VinSpinIn

VinSpinIn A chemical probe for the Spin family proteins

overview

Figure 1: Chemical structure of VinSpinIn and the inactive VinSpinIC.

The Spindlin proteins are tudor domain containing proteins. There are five spindlin family members (Spin1, Spin2A, Spin2B, Spin3, Spin4), which are expressed at various levels throughout the body. However, spindlin1 (Spin1) is expressed at higher levels compared to the other family members.1 Our current knowledge of the biological roles of spindlins is limited to that of Spin1.

The Spin proteins consist of three tudor-like Spin/Ssty domains, arranged in a clockwise orientation.2-4 Spin1 has been shown to bind to trimethylated lysine 4 of histone 3 (H3K4me3) via domain 2 (ITC Kd = 147 nM).3,5 However, increased binding is observed when a second epigenetic methylation mark is present on arginine 8 (asymmetrically dimethylated) (H3K4me3R8me2a; ITC Kd = 45 nM).2 Spin1 also binds to H4K20me3 via domain 2.6

Spin1 binding to methylated histones is associated with transcriptional activation. Spin1 was found to be overexpressed in various cancers and has been shown to drive cancer cell proliferation through activation of the Wnt/β-catenin, PI3K/Akt and RET signalling pathways.2,7-10

Conversely, Spin1 has been shown to facilitate the inactivation of p53 by sequestering the ribosomal protein uL18.11

Direct or indirect Spin1 knockdown resulted in cancer cell and xenograft tumor growth inhibition, and such studies suggest that small molecule inhibition of Spin1 may be a viable approach for the treatment of certain cancers.8-13

Therefore, the SGC has developed VinSpinIn as a potent, cell active chemical probe for the Spin family proteins. VinSpinIn, along with the structurally very similar inactive control compound VinSpinIC, will contribute significantly to the elucidation of the biological roles and functions of the spindlin proteins, and will aid with the validation of Spin1 as a chemotherapeutic target.

Potency Against Target Family
The SYPRO Orange thermal shift assay was employed to assess the potency of VinSpinIn and VinSpinIC against four of the five Spin family members (Table 1). VinSpinIn induced a large shift in the thermal stability in all of the Spin protein assessed, while VinSpinIC did not. ITC was also performed to determine the potency of VinSpinIn on four Spin family members as well as an additional Spin1 construct (Table 1). VinSpinIn had KDs ranging between approximately 10-130 nM across the family.

Spin Family Proteins

Thermal Shift Assay

(ΔTm°C)

ITC Assay

(KD nM)

VinSpinInVinSpinICVinSpinIn
Spin149-262Not testedNot tested9.9
SPIN126-26213.171.02111.1
SPIN2B22-25810.470.2946.1
SPIN321-25814.122.34131.1
SPIN436-2496.530.2518.1

Table 1: Potency Against Target Family

Selectivity
VinSpinIn and VinSpinIC were screened against a panel of methyl binding domains (MBDs) using the thermal shift assay (Table 2). No significant thermal shift was observed for any of the MBDs screened.
A Scintillation Proximity Assay (SPA) was employed to screen VinSpinIn and VinSpinIC against a panel of methyltransferases and IC50s were determine for selected targets (Table 3). The lowest IC50 of VinSpinIn (PRMT4) was approximately 300 times greater than the AlphaScreen IC50 on Spin1 (30 nM).

Methyl Lysine Binders

VinSpinIn

(ΔTm°C)

VinSpinIC

(ΔTm°C)

UHRF1139-298-0.04-0.14
53BP11483-1606-0.65-0.41
TDRD3525-6110.15-0.95
SND1650-910-0.09-1.62
SETDB1197-403-0.04-0.34
SGF29129-293-0.05-0.92
CCDC101114-293-0.040.3
CHD1269-446-0.652.37
FALZ2736-27930.150.31
ING2211-266-0.090.25
JARID1A1542-1660-0.04-0.04
MLL1558-1773-0.050.63
MLL5113-171-0.05-0.05
PHF21-640.020.19
PHF81-630.23-0.11
TAF31086-1153-0.21-0.04

Table 2: Screening on a panel of MBDs.

Methyltransferase Panel

VinSpinIn

(IC50 µM)

VinSpinIC

(IC50 µM)

PRMT492
SETD2205
PRMT7198
SUV39H13418
PRMT62719
PRC22127
SMYD22244
PRDM92544
PRMT14759
PRMT84566

Table 3: IC50s on methyltransferases.

Dosage
Use between 0.5 and 3 µM for Cellular Assays and 1 µM for screening at a single shot, for both VinSpinIn & VinSpinIC.

In vitro Activity
In a number of biophysical assays, VinSpinIn was shown to be a potent Spin1 inhibitor and has an ITC KD which is approximately 130 times more potent than that of the inactive control VinSpinIC (Table 4).

Compound

AlphaScreen

(Spin126-262)

(IC50)

Octet BLI

(Spin126-262)

(KD)

ITC

(Spin126-262)

(KD)

ITC

(Spin149-262)

(KD)

Tm Shift

(Spin126-262)

(ΔTm°C)

VinSpinIn30 nM55 nM111.1 nM9.9 nM13.2
VinSpinIC3.64 µMnot testednot tested1.3 µM1.0

Table 4: In vitro biophysical binding assay results of VinSpinIn & VinSpinIC.

Cellular Activity
In a NanoBRET cellular target engagement assay VinSpinIn displayed dose dependant inhibition of the Spin1-H3 interaction; the inactive VinSpinIC showed no inhibition (Figure 2).


Figure 2: NanoBRET cellular engagement assay. Click on the ‘Cell-based Assay Data’ tab above for more details.

properties

Click here to download the SDF file.

VinSpinIn
Physical and chemical properties
Molecular weight738.96 g/mol
Molecular formulaC42H58N8O4
IUPAC name2-[4-(2-{[2-(3-{[2-amino-5-(cyclopropylmethoxy)-3,3-dimethyl-3H-indol-6-yl]oxy}propyl)-2,3-dihydro-1H-isoindol-5-yl]oxy}ethyl)-1H-1,2,3-triazol-1-yl]-1-{4-[2-(pyrrolidin-1-yl)ethyl]piperidin-1-yl}ethan-1-one
logP3.85
TPSA123.57
No. of chiral centres0
No. of rotatable bonds17
No. of hydrogen bond acceptors2
No. of hydrogen bond donors12
Storage+4 °C
DissolutionDMSO (up to at least 50 mM)

Click here to download the SDF file.

VinSpinIC
Physical and chemical properties
Molecular weight738.96 g/mol
Molecular formulaC42H58N8O4
IUPAC name2-[4-({[2-(4-{[2-amino-5-(cyclopropylmethoxy)-3,3-dimethyl-3H-indol-6-yl]oxy}butyl)-2,3-dihydro-1H-isoindol-5-yl]oxy}methyl)-1H-1,2,3-triazol-1-yl]-1-{4-[2-(pyrrolidin-1-yl)ethyl]piperidin-1-yl}ethan-1-one
clogP3.99
TPSA123.57
No. of chiral centres0
No. of rotatable bonds17
No. of hydrogen bond acceptors2
No. of hydrogen bond donors12
Storage+4 °C
DissolutionDMSO (up to at least 50 mM)

SMILES:
VinSpinIn: CC1(C)C(N)=Nc2cc(OCCCN3Cc4ccc(OCCc5cn(CC(=O)N6CCC(CCN7CCCC7)CC6)nn5)cc4C3)c(OCC3CC3)cc21
VinSpinIC:  CC1(C)C(N)=Nc2cc(OCCCCN3Cc4ccc(OCc5cn(CC(=O)N6CCC(CCN7CCCC7)CC6)nn5)cc4C3)c(OCC3CC3)cc21

InChI:
VinSpinIn: InChI=1/C42H58N8O4/c1-42(2)36-23-38(54-29-31-6-7-31)39(24-37(36)44-41(42)43)53-20-5-16-48-25-32-8-9-35(22-33(32)26-48)52-21-13-34-27-50(46-45-34)28-40(51)49-18-11-30(12-19-49)10-17-47-14-3-4-15-47/h8-9,22-24,27,30-31H,3-7,10-21,25-26,28-29H2,1-2H3,(H2,43,44)/f/h43H2
VinSpinIC: InChI=1/C42H58N8O4/c1-42(2)36-22-38(54-28-31-7-8-31)39(23-37(36)44-41(42)43)52-20-6-5-16-48-24-32-9-10-35(21-33(32)25-48)53-29-34-26-50(46-45-34)27-40(51)49-18-12-30(13-19-49)11-17-47-14-3-4-15-47/h9-10,21-23,26,30-31H,3-8,11-20,24-25,27-29H2,1-2H3,(H2,43,44)/f/h43H2

InChIKey:
VinSpinIn: XPEJZXWPKDAYFX-YDFFNMJFNA-N
VinSpinIC: FOBGCBSESHUBEI-YDFFNMJFNA-N

Supplemental Material

Experimental_VinSpinIn, VinSpinIn Spectra

selectivity profile

The SYPRO Orange thermal shift assay was employed to screen VinSpinIn and VinSpinIC against a panel of methyl binding domains (MBDs), which included four of the five Spin family members (Table 1). VinSpinIn induced a large shift in thermal stability of Spin1, Spin2B, Spin3, Spin4. No other significant shift in thermal stability was observed for VinSpinIn or the inactive VinSpinIC.

Methyl Lysine Binders

VinSpinIn

(ΔTm°C)

VinSpinIC

(ΔTm°C)

a UHRF1139-298-0.04-0.14
a 53BP11483-1606-0.65-0.41
a TDRD3525-6110.15-0.95
a SND1650-910-0.09-1.62
a SETDB1197-403-0.04-0.34
a SGF29129-293-0.05-0.92
b CCDC101114-293-0.040.3
b CHD1269-446-0.652.37
b FALZ2736-27930.150.31
b ING2211-266-0.090.25
b JARID1A1542-1660-0.04-0.04
b MLL1558-1773-0.050.63
b MLL5113-171-0.05-0.05
b PHF21-640.020.19
b PHF81-630.23-0.11
b TAF31086-1153-0.21-0.04
b SPIN126-26213.171.02
b SPIN2B22-25810.470.29
b,c SPIN321-25814.122.34
b SPIN436-2496.530.25

Table 1: Screening on a panel of MBDs.a protein = 0.05 to 0.2 mg/ml, compound = 200 µM; b protein = 2 µM, compound = 20 µM; c Intrinsic tryptophan fluorescence used

A fluorescence polarization displacement assays was employed to screen the compounds against L3MBTL1 and L3MBTL3 (MBT methyl lysine readers), which gave respective Kdisp.s of 27 and 8 µM for VinSpinIn, and 28 and 14 µM for the inactive VinSpinIC. Therefore, VinSpinIn is approximately 267 times less potent towards L3MBTL1 than Spin1.

Selectivity screening was also performed against a panel of methyl transferases domains (MTDs) including protein, DNA and RNA methyltransferases using a Scintillation Proximity Assay (SPA) (Figure 1).

Figure 1: (Top) Methyltransferase activity in presence of VinSpinIn at 10 & 50 µM
(Bottom) Methyltransferase activity in presence of VinSpinIC at 10 and 50 µM.

For selected targets IC50s were determined (Table 2). The lowest IC50 of VinSpinIn (PRMT4) was approximately 300 times greater than the AlphaScreen IC50 on Spin1.

Methyltransferase Panel

VinSpinIn

(IC50 µM)

VinSpinIC

(IC50 µM)

PRMT492
SETD2205
PRMT7198
SUV39H13418
PRMT62719
PRC22127
SMYD22244
PRDM92544
PRMT14759
PRMT84566
DNMT3A/3La9
DNMT3B/3La5
G9aa9
GLPa21
SETDB1a6

Table 2: IC50s of selected methyltransferases.a Not determined

Materials and Methods

Effects of VinSpinIn  and VinSpinIC on methyltransferase activity of G9a, GLP, SUV39H1, SUV39H2, SETDB1, SETD8, SUV420H1, SUV420H2, SETD7, MLL1 trimeric complex, MLL3 pentameric complex, EZH2 trimeric complex, PRMT1, PRMT3, PRMT4, PRMT5-MEP50 complex, PRMT6, PRMT7, PRMT8, PRMT9, PRDM9, SETD2, SMYD2, SMYD3, and DNMT1 was assessed by monitoring the incorporation of tritium-labeled methyl group to lysine or arginine residues of peptide substrates using Scintillation Proximity Assay (SPA) as previously described14.  Assays were performed in a 10 µl reaction mixture containing 3H-SAM (Cat.# NET155V250UC; Perkin Elmer; www.perkinelmer.com) at substrate concentrations close to Km values for each enzyme.  Two concentrations (10µM and 50 µM) of VinSpinIn or  VinSpinIC  were used in all selectivity assays.  To stop the enzymatic reactions, 10 µl of 7.5 M guanidine hydrochloride was added, followed by 180 µl of buffer (20 mM Tris, pH 8.0), mixed and then transferred to a FlashPlate (Cat.# SMP103; Perkin Elmer; www.perkinelmer.com). After mixing, the reaction mixtures in Flash plates were incubated for 2 hours and the CPM were measured using Topcount plate reader (Perkin Elmer, www.perkinelmer.com). The CPM counts in the absence of compound for each data set were defined as 100% activity. In the absence of the enzyme, the CPM counts in each data set were defined as background (0%).

For DOT1L, NSD1, NSD2, NSD3, ASH1L, DNMT3A/3L, and DNMT3B/3L, a filter-based assay was used. In this assay, 10 µl of reaction mixtures were incubated at 23 oC for 1 hour, 50 µl of 10% trichloroacetic acid (TCA) was added, mixed and transferred to filter-plates (Millipore; cat.# MSFBN6B10; www.millipore.com). Plates were centrifuged at 2000 rpm (Allegra X-15R - Beckman Coulter, Inc.) for 2 min followed by 2 additional 10% TCA wash and one ethanol wash followed by centrifugation. Plates were dried and 30 µl MicroO (MicroScint-O; Cat.# 6013611, Perkin Elmer; www.perkinelmer.com) was added to each well, centrifuged and removed. 50 µl of MicroO was added again and CPM was measured using Topcount plate reader.

IC50 Determinations:
IC50 values were determined for inhibition of methyltransferase activity of the following enzymes:

  • PRMT4 (20 nM PRMT4, 1 µM of biotinlated-H3 1-25, 2 µM of 3H-SAM)
  • SETD2 (150 nM SETD2, 0.5 µM of biotinylated-H3 21-44, 5 µM of 3H-SAM)
  • PRMT7 (25 nM PRMT7, 0.3 µM of biotinylated-H2B(23-37), 1 µM of 3H-SAM)
  • SUV39H1 (20 nM SUV30H1, 0.2 µM of biotinlated-H3 1-25, 5 µM of 3H-SAM)
  • PRMT6 (20 nM PRMT6, 1 µM of biotinlated-H4 1-24, 2 µM of 3H-SAM)
  • PRC2 (20 nM PRC2, 1 µM of biotinlated-H3 21-44, 2 µM of 3H-SAM)
  • SMYD2 (30 nM SMYD2, 3 µM of biotinlated-P53peptide, 0.3 µM of 3H-SAM)
  • PRDM9 (5 nM PRDM9, 4 µM of biotinlated-H3 1-25, 71 µM of SAM)
  • PRMT1 (15 nM PRMT1, 0.13 µM of biotinlated-H4 1-24, 5 µM of 3H-SAM)
  • PRMT8 (20 nM PRMT8, 1 µM of biotinlated-H4 1-24, 2 µM of 3H-SAM)
  • DNMT3A/3L (20 nM DNMT3A/3L, 0.5 µM of poly DI-DC, 1 µM of 3H-SAM)
  • DNMT3B/3L (50 nM DNMT3B/3L, 0.5 µM of poly DI-DC, 1 µM of 3H-SAM)
  • G9a (5 nM G9A, 0.8 µM of biotinlated-H3 1-25, 10 µM of SAM)
  • GLP (5 nM Glp, 0.8 µM of biotinlated-H3 1-25, 10 µM of SAM).

To stop the enzymatic reactions, 7.5 M Guanidine hydrochloride was added, followed by 180 µL of buffer (20 mM Tris, pH 8.0), mixed and then transferred to a FlashPlate (Cat.# SMP103; Perkin Elmer; www.perkinelmer.com). After mixing, the reaction mixtures in Flash plate were incubated for 2 hour and the CPM counts were measured using Topcount plate reader (Perkin Elmer, www.perkinelmer.com). The CPM counts in the absence of compound for each data set were defined as 100% activity. In the absence of the enzyme, the CPM counts in each data set were defined as background (0%). The IC50 values were calculated using GraphPad Prism 7 software.

in vitro potency
cell based assay data

In a NanoBRET cellular target engagement assay VinSpinIn displayed dose dependant inhibition of the Spin1-H3 interaction, with an IC50 of 270 nM. The inactive VinSpinIC showed no inhibition (Figure 1).

Figure 1: NanoBRET cellular target engagement assay performed in U2OS cells. Full length SPIN1 with N-terminal NanoLuc; Histone 3.3 with C-terminal Halotag; 24h incubation with VinSpinIn and VinSpinIC

Materials and Methods

U20S cell (2.8 x 105) were plated in each well of a 6-well plate after 6h cells were co-transfected with C-terminal HaloTag-Histone 3.3 (NM_002107) and an N-terminal NanoLuciferase fusion of full length SPIN1 at a 1:500 (NanoLuc® to HaloTag®) ratio respectively with FuGENE HD transfection regent15.

Sixteen hours post-transfection, cells were collected, washed with PBS, and exchanged into media containing phenol red-free DMEM and 4% FBS in the absence (control sample) or the presence (experimental sample) of 100 nM NanoBRET 618 fluorescent ligand (Promega). Cells were then re-plated in a 384-well assay white plate (Greiner #3570) at 2.7x103 cells per well.  VinSpinIn and VinSpinIC were then added directly to media at final concentrations 0-30μM or an equivalent amount of DMSO as a vehicle control, and the plates were incubated for 24 h at 37oC in the presence of 5% CO2.

NanoBRET Nano-Glo substrate (Promega) was added to both control and experimental samples at a final concentration of 10 µM.  Readings were performed within 10 minutes using a ClarioSTAR (BMG labtech) equipped with 460 nm and 610 nm filters.  A corrected BRET ratio was calculated and is defined as the ratio of the emission at 610 nm/460 nm for experimental samples minus the emission at 610 nm/460 nm for control samples (without NanoBRET fluorescent ligand). BRET ratios are expressed as milliBRET units (mBU), where 1 mBU corresponds to the corrected BRET ratio multiplied by 1000.

references
  1. https://www.proteinatlas.org/search/Spindlin.
  2. Su, X. et al. Molecular basis underlying histone H3 lysine-arginine methylation pattern readout by Spin/Ssty repeats of Spindlin1. Genes Dev 28, 622-636, doi: 10.1101/gad.233239.113 (2014).
  3. Yang, N. et al. Distinct mode of methylated lysine-4 of histone H3 recognition by tandem tudor-like domains of Spindlin1. Proc Natl Acad Sci U S A 109, 17954-17959, doi: 10.1073/pnas.1208517109 (2012).
  4. Zhao, Q. et al. Structure of human spindlin1. Tandem tudor-like domains for cell cycle regulation. J Biol Chem 282, 647-656, doi: 10.1074/jbc.M604029200 (2007).
  5. Wang, W. et al. Nucleolar protein Spindlin1 recognizes H3K4 methylation and stimulates the expression of rRNA genes. EMBO Rep 12, 1160-1166, doi: 10.1038/embor.2011.184 (2011).
  6. Shanle, E. K. et al. Histone peptide microarray screen of chromo and Tudor domains defines new histone lysine methylation interactions. Epigenetics Chromatin 10, 12, doi: 10.1186/s13072-017-0117-5 (2017).
  7. Wang, J. X. et al. SPINDLIN1 Promotes Cancer Cell Proliferation through Activation of WNT/TCF-4 Signaling. Mol Cancer Res 10, 326-335, doi: 10.1158/1541-7786.Mcr-11-0440 (2012).
  8. Franz, H. et al. The histone code reader SPIN1 controls RET signaling in liposarcoma. Oncotarget 6, 4773-4789, doi: 10.18632/oncotarget.3000 (2015).
  9. Chen, X. et al. Suppression of SPIN1-mediated PI3K-Akt pathway by miR-489 increases chemosensitivity in breast cancer. J Pathol 239, 459-472, doi: 10.1002/path.4743 (2016).
  10. Li, Y., Ma, X., Wang, Y. & Li, G. miR-489 inhibits proliferation, cell cycle progression and induces apoptosis of glioma cells via targeting SPIN1-mediated PI3K/AKT pathway. Biomed Pharmacother 93, 435-443, doi: 10.1016/j.biopha.2017.06.058 (2017).
  11. Fang, Z. et al. SPIN1 promotes tumorigenesis by blocking the uL18 (universal large ribosomal subunit protein 18)-MDM2-p53 pathway in human cancer. Elife 7, doi: 10.7554/eLife.31275 (2018).
  12. Chen, X. et al. Long noncoding RNA MHENCR promotes melanoma progression via regulating miR-425/489-mediated PI3K-Akt pathway. Am J Transl Res 9, 90-102 doi: 10.1042/BSR20170682 (2017).
  13. Drago-Ferrante, R. et al. Suppressive role exerted by microRNA-29b-1-5p in triple negative breast cancer through SPIN1 regulation. Oncotarget 8, 28939-28958, doi: 10.18632/oncotarget.15960 (2017).
  14. Barsyte-Lovejoy, D. et al. (R)-PFI-2 is a potent and selective inhibitor of SETD7 methyltransferase activity in cells, PNAS 111, 12853-12858. doi: 10.1073/pnas.1407358111 (2014).
  15. Machleidt, T. et al. NanoBRET--A Novel BRET Platform for the Analysis of Protein-Protein Interactions. ACS Chem Biol 10, 1797-1804, doi: 10.1021/acschembio.5b00143 (2015).
pk properties
co-crystal structures

The co-crystal structure of Spin1 with VinSpinIn confirms that VinSpinIn binds to both domain 1 and 2 of Spin1, and therefore is referred to as a bidentate inhibitor (Figure 1).

Figure 1: Co-crystal structure of Spin1 (domain 1 = purple; domain 2 = green; domain 3 = yellow) with VinSpinIn (Cyan).

synthetic schemes
materials and methods