CDK12 (IC50 = 158 nM); CDK13 (IC50 = 69 nM); CDK7 (IC50 = 8.5 μM); CDK9 (IC50 = 10.5 μM)

THZ531 potently inhibits CDK12 and CDK13, with IC50s of 158 nM and 69 nM, respectively, according to the results of kinase assays; in contrast, the inhibition of CDK7 and CDK9 is more than 50-fold weaker, with IC50s of 8.5 and 10.5 μM, respectively. With an IC50 of 50 nM, THZ531 treatment causes a significant and permanent reduction in Jurkat cell proliferation. The number of cells exhibiting sub-G1 content increases in a dose- and time-dependent manner after treatment with increasing doses of THZ531, according to FACS cell cycle analysis. Over the course of the experiment, there is no increase in the percentage of apoptotic cells at 50 nM THZ531 compared to the DMSO control. With 30 to 40% of cells exhibiting Annexin V positivity after 72 hours, higher dosages of THZ531 produce a noticeable Annexin V signal. THZ531 treatment is also associated with a marked decrease in elongating Pol II[1].

In addition, combination treatment of THZ531 with Olaparib significantly reduced tumor burden in animal models. [2]
Next, the combination was tested in vivo in immune-deficient NSG mice. The effects of Olaparib (30 mg/kg by oral gavage once daily, five days per week) and/or THZ 531 (10 mg/kg i.p., once daily, five days per week) was tested against KMS28 subcutaneous tumors (Figure 4B). On the third week of treatment, tumors were weighed (Figure 4C) and tumor images have been provided in Supplementary Figure S3. Significant synergy was observed in combination treated group. Slight weight loss occurred in mice treated with THZ531, but all mice were healthy and active, not showing signs of toxicities. [2]

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The combination of these molecules with PARP inhibitors showed striking synergy in cell viability and DNA damage assays in vitro and in multiple models of Ewing sarcoma, including a PDX, in vivo without hematopoietic toxicity. [3]
Because THZ531 is not optimized for in vivo studies, we used the parental compound, THZ1. We first confirmed synergy between the parental molecule and olaparib in vitro (Figure S5A). We then established an A673 xenograft mouse model of Ewing sarcoma and performed a 4-arm study with twice-daily (BID) treatment of vehicle, 10 mg/kg THZ1, 50 mg/kg olaparib, and the combination of olaparib+THZ1 delivered for 40 days. Olaparib and THZ1 as single agents each significantly decreased tumor growth rate and extended the survival of the mice (median survival = 18 days vehicle, 32 days THZ1 and 28 days in the olaparib groups) (Figure 6A, B). A striking decreased tumor growth rate was observed when both drugs were used in combination (Figure 6A), and the combination treatment significantly extended survival compared to the vehicle control or either single agent (Figure 6B). Seventy percent of mice in the combination arm were still alive by day 40 when treatment was ended (Figure 6B). Mice were followed out to day 150 without further treatment. At day 60, 40% (4 out of 10) of the mice in the combination group were still alive, whereas all of the mice in the vehicle and single agent arms had been sacrificed. Furthermore 20% of tumors in the combination arm had regressed to where they were no longer palpable (Figure 6B). In order to test the generalizability of this treatment strategy in Ewing sarcoma, we performed combination in vivo studies in a second xenograft mouse model using TC71 cells as well as a patient-derived xenograft (PDX) mouse model of this disease. In the TC71 xenograft study, both THZ1 and olaparib significantly reduced the tumor growth rate and extended survival as single agents (Figure 6C, D), and the combination of THZ1 and olaparib significantly reduced tumor growth rate compared to either agent used alone (Figure 6C). The median survival of mice in the vehicle treated group was 30.5 days, compared to 42 days in the olaparib group, 38.5 days in the THZ1 group and 66.5 days in the combination group (Figure 6D). Furthermore, we established a PDX mouse model of Ewing sarcoma with a type I EWS/FLI fusion from a tumor resected from the fibula of a 12 year old patient at diagnosis (HSJD-ES-002). In this model, THZ1 as a single agent did not significantly reduce the tumor progression (Figure 6E) but did significantly increase overall survival (Figure 6F). Importantly, the combination of THZ1 and olaparib strikingly reduced tumor progression and increased survival in this PDX (Figure 6E, F): vehicle, 23 days; olaparib, 42 days; THZ1, 44 days; and combination, 56 days. Taken together, this drug combination resulted in a more profound and durable response than treatment with either drug alone.[3]

THZ531 or DMSO are applied to the cells for six hours. Cells are treated, then twice-washed in cold PBS before being lysed in the lysis buffer provided below: 50 mM Hepes pH 7.4, 150 mM NaCl, 1% Nonidet P40 substitute, 5 mM EDTA, 1 mM DTT, and protease/phosphatase cocktails. After clearing, lysates are subjected to an overnight pulldown at 4°C using either bio-THZ1 or bio-TH531. For three hours, lysates are further incubated at room temperature in order to maximize the formation of covalent bonds. After that, lysates are incubated for a further two to three hours at 4°C with streptavidin agarose for pulldown[1].

THZ531 or DMSO at the recommended concentrations are applied to Jurkat cells for 72 hours after they are plated at 20,000 cells per well in fresh media in 96-well plates. Following their 24-hour inoculation with fresh media at the indicated concentrations for 72 hours, HAP1 cells are seeded in 96-well plates at a density of 12,000 cells/well. Evaluation is done on THZ531's anti-proliferative impact. Cells treated with THZ531 or DMSO for 6 hours are used to measure the impact of inhibitor washout on the anti-proliferation of Jurkat cells. The medium containing the inhibitor is then removed, and THZ531 or not is added during a 66-hour incubation. Evaluation is done on THZ531's anti-proliferative impact. There are three biological duplicates used for every proliferation assay. Fitting a non-linear regression curve is used to determine IC50s[1].

All animal studies were in accordance with protocols approved by Institutional Animal Care and Use Committee (IACUC) at National University of Singapore. NSG mice were purchased from InVivos, Singapore. The mice were kept at Animal Research Facilities at National University of Singapore in a sterile condition at 20–26 °C temperature, 50% humidity, and in a light-controlled environment (12 h light, 12 h dark). Mice were provided food and water ad libitum. They were monitored daily by trained comparative medicine staff for their health and well-being. For the xenograft experiments, MM cell line KMS-28 (3 × 106 cells) were injected subcutaneously into six-week-old NSG mice. Mice were randomly divided into four groups: (1) Oral treatment with Olaparib (30 mg/kg); (2) Intraperitoneal injection with THZ 531 (10 mg/kg); (3) Treatment with both Olaparib and THZ531; (4) Diluent control. The mice received the drug/diluent control five times per week for 3 weeks.[2]

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For the in vivo studies, THZ1 was solubilized in 10% DMSO, 90% D5W, and olaparib was solubilized in 10% DMSO, 90% HPBCD.[3]
THZ1 and olaparib combination study Three million A673 or TC71 cells were subcutaneously implanted into the right flanks of 7–8 week old nude female mice. For PDX studies, 1 mm3 viably frozen tumor chunks were dipped in matrigel and implanted into the right flanks via minor surgery. When tumors measured 100–150 mm3, mice were divided into four groups: vehicle control, THZ1, olaparib, and THZ1 and olaparib in combination. THZ1 was administered IP BID at 10 mg/kg, and olaparib was administered PO BID at 50 mg/kg. Tumors were measured twice per week and mice were followed for survival. Statistical significance was determined by log-rank test for survival curves.[3]

[1]. Covalent targeting of remote cysteine residues to develop CDK12 and CDK13 inhibitors. Nat Chem Biol. 2016 Oct;12(10):876-84.

[2]. THZ531 Induces a State of BRCAness in Multiple Myeloma Cells: Synthetic Lethality with Combination Treatment of THZ 531 with DNA Repair Inhibitors. Int J Mol Sci. 2022 Feb; 23(3): 1207.
[3]. EWS/FLI Confers Tumor Cell Synthetic Lethality to CDK12 Inhibition in Ewing Sarcoma. Cancer Cell . 2018 Feb 12;33(2):202-216.e6.

THZ531 is a member of the class of indoles that is 5-chloro-4-(1H-indol-3-yl)-N-[(3R)-piperidin-3-yl]pyrimidin-2-amine in which the piperidine NH group is substituted by a 4-{[(2E)-4-(dimethylamino)but-2-enoyl]amino}benzoyl group. It is a first-in-class CDK12 and CDK13 covalent kinase inhibitor with IC50 of 158 nM and 69 nM, respectively. It has a role as an apoptosis inducer, an antineoplastic agent and an EC 2.7.11.22 (cyclin-dependent kinase) inhibitor. It is a member of indoles, an organochlorine compound, a N-acylpiperidine, an aminopyrimidine, an enamide, a secondary carboxamide and a secondary amino compound.

C30H32CLN7O2

558.0738

557.23

C, 64.57; H, 5.78; Cl, 6.35; N, 17.57; O, 5.73

1702809-17-3

1702809-17-3

118025540

White to light yellow solid powder

4.1

3

6

8

40

880

1

CN(C)C/C=C/C(=O)NC1=CC=C(C=C1)C(=O)N2CCC[C@H](C2)NC3=NC=C(C(=N3)C4=CNC5=CC=CC=C54)Cl

RUBYHLPRZRMTJO-MOVYNIQHSA-N

InChI=1S/C30H32ClN7O2/c1-37(2)15-6-10-27(39)34-21-13-11-20(12-14-21)29(40)38-16-5-7-22(19-38)35-30-33-18-25(31)28(36-30)24-17-32-26-9-4-3-8-23(24)26/h3-4,6,8-14,17-18,22,32H,5,7,15-16,19H2,1-2H3,(H,34,39)(H,33,35,36)/b10-6+/t22-/m1/s1

(E)-N-[4-[(3R)-3-[[5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl]amino]piperidine-1-carbonyl]phenyl]-4-(dimethylamino)but-2-enamide

THZ-531; THZ531; THZ 53; (R,E)-N-(4-(3-((5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl)amino)piperidine-1-carbonyl)phenyl)-4-(dimethylamino)but-2-enamide; CHEMBL4163879; THZ 531; THZ-531; (E)-N-[4-[(3R)-3-[[5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl]amino]piperidine-1-carbonyl]phenyl]-4-(dimethylamino)but-2-enamide; THZ531 HCl;

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: 100~250 mg/mL (179.2~448 mM)
Ethanol: ~4 mg/mL (~7.2 mM)

Solubility in Formulation 1: ≥ 1.43 mg/mL (2.56 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 14.3 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: 1.43 mg/mL (2.56 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 14.3 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: ≥ 1.43 mg/mL (2.56 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 14.3 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.)