PRMT5-MEP50 complex (IC50 not specified; KD = 6 ± 2 nM by SPR)
G9a, GLP, SUV39H1, SUV39H2, SETDB1, SETD8, SUV420H1, SUV420H2, SETD7, MLL1 trimeric complex, MLL3 pentameric complex, EZH2 trimeric complex, PRMT1, PRMT3, PRMT4, PRMT6, PRMT7, PRMT8, PRMT9, PRDM9, SETD2, SMYD2, SMYD3, DOT1L, NSD1, NSD2, NSD3, ASH1L, DNMT3A/3L, DNMT3B/3L: no inhibition at 1 μM and 10 μM [1]

LLY-284 (compound 1) showed no significant inhibition against a panel of methyltransferases (G9a, GLP, SUV39H1, SUV39H2, SETDB1, SETD8, SUV420H1, SUV420H2, SETD7, MLL1, MLL3, EZH2, PRMT1, PRMT3, PRMT4, PRMT6, PRMT7, PRMT8, PRMT9, PRDM9, SETD2, SMYD2, SMYD3, DOT1L, NSD1, NSD2, NSD3, ASH1L, DNMT3A/3L, DNMT3B/3L) at 1 μM and 10 μM concentrations, with activity remaining at 100% relative to control. [1]
In PRMT5 cellular assay using MCF7 cells treated with LLY-284 for 48 hours, the levels of symmetrically dimethylated SmBB' (Rme2s) were reduced as measured by Western blot. [1]
In A375 melanoma cells, LLY-284 was tested in an MDM4 exon5/6 PCR assay (splicing assay) to determine EC50 for fold change in exon skipping, but no specific EC50 value was provided in the text; the assay method was described. [1]
In a 7-day proliferation assay using various cancer cell lines (not specified which), LLY-284 was tested to yield IC50 values, but no numerical IC50 was provided in the text. [1]

Scintillation Proximity Assay (SPA) for methyltransferase selectivity: The effect of LLY-284 on methyltransferase activity of 24 enzymes (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) was assessed by monitoring incorporation of tritium-labeled methyl group to peptide substrates. Reactions were performed in 10 μl volume containing 3H-SAM (specific activity 12-18 Ci/mmol) at substrate concentrations near apparent Km. Two concentrations (1 μM and 10 μM) of compound were tested. Reactions were stopped with 10 μl of 7.5 M guanidine hydrochloride, then 180 μl of buffer (20 mM Tris-HCl, pH 8.0) was added, transferred to a FlashPlate, incubated for 2 hours, and CPM measured using a TopCount NXT. CPM without compound = 100% activity; without enzyme = background (0%). [1]
Filter-based assay for DOT1L, NSD1, NSD2, NSD3, ASH1L, DNMT3A/3L, DNMT3B/3L: 10 μl reaction mixtures were incubated at 23°C for 1 hour, then 50 μl of 10% trichloroacetic acid (TCA) was added, mixed, transferred to filter-plates, centrifuged at 2000 rpm for 2 min, washed twice with 10% TCA and once with ethanol, dried, added 30 μl MicroScint-O, centrifuged, removed, added 50 μl MicroScint-O, and CPM measured using a Topcount plate reader. [1]
PRMT5-MEP50 SPA assay for mechanism of action: Reaction mixture contained 20 mM Tris-HCl (pH 8.0), 0.01% Tween-20, 5 mM TCEP, 15 nM PRMT5-MEP50, 0.07 μM biotinylated H4(1-24) peptide (SGRGKGKGKLGKGGAKRHRKVLDRD-Biotin), and 0.6 μM 3H-SAM. To assess competition with SAM, IC50 values of LLY-284 were determined at saturating peptide (2 μM) and varying SAM concentrations (0.5, 1, 4, 8, 12, 16, 20, and 25× apparent Km). To assess competition with peptide, SAM concentration was kept at saturation (3.5 μM) and IC50 values were determined at varying peptide concentrations (0.5, 1, 4, 8, 12, 16, 20, and 25× apparent Km). No dramatic change in IC50 was observed, indicating non-competitive inhibition with respect to both substrates. [1]
Surface Plasmon Resonance (SPR): SPR studies were performed using a Biacore T200 at 20°C. Approximately 4400 response units (RU) of biotinylated PRMT5 was captured onto a streptavidin-conjugated SA chip. LLY-284 was dissolved in 100% DMSO (20 mM stock), serially diluted 3-fold in DMSO to 8 μM, then diluted 1:20 in HBS-EP (20 mM Hepes pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% Tween-20) giving final 5% DMSO. Kinetic determination used single cycle kinetics with on time 90 sec, off time 300 sec, flow rate 100 μl/min. KD was calculated with 1:1 binding model. Results: KD = 6 ± 2 nM (n=3), kon = 3.9 ± 0.4 × 10^5 M^-1 s^-1, koff = 2.2 ± 0.8 × 10^-3 s^-1. [1]

PRMT5 cellular assay: MCF7 cells were grown in DMEM with 10% FBS, penicillin (100 units/ml) and streptomycin (100 μg/ml). 40% confluent cells were treated with different concentrations of LLY-284 or DMSO control for 48 hours. The levels of SmBB' and symmetrically methylated SmBB' were analyzed by Western blot. [1]
Western blot procedure: Cells were lysed in total lysis buffer (20 mM Tris-HCl pH 8, 150 mM NaCl, 1 mM EDTA, 10 mM MgCl2, 0.5% Triton X-100, 12.5 U/mL benzoase, protease inhibitor cocktail) for 3 min at RT, then SDS added to 1% final. Total lysates were resolved in 4-12% Bis-Tris Protein Gels with MOPS buffer, transferred onto PVDF membrane in Tris-Glycine transfer buffer containing 20% MeOH and 0.05% SDS for 1.5 h at 80 V. Blots blocked in 5% milk in 0.1% Tween-20 PBS for 1 h, incubated with primary antibodies (anti-smBB' 1:1000, anti-Rme2s 1:2000, anti-β-actin) overnight at 4°C, washed, incubated with goat-anti-rabbit IR800 and donkey-anti-mouse IR680 secondary antibodies (1:5000) in Odyssey Blocking Buffer for 1 h at RT, washed, and signal read on Odyssey scanner at 800 nm and 700 nm. [1]
Knockdown in MCF7 cells: MCF7 cells were grown in DMEM with 10% FBS, penicillin (100 units/mL) and streptomycin (100 μg/mL). 40% confluent cells were transfected with 50 nM siRNAs (control, PRMT6, PRMT5, PRMT1, PRMT4, PRMT3, PRMT7) using jetPRIME transfection reagent. Protein levels were analyzed by Western blot 3 days post-transfection. [1]
MDM4 Exon5/6 PCR Assay in A375 tumor cells: A375 cells were cultured to 70-90% confluence, trypsinized, seeded into 96-well plate at 5000/well, incubated overnight, then treated with LLY-284 at serial dilutions (1:3 10-point, final DMSO 0.2%) for 72 hr. Cells were washed with cold PBS, lysed with Lysis Working Reagent (from TAQMAN Gene Expression Cells-to-CT Kit), incubated 5 min at RT, stop solution added, then reverse transcription performed. qPCR for MDM4 exon 5 and exon 6 primers was performed separately. CT data were used to calculate fold change = 2^(-ΔCT) where ΔCT = (Exon6 CT CPD - Exon6 CT DMSO) - (Exon5 CT CPD - Exon5 CT DMSO). EC50 values were calculated using 4-parameter logistic fit in Genedata software (no specific EC50 value provided). [1]
7-day Proliferation Assay: Cancer cell lines were plated at appropriate density in 96-well plates, incubated 18-24 hr. Test compound LLY-284 was serially diluted 1:2 (10-point) in medium from 10 mM DMSO stock, added to cells (0.2% DMSO final). Incubated for 7 days at 37°C/5% CO2. Media was removed, 25 μl Cell Titer-Glo reagent added per well, incubated 10-20 min at RT, luminescence measured. IC50 values were calculated by 4-parameter curve fit (no specific IC50 provided). [1]

A375 xenograft tumor model in nude mice: A375 melanoma cells were grown in DMEM/High glucose with 10% HI FBS, harvested, and injected subcutaneously onto the rear flank of nude mice (5 × 10^6 cells/animal, mixed 1:1 with Matrigel). When tumors reached approximately 200 mm^3 (~18-21 days after implant), animals were randomized into control and test groups. LLY-284 was formulated in 1% HEC/0.25% Tween-80/0.05% Antifoam and administered by oral gavage. Tumor volume was measured twice a week with calipers, and percent inhibition of tumor volume versus vehicle control was reported (no numerical efficacy data provided). [1]
PK study in mouse: LLY-284 was administered orally (PO) at 10 mg/kg. Blood samples were collected for PK analysis. Formulation: not explicitly described for PK, but likely same as above. [1]

ADME/PK properties of LLY-284 in mouse (from summary table): MW = 342; HTSA pH7.4 solubility = 0.677 mg/ml; MDCK % A-B Transport = Moderate; CYP2D6, 2C9, 3A4 % Inhibition = 3, 5, 3 respectively; Human, Dog, Rat, Mouse % Metab = 28, 13, 26, 4.5 respectively; AUC (ng*hr/ml) = 7943; Cmax (ng/ml) = 3646; T1/2 (Hours) = 3.41; Unbound Cmax (nM) = 5754; CL (ml/min/kg) = 12.6; Vd,ss (ml/kg) = 1819; Fraction Unbound (mouse) = 0.54; Bioavailability (%F) = 50; clogP = 0.8; clogD = 0.3. [1]
Additional PK description: Plasma clearance was slow at approximately 0.25× hepatic blood flow (Qh, mouse = 90 ml/min/kg), consistent with metabolic stability. Volume of distribution was less than 5 L/kg, consistent with physicochemical properties. Oral bioavailability was 50%, suggesting absorption and no significant first-pass metabolism. Exposures were similar following IP and PO dosing, suggesting absorption and no gut metabolism. Exposures increased proportionally with dose following IP and PO administration. [1]

ACS Med. Chem. Lett. (2018)DOI:10.1021/acsmedchemlett.8b00014

LLY-284 (compound 1) is a potent PRMT5-MEP50 inhibitor with a non-competitive mechanism of action with respect to both SAM and peptide substrates, as shown by IC50 determination at varying concentrations of each substrate (up to 25× Km) where no dramatic change in IC50 was observed. The absolute configuration was established by single crystal X-ray crystallography of a para-bromobenzoate derivative. The compound has molecular formula C17H18N4O4 and molecular weight 342.13. [1]

C17H18N4O4

342.36

342.132

2226515-75-7

LLY-283;2040291-27-6

136213035

White to off-white solid powder

0.2

4

7

3

25

464

5

C1=CC=C(C=C1)[C@@H]([C@@H]2[C@H]([C@H]([C@@H](O2)N3C=CC4=C(N=CN=C43)N)O)O)O

WWOOWAHTEXIWBO-CNUKPYSBSA-N

InChI=1S/C17H18N4O4/c18-15-10-6-7-21(16(10)20-8-19-15)17-13(24)12(23)14(25-17)11(22)9-4-2-1-3-5-9/h1-8,11-14,17,22-24H,(H2,18,19,20)/t11-,12-,13+,14+,17+/m0/s1

(2R,3R,4S,5R)-2-(4-aminopyrrolo[2,3-d]pyrimidin-7-yl)-5-[(S)-hydroxy(phenyl)methyl]oxolane-3,4-diol

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)