivity Within Target Family

Protein IC50/nM (Activity) Tm shift °C 1
G9a (EHMT2) <15 4
GLP (EHMT1) 19 ± 1 8
SETD7 >10,000 nt
SETD8 >10,000 nd
PRMT3 >10,000 nd
SUV39H2 >10,000 nt
DOT1L nt nd
PRDM1 nt nd
PRDM10 nt nd
PRDM12 nt nd
SMYD3 nt nd
JMJD2E 4660 (AlphaScreen) nt
HTATIP nt nd

(nt=not tested, nd=not detected, 1 singlicate determination @ 100 µM)

Target IC50 / nM (Activity)
DNMT1 1287**
MLL >10,000**
EZH2 >10,000**
PRMT1 >10,000**
SUV39H2 >10,000**
G9a 91**

**Screened at BPS Bioscience using different format

Tm shift °C
Protein Screening Methods UNC0638 µM
1 10 100500
EHMT1 DSF 5 8 6
G9a DSF 2 4 4

Blank box indicates Tm shift of <2 °C

Selectivity Beyond Target Family

Radioligand binding performed at Ricerca

>30% Inhib @ 1 µM

Receptor %Inhib
Adrenergic alpha1A 90
Adrenergic alpha1B 69
Muscarinic M2 64

<30% Inhib @ 1 µM

Transporter, Norepinephrine (NET) Glutamate, NMDA, Phencyclidine
Nicotinic Acetylcholine Alpha1, Bungarotoxin Adenosine A2A
Dopamine D2S Calcium Channel L-Type, Dihydropyridine
GABAA, Flunitrazepam, CentralGABAA, Muscimol, Central
Sodium Channel, Site 2 Histamine H1
Potassium Channel hERGAdenosine A1
Cannabinoid CB1 Rolipram
Dopamine D1Potassium Channel[KATP]
Nicotinic AcetylcholineAdrenergic beta1
Adrenergic beta2 Prostanoid EP4
Muscarinic M3 Serotonin (5-Hydroxytryptamine) 5-HT2B
Opiate mu (OP3, MOP) Phorbol Ester
Adrenergic alpha2A Sigma1
Peptide Displacement Measured by FP Peptide Displacement Measured by FP

Materials and Methods

Activity Assay

Histone methyltransferase assay was performed using a coupled assay originally developed by Collazo et al. 2005. In this assay SAHH (S-adenosylhomocysteine hydrolase) and adenosine deaminase convert the methyltransferase reaction product (S-adenosylhomocysteine) to homocysteine and inosine. Homocysteine can be quantified using Thioglo-1 (Calbiochem). Substrate peptides used in this assay were: the first 25 residues of histone 3 [H3 (1-25)] for G9a, EHMT1 and SETD7 at 10, 20 and 100 µM respectively; the first 24 residues of histone 4 [H4 (1-24)] at 10 and 500 µM for PRMT3 and SETD8 respectively; and H3K9Me1 [H3 (1-15), monomethylated at lysine 9] at 200 µM for SUV39H2. The assay mixtures were prepared in 25 mM potassium phosphate buffer pH 7.5, 1 mM EDTA, 2 mM MgCl2, 0.01% Triton X-100 with 5 µM SAHH , 0.3 U/ml of adenosine deaminase from Sigma, 25 µM SAM, and 15 µM Thioglo-1. G9a (25 nM), EHMT1 (100 nM), SUV39H2 (100 nM), SETD7 (200 nM) and PRMT3 (1 µM) were assayed in the presence of UNC0638 at concentrations ranging from 4 nM to 16 µM. After 2 min incubation, reactions were initiated by the addition of above mentioned histone peptides. The methylation reactions were followed by monitoring the increase in fluorescence using BioTek Synergy2 plate reader with 360/40 nm excitation filter and 528/20 nm emission filter for 20 min in 384-well format. SETD8 (250 nM) was assayed under the same conditions; however the Thioglo-1 was added at the end of the reaction for quantification. The peptide and protein background were subtracted. IC50 values were calculated using Sigmaplot and the standard deviations were calculated from two independent experiments. SAHH clone was provided by Dr. Trievel, University of Michigan.

Differential Scanning Fluorimetry (DSF)

DOT1L, PRDM1, PRDM10, PRDM12, SMYD3, HIATIP, G9a, EHMT1 and SETD7 were screened for binding to UNC0638 by DSF. A real-time PCR device (RTPCR 480 II) from Roche was used to monitor protein unfolding by monitoring the increase in the fluorescence of the fluorophor SYPRO Orange (Invitrogen, Carlsbad, CA) as described before (Niesen et al. 2007; Vedadi et al. 2006). Protein samples ranging from 0.05 to 0.2 mg/mL in 100 mM Hepes buffer (pH 7.5) containing 150 mM NaCl, and 0, 1, 10 and 100 µM of the compound were screened. Compound dilutions were made from stock solutions of 100% DMSO. The final concentration of DMSO was kept at 0.2% throughout the dilutions. All these solutions contained 5x Sypro Orange. 20 µL aliquots were transferred to a 384-well PCR plate and scanned at a heating rate of 1 °C/min from 20 to 95 °C. Fluorescence intensities were plotted as a function of temperature by using an internally developed software package (Vedadi et al. 2006).

Differential Static Light Scattering (DSLS)

SETD8 and PRMT3 were screened for binding to UNC0638 by DSLS. Temperature-dependent aggregation was measured by using static light scattering (StarGazer) (Vedadi et al. 2006, Senisterra et al. 2006). Fifty microliters of protein (0.4 mg/ml) was heated from 27°C to 85°C at a rate of 1°C per min in each well of a clear-bottom 384-well plate (Nunc, Rochester, NY) in the presence of 0, 1, 10 and 100 µM of UNC0638. Incident light was shone on the protein drop from beneath at an angle of 30°. Protein aggregation was monitored by measuring the intensity of the scattered light every 30 s with a CCD camera. The pixel intensities in a preselected region of each well were integrated to generate a value representative of the total amount of scattered light in that region. These total intensities were then plotted against temperature for each sample well and fitted to the Boltzman equation by nonlinear regression. The resulting point of inflection of each resulting curve was defined as the Tagg.

Peptide Displacement

Fluorescence polarization (FP) measurements were performed in 384 well-plates, using Synergy 4 microplate reader from BioTek. H3 (1-15) peptide (ARTKQTARKSTGGKA) was synthesized, N-terminally labeled with fluorescein [F-H3 (1-15)] and purified by Tufts University Core Services (Boston, MA, USA). Displacement of F-H3 (1-15) peptide was monitored using the fluorescence polarization signal obtained upon peptide binding to G9a protein. G9a (4 µM in 20 mM Tris pH 8.0, 250 mM NaCl, 500 µM SAH, and 0.01% Triton) was incubated with 40 nM F-H3 (1-15) peptide, and different concentrations of BIX-01294 (purchased from Sigma-Aldrich), UNC0224, UNC0638 and unlabeled H3 (1-25) peptide from 0.1 to 100 µM were added. Displacement of peptide was monitored by following the decrease in FP signal. Data were normalized and plotted as percentage, and fit to a hyperbolic function using Sigma Plot software.

Cell-based Assay Data for UNC0638

UNC0638 released on June 1, 2010

Cellular Activity
(BIX-01294 reported by Kubicek et al, 2007 Molecular Cell 25, 473-481)

H3K9Me2 immunostaining and cell viability measured in MDA-MB231 cells after 48h exposure to BIX-01294 or UNC0638. UNC0638 exhibits a dose-dependant reduction in H3K9 dimethylation, while being more potent and efficacious than BIX-01294.

Cellular Toxicity

UNC0638 clearly shows an improved toxicity profile both in absolute terms and relative to its cell-based activity. This property will enable the use of UNC0638 without concern about possible complications of cellular toxicity.

In vitro G9a IC50(nM) H3K9Me2 48h IC50(nM) Tox 48h EC50(nM) Tox/Func Ratio
BIX-01294 133 ± 15 500 ± 43 2805 5.6
UNC0638 <15 81 ± 9 11190 138

Poor separation of functional and toxic effects

Good separation of functional and toxic effects

Materials and Methods

MDA-MB231 cells were cultured in RPMI with 10% FBS and MCF7 cells cultured in DMEM with 10% FBS.

MTT Toxicity Assay

Cells were grown in the presence or absence of inhibitors for stated amount of time. The media was removed and replaced with DMEM 10% FBS without phenol red supplemented with 1mg/ml of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) and incubated for 1-2h. Live cells reduce yellow MTT to purple formazan. The resulting formazan was solubilized in acidified isopropanol and 1% Triton and absorbance measured at 570nm, corrected for 650nm background.

In-Cell Western (ICW)

Cells were grown in 96-well plates in the presence of inhibitors as stated in figures. Media was removed by flicking and 2% formaldehyde in PBS added for 15min. After five washes with 0.1% Triton X100 in PBS, cells were blocked for 1h with 1% BSA in PBS. Three out of four replicates were exposed to primary H3K9m2 antibody, Abcam #1220 at 1/800 dilution in 1% BSA, PBS for 2h. One replicate was reserved for background control. The wells were washed five times with 0.1% Tween 20 in PBS, then secondary IR800 conjugated antibody (LiCor) and DNA-intercalating dye, DRAQ5 (LiCor) added for 1h. After 5 washes with 0.1% Tween 20 in PBS, the plates were read on Odyssey (LiCor) scanner at 800nm (H3K9m2 signal; 764nm excitation) and 700nm (DRAQ5 signal; 683nm excitation). Fluorescence intensity was quantified, normalized to background and DRAQ5 signal expressed as percentage of control.

Gene knockdown

shRNAs were obtained from The RNAi Consortium (TRC) (Dr J Moffat) and processed and used as outlined in the TRC protocols.
G9a shRNA: NM_025256.4-3163s1c1; EHMT1 shRNA NM_024757.3-346s1c1; Control promegaLuc_221s1c1

References and Relevant Links

UNC0638 released on June 1, 2010


  1. Vedadi, M.; Barsyte-Lovejoy, D.; Liu, F.; Rival-Gervier, S.; Allali-Hassani, A.; Labrie, V.; Wigle, T. J.; DiMaggio, P. A.; Wasney, G. A.; Siarheyeva, A.; Dong, A.; Tempel, W.; Wang, S. C.; Chen, X.; Chau, I.; Mangano, T. J.; Huang, X.; Simpson, C. D.; Pattenden, S. G.; Norris, J. N.; Kireev, D. B.; Tripathy, A.; Edwards, A.; Roth, B. L.; Janzen, W. P.; Garcia, B. A.; Petronis, A.; Ellis, J.; Brown, P. J.; Frye, S. V.; Arrowsmith C. H.*; & Jin, J.*. A chemical probe selectively inhibits G9a and GLP methyltransferase activity in cells. Nature Chemical Biology 2011, 7, 566-574.
  2. Bernstein, B. E.; Meissner, A.; Lander, E. S. The mammalian epigenome. Cell 2007, 128, 669-681.
  3. Gelato, K. A.; Fischle, W. Role of histone modifications in defining chromatin structure and function. Biol. Chem. 2008, 389, 353-363.
  4. Strahl, B. D.; Allis, C. D. The language of covalent histone modifications. Nature 2000, 403, 41-45.
  5. Jenuwein, T.; Allis, C. D. Translating the histone code. Science 2001, 293, 1074-1080.
  6. Esteller, M. Epigenetics in cancer. N. Engl. J. Med. 2008, 358, 1148-1159.
  7. Lyko, F.; Brown, R. DNA methyltransferase inhibitors and the development of epigenetic cancer therapies. J. Natl. Cancer Inst. 2005, 97, 1498-1506.
  8. Martin, C.; Zhang, Y. The diverse functions of histone lysine methylation. Nat. Rev. Mol. Cell. Biol. 2005, 6, 838-849.
  9. Rea, S.; Eisenhaber, F.; O'Carroll, D.; Strahl, B. D.; Sun, Z. W.; Schmid, M.; Opravil, S.; Mechtler, K.; Ponting, C. P.; Allis, C. D.; Jenuwein, T. Regulation of chromatin structure by site-specific histone H3 methyltransferases. Nature 2000, 406, 593-599.
  10. Kouzarides, T. Chromatin modifications and their function. Cell 2007, 128, 693-705.
  11. Copeland, R. A.; Solomon, M. E.; Richon, V. M. Protein methyltransferases as a target class for drug discovery. Nat. Rev. Drug Discov. 2009, 8, 724-732.
  12. Spannhoff, A.; Sippl, W.; Jung, M. Cancer treatment of the future: inhibitors of histone methyltransferases. Int. J. Biochem. Cell Biol. 2009, 41, 4-11.
  13. Fog, C. K.; Jensen, K. T.; Lund, A. H. Chromatin-modifying proteins in cancer. APMIS 2007, 115, 1060-1089.
  14. Spannhoff, A.; Hauser, A. T.; Heinke, R.; Sippl, W.; Jung, M. The emerging therapeutic potential of histone methyltransferase and demethylase inhibitors. ChemMedChem 2009, 4, 1568-1582.
  15. Li, Y.; Reddy, M. A.; Miao, F.; Shanmugam, N.; Yee, J.-K.; Hawkins, D.; Ren, B.; Natarajan, R. Role of the Histone H3 Lysine 4 Methyltransferase, SET7/9, in the Regulation of NF-{kappa}B-dependent Inflammatory Genes: RELEVANCE TO DIABETES AND INFLAMMATION. J. Biol. Chem. 2008, 283, 26771-26781.
  16. Maze, I.; Covington, H. E., 3rd; Dietz, D. M.; LaPlant, Q.; Renthal, W.; Russo, S. J.; Mechanic, M.; Mouzon, E.; Neve, R. L.; Haggarty, S. J.; Ren, Y.; Sampath, S. C.; Hurd, Y. L.; Greengard, P.; Tarakhovsky, A.; Schaefer, A.; Nestler, E. J. Essential role of the histone methyltransferase G9a in cocaine-induced plasticity. Science 2010, 327, 213-216.
  17. Schaefer, A.; Sampath, S. C.; Intrator, A.; Min, A.; Gertler, T. S.; Surmeier, D. J.; Tarakhovsky, A.; Greengard, P. Control of Cognition and Adaptive Behavior by the GLP/G9a Epigenetic Suppressor Complex. Neuron 2009, 64, 678-691.
  18. Imai, K.; Togami, H.; Okamoto, T. Involvement of histone H3 Lysine 9 (H3K9) methyl transferase G9a in the maintenance of HIV-1 latency and its reactivation by BIX01294. J. Biol. Chem. In press, doi/10.1074/jbc.M110.103531.
  19. Tachibana, M.; Sugimoto, K.; Nozaki, M.; Ueda, J.; Ohta, T.; Ohki, M.; Fukuda, M.; Takeda, N.; Niida, H.; Kato, H.; Shinkai, Y. G9a histone methyltransferase plays a dominant role in euchromatic histone H3 lysine 9 methylation and is essential for early embryogenesis. Genes Dev. 2002, 16, 1779-1791.
  20. McGarvey, K. M.; Fahrner, J. A.; Greene, E.; Martens, J.; Jenuwein, T.; Baylin, S. B. Silenced tumor suppressor genes reactivated by DNA demethylation do not return to a fully euchromatic chromatin state. Cancer Res. 2006, 66, 3541-3549.
  21. Kondo, Y.; Shen, L.; Ahmed, S.; Boumber, Y.; Sekido, Y.; Haddad, B. R.; Issa, J. P. Downregulation of histone H3 lysine 9 methyltransferase G9a induces centrosome disruption and chromosome instability in cancer cells. PLoS ONE 2008, 3, e2037.
  22. Huang, J.; Dorsey, J.; Chuikov, S.; Zhang, X.; Jenuwein, T.; Reinberg, D.; Berger, S. L. G9A and GLP methylate lysine 373 in the tumor suppressor p53. J. Biol. Chem. In press, doi/10.1074/jbc.M109.062588.
  23. Kubicek, S.; O'Sullivan, R. J.; August, E. M.; Hickey, E. R.; Zhang, Q.; Teodoro, M. L.; Rea, S.; Mechtler, K.; Kowalski, J. A.; Homon, C. A.; Kelly, T. A.; Jenuwein, T. Reversal of H3K9me2 by a small-molecule inhibitor for the G9a histone methyltransferase. Mol. Cell. 2007, 25, 473-481.
  24. Shi, Y.; Do, J. T.; Desponts, C.; Hahm, H. S.; Scholer, H. R.; Ding, S. A combined chemical and genetic approach for the generation of induced pluripotent stem cells. Cell Stem Cell 2008, 2, 525-528.
  25. Shi, Y.; Desponts, C.; Do, J. T.; Hahm, H. S.; Scholer, H. R.; Ding, S. Induction of pluripotent stem cells from mouse embryonic fibroblasts by Oct4 and Klf4 with small-molecule compounds. Cell Stem Cell 2008, 3, 568-574.
  26. Greiner, D.; Bonaldi, T.; Eskeland, R.; Roemer, E.; Imhof, A. Identification of a specific inhibitor of the histone methyltransferase SU(VAR)3-9. Nat. Chem. Biol. 2005, 1, 143-145.
  27. Cole, P. A. Chemical probes for histone-modifying enzymes. Nat. Chem. Biol. 2008, 4, 590-597.
  28. Frye, S. V.; Heightman, T.; Jin, J. Targeting Methyl Lysine. Annual Reports in Medicinal Chemistry 2010, in press.
  29. Frye, S. V. The art of the chemical probe. Nat. Chem. Biol. 2010, 6, 159-161.
  30. Chang, Y.; Zhang, X.; Horton, J. R.; Upadhyay, A. K.; Spannhoff, A.; Liu, J.; Snyder, J. P.; Bedford, M. T.; Cheng, X. Structural basis for G9a-like protein lysine methyltransferase inhibition by BIX-01294. Nat. Struct. Mol. Biol. 2009, 16, 312-317.
  31. Quinn, A. M.; Allali-Hassani, A.; Vedadi, M.; Simeonov, A. A chemiluminescence-based method for identification of histone lysine methyltransferase inhibitors. Molecular BioSystems 2010, DOI:10.1039/B921912A.
  32. Liu, F.; Chen, X.; Allali-Hassani, A.; Quinn, A. M.; Wasney, G. A.; Dong, A.; Barsyte, D.; Kozieradzki, I.; Senisterra, G.; Chau, I.; Siarheyeva, A.; Kireev, D. B.; Jadhav, A.; Herold, J. M.; Frye, S. V.; Arrowsmith, C. H.; Brown, P. J.; Simeonov, A.; Vedadi, M.; Jin, J. Discovery of a 2,4-diamino-7-aminoalkoxyquinazoline as a potent and selective inhibitor of histone lysine methyltransferase G9a. J. Med. Chem. 2009, 52, 7950-7953.
  33. Collazo, E.; Couture, J. F.; Bulfer, S.; Trievel, R. C. A coupled fluorescent assay for histone methyltransferases. Anal. Biochem. 2005, 342, 86-92.
  34. Min, J.; Allali-Hassani, A.; Nady, N.; Qi, C.; Ouyang, H.; Liu, Y.; MacKenzie, F.; Vedadi, M.; Arrowsmith, C. H. L3MBTL1 recognition of mono- and dimethylated histones. Nat. Struct. Mol. Biol. 2007, 14, 1229-1230.
  35. Wigle, T. J.; Provencher, L. M.; Norris, J. L.; Jin, J.; Brown, P. J.; Frye, S. V.; Janzen, W. P. Accessing Protein Methyltransferase and Demethylase Enzymology Using Microfluidic Capillary Electrophoresis. Chemistry & Biology In press.
  36. Morrison, J. F. Kinetics of the reversible inhibition of enzyme-catalysed reactions by tight-binding inhibitors. Biochim. Biophys. Acta. 1969, 185, 269-286.
  37. Williams, J. W.; Morrison, J. F. The kinetics of reversible tight-binding inhibition. Methods Enzymol. 1979, 63, 437-467.
  38. Sakurai, M.; Rose, N. R.; Schultz, L.; Quinn, A. M.; Jadhav, A.; Ng, S. S.; Oppermann, U.; Schofield, C. J.; Simeonov, A. A miniaturized screen for inhibitors of Jumonji histone demethylases. Mol. Biosyst. 2010, 6, 357-364.
  39. Niesen, F. H.; Berglund, H.; Vedadi, M. The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nature protocols 2007, 2, 2212-2221.
  40. Vedadi, M.; Niesen, F. H.; Allali-Hassani, A.; Fedorov, O. Y.; Finerty, P. J., Jr.; Wasney, G. A.; Yeung, R.; Arrowsmith, C.; Ball, L. J.; Berglund, H.; Hui, R.; Marsden, B. D.; Nordlund, P.; Sundstrom, M.; Weigelt, J.; Edwards, A. M. Chemical screening methods to identify ligands that promote protein stability, protein crystallization, and structure determination. Proceedings of the National Academy of Sciences of the United States of America 2006, 103, 15835-15840.
  41. Allali-Hassani, A.; Wasney, G. A.; Chau, I.; Hong, B. S.; Senisterra, G.; Loppnau, P.; Shi, Z.; Moult, J.; Edwards, A. M.; Arrowsmith, C. H.; Park, H. W.; Schapira, M.; Vedadi, M. A survey of proteins encoded by non-synonymous single nucleotide polymorphisms reveals a significant fraction with altered stability and activity. Biochem. J. 2009, 424, 15-26.