PKMYT1 (IC50 = 14 nM)

In the HCC1569 breast cancer cell line, RP-6306 (500 nM; 24 h duration) treatment produced pan-γH2AX, suggesting that tumor-derived CCNE1 also renders cells vulnerable to DNA damage induction following PKMYT1 transcription [2]. In cells that overexpress CCNE1, CDK1 is suddenly and selectively activated, which encourages early mitosis in cells that start DNA synthesis [2].

In a prenatal xenograft model of CCNE1 (OVCAR3), RP-6306 (15, 50, and 300 ppm; oral; daily; for 21 days) resulted in a significant dose-dependent reduction in OVCAR3 tumor growth [1] .

PKMYT1 enzymatic assay (ADP GLO)[1]
To determine the IC50 of PKMYT1 inhibitor compounds, the ADP-GLO assa was used. First, human recombinant PKMYT1 enzyme was diluted in the Enzyme Assay Buffer (70 mM Hepes, 3 mM MgCl2, 3 mM MnCl2, 50 μg/mL PEG20000, 3 μM Na-Orthovanadate (added fresh), 1.2 mM Dithiothreitol (added fresh) in a 5 μL volume and plated in white 384-well plates. Then, 5 μL of inhibitor or DMSO control was diluted in Enzyme Assay Buffer and added to the plate. The enzyme/compound mix was then incubated at room temperature for 15 minutes. Finally, the enzymatic reaction was started by the addition of 5 μL of ATP (diluted in Enzyme Assay Buffer) so that the final ATP concentration is 10 μM and the final PKMYT1 enzyme concentration was 18.5 nM. The enzymatic reaction was then incubated in a 30C incubator for 1 hour. At the end of the incubation period, 15 μL of ADP-GLO Reagent was added and the plate was incubated at room temperature for 40 minutes. Finally, 30 μL of the Kinase Detection Reagent was added and the plate was incubated at room temperature for 30 minutes after which the luminescence was measured using the Envision Plate reader. The IC50 was then determined for each compound screened in the assay. Reported IC50 in this manuscript are the geometrical mean of at least n=3 replicates.[1]

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PKMYT1/Kinases NanoBret Assay[1]
To determine the affinity of compounds in the NanoBRET target engagement assay, HEK293 T cells were transfected with NanoLuc fusion vector DNA (PKMYT1 NanoLuc Fusion Vector or other kinases of interest and Transfection carrier DNA using the Fugene HD Transfection reagent in Opti-MEM No Phenol Red buffer. After an overnight incubation in a 37 °C/5% CO2 incubator, the transfected HEK293 T cells were trypsinized, counted and resuspended in Opti-MEM No Phenol Red buffer at a concentration of 200000 cells/mL. White 96-well plates were then plated with 85 μL of cells (17000 cells/well) to which 5 μL of the 20X tracer solution diluted in tracer dilution buffer was added. Finally, 10 μL of the 10X compounds diluted in Opti-MEM No Phenol Red buffer was added and the plates were then incubated in a 37 °C/5% CO2 incubator for 2 hours. After this incubation, a 50 μL 3X solution of the Substrate/Inhibitor mix was added to the cells. The plate was then transferred to the Perkin Elmer EnVision Multimode plate reader where the Acceptor emission (610 nm) and the Donor emission (450 nm) are measured. Reported IC50 in this manuscript are the geometrical mean of at least n=3 replicates.

PKMYT1 cell-based activity assay (CDK1 pThr14 AlphaLISA)[1]
To determine compound IC50, FUOV1 cells were plated into a 96-well TC-treated culture plate at 50000 cells/well in a final volume of 100 μL of media. The plates were then allowed to equilibrate in a biological safety cabinet for 30 minutes before being placed in a humidified incubator at 37 °C and 5% CO2 overnight. The next day, 2 μL of PKMYT1 inhibitors or DMSO were diluted in 400 μL of warmed culture media in a 96-well block using a Biomek FX liquid handler. Compounds were mixed in media and then 25 μL was dispensed into each well of the 96-well cell plate. Plates were centrifuged at 300 g for 10 seconds and then placed in the incubator for 2 hours. After the 2-hour incubation with compound, media was removed via aspiration using a multichannel pipette. 30 μL of 1X AlphaLISA lysis buffer (Perkin Elmer) supplemented with protease and phosphatase inhibitors as well as 1 mM PMSF, was added to each well. Plates were rotated at 500g for 20 minutes to facilitate lysis. Plates were then sealed with aluminum foil and frozen at −80 °C for at least 1 hour. Lysates were thawed at 37 °C for 10 minutes, then 10 μL of each lysate was transferred in duplicate to a white 384-well assay plate. Antibody mixture was prepared in 1X AlphaLISA assay buffer containing antibodies (5 nM final concentration for rabbit pThr14-CDK1 and mouse total CDK1. 5 μL of antibody mixture was added to each well of the assay plate. Assay plate was sealed and stored at 4 °C overnight. The next day, AlphaLISA bead mixture was prepared in 1X AlphaLISA assay buffer. Anti-rabbit IgG Acceptor and anti-mouse IgG Donor beads were prepared to a concentration of 80 μg/ml in assay buffer. 5 μL of bead mixture was added to each well of the assay plate (20 μg/ml final concentration for each bead). The plate was protected from light and incubated for 2 hours at room temperature. After a 2 h incubation with beads, the plate was read using the Perkin Elmer EnVision Multimode plate reader with excitation at 680 nm and emission at 615 nm. [1]

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Cell proliferation assays[2]
RPE1-hTERT Cas9 TP53−/−, FT282-hTERT TP53R175H and their respective CCNE1-high isogenic pairs were seeded in 96-well plates (Corning Costar cat. no. 5595) at a density of 150 cells per well for RPE1-hTERT Cas9 TP53−/− CCNE1 (C2) or 100 cells per well for all others. After 24 h, cells were treated using an automated D300e digital dispenser at drug concentrations ranging from 0.15 nM to 3 µM. Medium and drugs were refreshed every 3–4 days and cellular confluency was monitored up to 6 population doublings using an IncuCyte S3 Live-Cell Imager. Per cent confluence relative to a non-treated control was used to evaluate growth inhibition induced by test compounds. Synergy between RP-6306 and hydroxyurea or gemcitabine was analysed using the online SynergyFinder v2.0 tool using the ZIP model (https://synergyfinder.fimm.fi).

Animal/Disease Models: OVCAR3-carrying mice [1]
Doses: 15, 50, and 300 ppm (equivalent to approximately 3, 10, and 60 mg/kg/day)
Route of Administration: Oral; daily; for 21 days
Experimental Results: Caused OVCAR3 tumors There was a statistically significant and dose-dependent reduction in growth.

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OVCAR3 cells were implanted at 5×106 cells per mouse into the right flanks of female SCID-beige mice (5–7 weeks old; Charles River), in 1:1 matrigel: media. When tumors reached the target size of 100–150 mm3, (n=8) mice were randomized to treatment groups and treatment with RP-6306 was initiated. In vivo studies involving cell-derived xenografts were performed at Repare Therapeutics, in a CCAC (Canadian Council on Animal Care)-accredited vivarium with an Institutional Animal Care Committee-approved protocol. RP-6306 was formulated in chow at 15–300 ppm or in 0.5% methylcellulose and orally administered twice daily (BID, 0–8 h) for a maximum of 21 days. Chow treated mice were acclimatized to blank chow prior to drug-formulated chow for 3–5 days. Tumor volume was measured using a digital caliper and calculated using the formula 0.52×L×W2. TGI was defined as the formula: % TGI= ((TVvehicle/last – TVvehicle/day0) - (TVtreated/last – TVtreated/day0)) / (TVvehicle/last – TVvehicle/day0) x100 calculated based on the means of the treatment groups at day 0 and last day of measurement. Change in body weight (BW) was calculated using the formula: % BW change = (BWlast-BWday0/ BWday0) x100. BW change was calculated based on individual body weight changes relative to day 0. Statistical significance relative to vehicle control or other test groups was established by one-way ANOVA followed by Fisher’s LSD test for multiple groups and unpaired t-test for two group comparisons (GraphPad Prism v9.0).[1]

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HCC1569, OVCAR3 and SUM149PT cells were implanted at 5 × 106 cells per mouse into the right flanks of female CB17-SCID, SCID-beige and NOD-SCID mice respectively (5–7 weeks old; Charles River), in 1:1 Matrigel:medium). When tumours reached the target size of 100–150 mm3, mice (n = 8) were randomized to treatment groups according to tumour volume and body weight using the ‘stratified’ method in Studylogv4.4 software and treatment with RP-6306 was initiated.[2]
Fresh primary human tumour tissue was collected and cut into small pieces (approximately 2–3 mm in diameter). These tumour fragments were inoculated subcutaneously into the right flank of female BALB/c nude mice (5–7 weeks old) for tumour development and subsequently passaged by implantation into the cohort of mice enrolled in the efficacy study. Mice were randomized according to growth rate into treatment groups (n = 6) when the mean tumour size reached approximately 150 (100–200) mm3 using the stratified method in Studylogv4.4 software. The procedures involving the care and use of animals in for the PDX model were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of CrownBio prior to execution. During the study, the care and use of animals were conducted in accordance with the regulations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).[2]
RP-6306 was formulated in 0.5% methylcellulose and orally administered twice daily (BID, 0–8 h) for a maximum of 21 days. Gemcitabine was administered once weekly intraperitoneally in saline. Animals were monitored for tumour volume, clinical signs and body weight three times per week. Tumour volume was measured using a digital calliper and calculated using the formula 0.52 × L × W2, where L is length and W is width. Response to treatment was evaluated for tumour growth inhibition (% TGI). Tumour growth inhibition (TGI) was defined as: TGI = ((TVvehicle/last − TVvehicle/day0) − (TVtreated/last − TVtreated/day0))/(TVvehicle/last − TVvehicle/day0) × 100% calculated based on the means of the treatment groups at day 0 and last day of measurement. TV is tumour volume and subscripts indicate treatment group and time of sampling. According to NIACC and IACUC approved animal protocols, mice were euthanized as soon as their tumour volume exceeded 2,000 mm3. Change in body weight (BW) was calculated using the formula: %BW change = (BWlast − BWday0/BWday0) × 100. BW change was calculated based on individual body weight changes relative to day 0. Statistical significance relative to vehicle control or other test groups was established by one-way ANOVA followed by Fisher’s least significant difference test for multiple groups and unpaired t-test for two group comparisons (GraphPad Prism v9.0). Investigators were not blinded during data collection and analysis.[2]

[1]. Discovery of an Orally Bioavailable and Selective PKMYT1 Inhibitor, RP-6306. J Med Chem. 2022 Aug 11;65(15):10251-10284.

[2]. CCNE1 amplification is synthetic lethal with PKMYT1 kinase inhibition. Nature. 2022 Apr;604(7907):749-756.

Lunresertib is an orally bioavailable inhibitor of the human membrane-associated tyrosine- and threonine-specific cdc2-inhibitory kinase (PKMYT1), with potential antineoplastic activity. Upon oral administration, lunresertib targets, binds to and inhibits the activity of PKMYT1. This results in the inhibition of CDK1 phosphorylation, which may promote both premature mitosis and a prolonged mitotic arrest, and lead to the accumulation of unrepaired DNA damage and apoptosis in susceptible tumor cells, such as CCNE1-overexpressing tumor cells. PKMYT1 phosphorylates CDK1 specifically when CDK1 is complexed to cyclins, which blocks progression from G2 into mitosis.

C18H20N4O2

324.3770

324.158625

2719793-90-3

(Rac)-RP-6306;2719749-28-5;(R)-RP-6306;2719793-91-4

156869388

Off-white to gray solid powder

3.1

3

4

2

24

488

0

O([H])C1C([H])=C([H])C(C([H])([H])[H])=C(C=1C([H])([H])[H])N1C(=C(C(N([H])[H])=O)C2C([H])=C(C([H])([H])[H])C(C([H])([H])[H])=NC1=2)N([H])[H]

ARBRHWRTXPWZGN-UHFFFAOYSA-N

InChI=1S/C18H20N4O2/c1-8-5-6-13(23)10(3)15(8)22-16(19)14(17(20)24)12-7-9(2)11(4)21-18(12)22/h5-7,23H,19H2,1-4H3,(H2,20,24)

2-amino-1-(3-hydroxy-2,6-dimethylphenyl)-5,6-dimethylpyrrolo[2,3-b]pyridine-3-carboxamide

(Rac)-RP-6306; lunresertib; 2719793-90-3; (R)-RP-6306; 2719749-28-5; CHEMBL5199076; N95U3A7N57;

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 : ~50 mg/mL (~154.14 mM)

Solubility in Formulation 1: 5 mg/mL (15.41 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 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 2: ≥ 2.5 mg/mL (7.71 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 25.0 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 3: ≥ 2.5 mg/mL (7.71 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 25.0 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.)