PRMT5

In MTAP deletion HCT116 dividing wild-type cells, MRTX-1719 (10 days) suppresses PRMT5 activity with an IC50 of 8 nM [1]. In HCT116 cells lacking MTAP, the IC50 value of MRTX1719 (10 days) is 12 nM, but in HCT116 cells with wild-type phenotype, it is 890 nM [1].

MRTX1719 (12.5-100 mg/kg/day, face, 21 days) inhibits tumor growth in Lu-99 orthotopic xenografts [2]. Pharmacokinetic analysis [2] Model Route Dose (mg/kg) Cltotal ( mL/min/kg) Vdss (L/kg) t1/2 (h) CD-1 mice. iv 3 83 6.3 1.5 Beagle dog iv 2 14 3.4 4.8 Cynomolgus monkey iv 2 15 2.3 6.1 Model route dose (mg/kg) Cmax (ug/mL) AUCinf (h*ug/mL) F (%) CD-1 tiny mouse. po 30 1.16 4.85 80 Beagle dog po 10 1.40 7.47 59 Cynomolgus monkey po 10 / / 41

PRMT5 biochemical studies were conducted using the FlashPlate system at Reaction Biology Corporation using the PMRT5/MEP50 complex (#HMT-22-148: Human recombinant PRMT5 [GenBank Accession No NM_006109; amino acids 2–637 (end)], with N-terminal Flag-Tag and Human recombinant MEP50 [Gene GenBank Accession No. NM_024102; amino acids 2–342 (end)], with N-terminal His tag, coexpressed in Sf9 cell expression system). The FlashPlate substrate was a Biotin-Histone H4 (1–15) peptide. The methyl donor was S-Adenosyl-L-[methyl-3H]methionine. The reaction buffer was 50 mmol/L Tris-HCl (pH 8.5), 0.002% Tween20, 0.005% bovine serum albumin (BSA), 1 mmol/L TCEP, and 1% dimethyl sulfoxide (DMSO).[1]
The final reaction conditions consisted of 3 nmol/L PRMT5/MEP50, 40 nmol/L Histone H4)-Biotin peptide, and 1 μmol/L SAM. Substrate was prepared in a reaction buffer. Enzyme was delivered into the substrate solution at 40 μL/well. Compounds were delivered in DMSO into the reaction mixture and preincubated for 20 minutes at room temperature. Tritiated SAM was delivered into the reaction mixture to initiate the reaction at 10 μL/well, and the reaction was incubated for 2 hours at room temperature. To stop the reaction, 10 μL/well of 600 μmol/L cold SAM was delivered, and 50 μL/well of stopped reaction was transferred into the FlashPlate and incubated for 1 hour at room temperature. Plates were washed with PBS and counted on a TopCount HTS Microplate Scintillation and Luminescence Counter. A 10-dose IC50 with 3-fold serial dilution starting at 1 μmol/L was run in the presence or absence of 2 μmol/L MTA.[1]

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Biochemical Methyl Transferase Assay[2]
The assay uses purified human PRMT5 enzyme to convert S-adenosyl-l-[methyl-3H]methionine plus histone H4 l-arginine to S-adenosyl-l-homocysteine plus histone H4 [methyl-3H]-l-arginine. The assay was carried out using streptavidin-coated FlashPlates, which contain a scintillant embedded in the plastic of the plate. The histone H4 peptide substrate was conjugated with biotin, which binds to the streptavidin-coated well of the plate, placing the H4 peptide in close proximity to the side of the well and the scintillant. The transfer of the tritiated methyl group from S-adenosyl-l-[methyl-3H]methionine to the bound histone H4 peptide generated a radiolabeled histone H4, which was quantified by measuring radioactivity in a scintillation counter to determine the activity of PRMT5 enzyme in the presence and absence of compound. The assay reactions were also conducted in the presence and absence of MTA to determine whether the compounds exhibit MTA-cooperative activity. Briefly, compounds were solubilized in 100% DMSO at a top concentration of 10 mM. For IC50 determinations, the initial starting concentration for the serial dilutions of each compound was 50 μM. Control samples lacking compound, PRMT5/MEP50 complex, or various reaction components were also prepared and processed in parallel with compound test samples. SAH was used as a positive control for assay validation. To measure PRMT5 inhibitory activity, 3 nM PRMT5/MEP50 complex (Reaction Biology Corporation) was preincubated with test compound in assay buffer containing 40 nM histone H4 peptide (amino acids 1–15)–biotin conjugate for 20 min at room temperature. The enzymatic reaction was initiated by adding 1 μM tritiated SAM (final concentration), and the reaction was allowed to proceed for 20 min. The reaction was stopped, and the amount of bound, tritiated H4 peptide in each sample was determined using a scintillation counter. The IC50 value for each compound was calculated from each 10-point dose–response curve for samples plus and minus MTA using GraphPad Prism software.

HCT116 MTAP-WT and MTAP-del Viability Assay[2]
Viability assays were performed using HCT116 MTAP-WT and HCT116 MTAP-del cell lines. Control samples were analyzed in parallel. On day 0, 250 HCT116 MTAP-WT or HCT116 MTAP-del cells were seeded in 96-well plates in McCoy’s 5A supplemented with 10% fetal bovine serum and pen/strep, and the cells were incubated overnight at 37 °C plus 5% CO2. The following day, cells were treated with DMSO vehicle control or a dose–response of test compound and incubated at 37 °C plus 5% CO2 for 5 days. On day 6, the cells were trypsinized and split 1:20 into new 96-well plates with fresh medium containing the same concentration of test compound and incubated for an additional 5 days at 37 °C plus 5% CO2. On day 11, the viability of the cells was measured using a CTG assay kit in accordance with the manufacturer’s instructions. The IC50 values for each compound after 10 days of treatment were calculated using GraphPad PRISM software.

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HCT116 MTAP-WT and MTAP-del SYM11 In-Cell Western Assay[2]
In-Cell Western assays were performed using HCT116 MTAP-WT and HCT116 MTAP-del cell lines by measuring the PRMT5-dependent symmetric dimethyl arginine (SDMA) signal. Control samples were analyzed in parallel. On day 0, 2000 HCT116 MTAP-WT and HCT116 MTAP-del cells were seeded in 96-well plates in McCoy’s 5A supplemented with 10% fetal bovine serum and pen/strep, and the cells were incubated overnight at 37 °C plus 5% CO2. The following day, cells were treated with DMSO vehicle control or a dose–response of test compounds and incubated at 37 °C plus 5% CO2 for 4 days. After 4 days of treatment, the cells were fixed by adding 50 μL of a 4% paraformaldehyde solution to each well, and the cells were incubated for 20 min at room temperature. The paraformaldehyde solution was then removed, and 150 μL of ice-cold methanol was added and the plate stored at −20 °C for 10 min. After such time, the methanol was removed and 150 μL of Odyssey Blocking Buffer + 0.05% Tween-20 was added, and the plate was incubated at room temperature with shaking for 1 h. To each test well, 50 μL of SYM11 antibody (Millipore 07-413) diluted 1:500 in Odyssey Blocking Buffer + 0.05% Tween-20 was added, and the plate was placed at 4 °C overnight. The primary SYM11 antibody solution was removed by aspiration, and the wells were washed 3 times with phosphate buffered saline containing 0.1% Tween-20 (PBST). A 50 μL aliquot of a goat anti-rabbit IRDye 800CW secondary antibody diluted 1:800 and nuclear stain DRAQ5 diluted 1:10000 in Odyssey Blocking Buffer + DRAQ5 + 0.05% Tween-20 was added, and the plate was stored for 2 h in the dark at room temperature. The secondary antibody solution was removed by aspiration, and the wells were washed 3 times with PBST. The SYM11 signal and the DRAQ5 signal were quantified using a Li-Cor Odyssey machine reading at 800 nM and 700 nM, respectively. The SYM11/DRAQ5 ratio was used to calculate the inhibition of SDMA as percent of DMSO control.

Animal/Disease Models: Lu-99 (MTAP/CDKN2A deleted human lung cancer cell line) xenograft tumor model [2]
Doses: 12.5, 25, 50 and 100 mg/kg/d, 21-day
Route of Administration: Oral tube feeding
Experimental Results: tumor volume reduction, tumor growth inhibition (TGI) of 86% at 50 mg/kg and 88% at 100 mg/kg.

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In Vivo Studies[1]
All mouse studies were approved by the Institutional Animal Care and Use Committee based on guidelines from the NIH. Mice were maintained under pathogen-free conditions, and food and water were provided ad libitum.[1]
Six- to 8-week-old female Hsd:Athymic Nude-Foxn1nu mice were injected subcutaneously with tumor cells in 100 μL of PBS and Matrigel matrix in the right hind flank of each mouse with 5e6 cells (LU99) or 1e6 cells (HCT116 parental and HCT116 MTAP del) 50:50 cells:Matrigel. Mouse health was monitored daily, and caliper measurements began when tumors were palpable. Tumor volume measurements were determined utilizing the formula 0.5 × L × W2, in which L refers to the length and W refers to the width of each tumor. When tumors reached the desired average study start tumor volume (LU99: 179 mm3 or 116 mm3; HCT116 MTAP WT: 140 mm3; HCT116 MTAP del: 185 mm3), mice were randomized into treatment groups. MRTX1719 was formulated in 0.5% methylcellulose (4,000 cps) + 0.2% Tween80 in water once per week and stored at room temperature protected from light. GSK3326595 was formulated in 0.5% methylcellulose (4,000 cps) + 0.2% Tween80 in water once per week and stored at 4°C protected from light. JNJ-64619178 was formulated in 20% HP-β-CD in water once per week and stored at room temperature protected from light. Mice were orally administered vehicle, MRTX1719, GSK3326595, or JNJ-64619178 at the indicated doses and schedules. Mice were monitored daily, with tumor volumes and body weights measured 2 or 3 times per week.[1]

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The PXF 537, PXF 2197, PXF 1118, PXF 541, and PXF 2442 mesothelioma PDX model experiments were conducted at Charles River with similar study designs to the LU99 and HCT116 tumor models. Briefly, NMRI nu/nu mice were inoculated subcutaneously with a 2- to 3-mm3 PDX fragment and randomized for treatment initiation when the mean tumor volumes reached ∼120 to 140 mm3. Mice were treated with vehicle or MRTX1719 (n = 3 per group) at the indicated dose and duration.[1]

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Xenograft Study[2]
The Lu-99 cell line (RCB1900) was obtained from RIKEN BioResource Research Center (Tsukuba, Japan) and grown in RPMI 1640 media containing 10% fetal bovine serum, 10 mM HEPES, 1 mM sodium pyruvate, and 1% antibiotic–antimycotic. Mouse studies were conducted in compliance with all applicable regulations and guidelines of the Institutional Animal Care and Use Committee (IACUC) from the National Institutes of Health (NIH). Mice were maintained under pathogen-free conditions and food and water were provided ad libitum. 6–8-week-old female Hsd:Athymic Nude-Foxn1nu mice (Envigo, San Diego) were injected subcutaneously, with 5.0 × 106 Lu-99 tumor cells in 100 μL of PBS and Matrigel matrix in the right hind flank of each mouse (50:50 cells:Matrigel). Mouse health was monitored daily, and caliper measurements began when tumors were palpable. Tumor volume measurements were determined utilizing the formula 0.5 × L × W2, in which L refers to length and W refers to width of each tumor. When tumors reached an average tumor volume of ∼180 mm, mice were randomized into treatment groups. MRTX1719 was formulated in 0.5% methylcellulose (4000 cps) + 0.2% Tween 80 in water once per week and stored at room temperature protected from light. Mice were orally administered vehicle or MRTX1719 PO at the indicated doses and schedules. Mice were monitored daily, and tumor volumes and body weights were measured 2 or 3 times per week. Percent tumor growth inhibition (% TGI) was calculated using the following formula: (1 – (Final Drug Treated Tumor Volume – Initial Drug Treated Tumor Volume)/(Final Vehicle Treated Tumor Volume – Initial Vehicle Treated Tumor Volume)) × 100.[2]
Whole blood was collected to measure plasma concentrations of MRTX1719 and assess pharmacokinetic properties of MRTX1719 at specific time points postdose. Blood (∼50 μL) was collected via submandibular draw into a heparinized tube and immediately inverted to mix. Heparinized blood was then centrifuged at 4000 rpm for 6 min to separate plasma and cellular fractions. The separated plasma was transferred to a polypropylene 96-well plate and immediately stored at −20 °C until analysis. At the end of study, mice were humanely sacrificed, and tumors were surgically removed and immediately cut in half. One half was transferred to a Lysing Matrix A tube, and the other half was transferred to a screw-top microtube. These tubes were immediately submerged in liquid nitrogen to snap freeze the tissue. Frozen tumor fragments were stored at −80 °C until analysis.

[1]. MRTX1719 is an MTA-cooperative PRMT5 inhibitor that exhibits synthetic lethality in preclinical models and patients with MTAP deleted cancer. Cancer Discov. 2023 Aug 8;CD-23-0669.

[2]. Fragment-Based Discovery of MRTX1719, a Synthetic Lethal Inhibitor of the PRMT5•MTA Complex for the Treatment of MTAP-Deleted Cancers. J Med Chem. 2022 Feb 10;65(3):1749-1766.

[3]. Targeting the genetic and immunological drivers of cancer.

PRMT5-MTA Inhibitor MRTX1719 is an orally bioavailable inhibitor of the protein arginine methyltransferase 5 (PRMT5)-methylthioadenosine (MTA) complex, with potential antineoplastic activity. Upon oral administration, PRMT5-MTA inhibitor MRTX1719 selectively binds to the PRMT5-MTA complex that is elevated in methylthioadenosine phosphoylase (MTAP)-deleted cancer cells, thereby specifically inhibiting the function of PRMT5 solely within MTAP-deleted cancer cells and not in normal, healthy cells. By inhibiting the methyltransferase activity of PRMT5, levels of both monomethylated and dimethylated arginine residues in histones H2A, H3 and H4 are decreased. This modulates the expression of genes involved in several cellular processes, including cellular proliferation. This may increase the expression of antiproliferative genes and/or decrease the expression of genes that promote cell proliferation, which may lead to decreased growth of rapidly proliferating cancer cells. MRTX1719 also causes dysregulated RNA splicing and decreased pRb. Together, this decreases proliferation and increases apoptosis specifically in MTAP-deleted cancer cells. PRMT5, a type II methyltransferase that catalyzes the formation of both omega-N monomethylarginine (MMA) and symmetric dimethylarginine (sDMA) on histones and a variety of other protein substrates involved in signal transduction and cellular transcription, is essential for the viability of cancer and normal cells. It is overexpressed in several neoplasms. Elevated levels are associated with decreased patient survival. MTAP is deleted in certain cancer cells leading to an accumulation of the metabolite MTA; MTA binds to and partially inhibits the activity of PRMT5.

C23H18CLFN6O2

464.87942647934

464.12

C, 59.42; H, 3.90; Cl, 7.63; F, 4.09; N, 18.08; O, 6.88

2630904-45-7

(S)-MRTX-1719;2630904-44-6;MRTX-1719 hydrochloride

156151242

White to light yellow solid powder

2.7

2

7

5

33

846

0

BZKIOORWZAXIBA-UHFFFAOYSA-N

InChI=1S/C23H18ClFN6O2/c1-31-22(20-15(8-26)19(33-12-3-4-12)7-17(24)21(20)25)16(10-28-31)11-2-5-13-14(6-11)18(9-27)29-30-23(13)32/h2,5-7,10,12H,3-4,9,27H2,1H3,(H,30,32)

2-(4-(4-(aminomethyl)-1-oxo-1,2-dihydrophthalazin-6-yl)-1-methyl-1H-pyrazol-5-yl)-4-chloro-6-cyclopropoxy-3-fluorobenzonitrile

MRTX1719; MRTX 1719; MRTX-1719;

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)

DMSO : ~100 mg/mL (~215.11 mM)

Solubility in Formulation 1: ≥ 2 mg/mL (4.30 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2 mg/mL (4.30 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2 mg/mL (4.30 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)