UBA1(IC50= 1 nM)

With varying EC50 values ranging from 0.006 μM to 1.31 μM, TAK-243 exhibits anti-proliferative effect on a panel of cell lines derived from hematologic and solid tumors[1].
Human AML cell lines (OCI-AML2, TEX, U937, and NB4) exhibit reduced growth and viability in response to TAK-243 in a concentration- and time-dependent manner, with IC50s ranging from 15 to 40 nM after 48 hours of treatment[3].

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TAK-243 forms a TAK-243–ubiquitin adduct and potently inhibits UAE in vitro. [1]
TAK-243 inhibits the turnover of short-lived proteins and disrupts cell cycle progression.[1]
TAK-243 induces irresolvable endoplasmic reticulum stress.[1]
TAK-243 treatment alters DNA damage repair.[1]
TAK-243 has anti-proliferative activity in human cancer cells.[1]

No toxicity was observed in mice (T/C=0.02) as TAK-243 significantly delays the growth of tumors without affecting body weight, serum chemistry, or organ histology. In both tested samples, TAK-243 lessens the primary AML tumor burden without causing any toxicity[3].

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Having demonstrated substantial TAK-243 exposure in tumors, we next evaluated TAK-243 target engagement with UAE and downstream pathway modulation in tumor samples. We developed immunohistochemistry (IHC) assays to analyze several pharmacodynamic (PD) biomarkers associated with UAE inhibition. The monoclonal antibody raised against the TAK-243–ubiquitin adduct (MIL90) was used to monitor UAE target engagement in tumors. Downstream pathway inhibition was evaluated using several biomarkers; antibodies were used to detect both polyubiquitylated and mono-ubiquitylated proteins (using the FK2 antibody, which detects all cellular forms of ubiquitin conjugates), mono-ubiquitylated histone H2B and cleaved caspase-3 (as a measure of apoptosis). Mice bearing either WSU-DLCL2 lymphoma or PHTX-132Lu (primary NSCLC) xenograft tumor tissues were dosed using a single intravenous injection of TAK-243 at its maximum tolerated dose. Both UAE target engagement, as assessed by the presence of the TAK-243–ubiquitin adduct, and downstream pathway inhibition were clearly evident in tumor tissue (Fig. 3a). Following a single dose, TAK-243 induced a rapid and prolonged PD response (Fig. 3b,c), consistent with pronounced UAE inhibition in tumor tissue. Of the PD biomarkers evaluated, adduct formation was detected most sensitively, and the adduct could be detected at very low TAK-243 doses (3 mg per kg body weight (mg/kg) was the lowest dose examined) that were insufficient to modulate downstream PD readouts (polyubiquitin or mono-ubiquitylated histone H2B) or antitumor efficacy (data not shown). The adduct could be detected 0.5–1.0 h before downstream biomarker modulation, a lag which could be due to a combination of differences in assay sensitivity, de novo UAE synthesis and drug washout in the tumor over time. Of note, at its maximally tolerated dose, TAK-243 showed negligible inhibition of NAE-dependent neddylated cullin levels in HCT-116 xenografts, despite profound inhibition of polyubiquitin formation and ubiquitylated histone H2B levels (Supplementary Fig. 11a,b). These data indicate TAK-243 profoundly affects downstream UAE biomarkers while having little-to-no impact on NAE-associated biomarkers in vivo.[1]

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TAK-243 has UAE-specific antitumor efficacy in vivo [1]
The antitumor activity of TAK-243 was determined with a panel of human-patient-derived and cell-line-derived xenograft (PDX and CDX, respectively) tumor models that represented both solid and hematological cancers. We established subcutaneous tumors in mice using the following CDX models: WSU-DLCL2 (diffuse, large B cell lymphoma), HCT-116 (colon carcinoma), THP-1 (acute myeloid leukemia), CWR22 (prostate cancer), Calu-6 (lung, non-small-cell adenocarcinoma (NSCLC)), HCC70 (triple-negative breast cancer) and MM1.S (multiple myeloma), as well as the following human primary PDX models: PHTX-24c (colon cancer), PHTX-132Lu (lung, NSCLC), PHTX-55B (triple-negative breast cancer), PHTX-235O (ovarian cancer), and HNM626 (neck cancer). Tumor-bearing mice were dosed for 3 weeks with TAK-243,which was administered intravenously on a twice-per-week schedule (for example, Monday and Thursday; denoted BIW), and tumor growth and animal body weight were monitored. TAK-243 treatment induced a marked and robust antitumor activity response in all of the models examined (Fig. 4, Table 2 and Supplementary Fig. 12a). The human cancer cell lines that were treated with TAK-243 both in vitro and in vivo retained a similar rank order in sensitivity. TAK-243 inhibited both mouse and human UAE, as both TAK-243–ubiquitin adduct formation and downstream PD readouts were detectable in mouse tissues (data not shown); therefore, mice could be used to preliminarily evaluate the therapeutic window for TAK-243. The dose-limiting toxicity observed in mice was a decrease in body weight. At the maximum tolerated dose in vivo (23–26 mg/kg), mean maximal body weight loss was <12% of the animals' body weight relative to that before the start of treatment (Supplementary Fig. 12b).

Biochemical assays.[1]
The E1–UBL-dependent pyrophosphate exchange (PPiX) activity was monitored over time in the presence of different concentrations of TAK-243 to assess the rate of E1 inactivation. Reactions were run in the presence of 1 mM ATP, and both the reaction protocol and curve fit analysis were performed similarly as that described previously34. The rates of recovery of UAE, NAE and SAE after TAK-243–UBL formation were assessed using the E1–E2 HTRF transthiolation assay, using a modified version of the recovery method described previously. The HTRF E2 transthiolation assay was used to determine the IC50 value of the UBA6 inhibitor against recombinantly purified UAE, NAE and SAE. This assay was run as previously described. An AlphaScreen E2 transthiolation assay format using similar conditions was used to measure the IC50 value of the UBA6 inhibitor against recombinant purified UBA6. Biotin-tagged USE1 enzyme was used instead of GST-tagged E2s. For the antibodies used in HTRF-based quantification, 6 μg/ml streptavidin-coupled donor beads and 15 μg/ml of anti-Flag-coupled acceptor beads were used for detection. UBA6i IC50 titrations were determined in the presence of concentrations of ATP at the KM for ATP for each respective E1 enzyme.

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In vitro E2∼UBL thioester assay.[1]
The HTRF E2 transthiolation assay was used for TAK-243 IC50 studies against recombinant, purified UAE, NAE, SAE and ATG7 enzymes. This assay was run as previously described5,14,35. An AlphaScreen E2 transthiolation assay format using similar conditions was used for UBA6 and UBA7. Biotinylated tagged E2 enzymes were used instead of GST-tagged E2s. Instead of HTRF detection antibodies, 6 μg/ml streptavidin-coupled donor beads), and 15 μg/ml of anti-Flag-coupled acceptor beads were used for detection. TAK-243 IC50 titrations were determined in the presence of concentrations of ATP at the KM for ATP for each respective E1 enzyme.

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Crystallization and structure determination.[1]
All protein constructs and reagents for crystallization studies were generated in a manner similar to that described previously5. Due to difficulties in crystallizing the human form of UAE, a humanized yeast version of UAE (also known as Uba1 from Saccharomyces cerevisiae, UniProtKB P22515) was used for all studies. Although there are sequence differences between yeast and human UAE, the active site residues are highly conserved. For example, 21 of 25 residues within 5 Å of the TAK-243-binding site are identical. In addition, the use of yeast UAE crystal structures as model systems to describe the enzymatic mechanism of human UAE is well documented in the literature. Based on a two-residue amino acid difference in the active site between human and yeast UAE, the substitutions Asn471Met and Lys519Arg (yeast numbering) were introduced into a truncated form of yeast UAE (residues 10–1,024). These two substitutions were required to bring the potency of TAK-243 in line with the human enzyme (data not shown). Crystals were formed by mixing humanized yeast UAE, TAK-243, ubiquitin and ATP–MgCl2. Crystals were briefly transferred into cryoprotectant (held on ice), which was comprised of 80% reservoir solution (0.2 M magnesium formate, 0.1 M Bis-Tris pH 6.5) and 20% polyethylene glycol (PEG)-400, and flash-cooled in liquid nitrogen. The structure was solved by molecular replacement using existing yeast UAE coordinates (Protein Data Bank (PDB) entry 3CMM) as a starting model. Manual rebuilding of the model was accomplished using the program Coot, and refinement was carried out with the CCP4i graphical interface to Refmac. Coordinates have been deposited in the PDB with structure code 5TR4. Refer to Supplementary Table 1 for crystallographic data collection and refinement statistics.

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Kinase panel.[1]
Inhibitory activities of TAK-243 (at 1 μM) against 319 kinases were measured in studies conducted at the Reaction Biology Corporation. Results are expressed as percentage inhibition of kinase activity (percent of control). Briefly, kinase-specific substrates were prepared in the following reaction buffer: 20 mM 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES) (pH 7.5), 10 mM MgCl2, 1 mM ethylene glycol tetra-acetic acid (EGTA), 0.02% Brij-35, 0.02 mg/ml bovine serum albumin (BSA), 0.1 mM Na3VO4, 2 mM dithiothreitol (DTT) and 1% DMSO. Cofactors unique to each assay were added to the substrate solution. The kinase to be tested was added to the reaction mixture, and [α-32P]ATP (specific activity 10 μCi/μl) was added to the reaction and mixed. The reaction was incubated for 120 min at room temperature, and the reactions were then spotted onto P81 ion-exchange paper. Filters were washed in 0.75% phosphoric acid, and radioactivity was measured and analyzed. Staurosporine was included as a control compound. Refer to Supplementary Table 2 for a full list of the kinases tested.

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Carbonic anhydrase assay.[1]
TAK-243 IC50 values were determined by titrating TAK-243 into reactions containing 25 nM human carbonic anhydrase (HCA)-I or 2.5 nM HCA-II with 25 μM fluorescein diacetate (assay buffer: 25 mM MOPS, pH 7.5 and 0.02% Triton-X100). The activity of the carbonic anhydrase enzymes were measured over time using a BMG Labtech Polarstar reader at an excitation wavelength of 485 nm and an emission wavelength of 520 nm. The assay is formatted as a gain-of-signal assay based on the release of acetate from fluorescein (relieving the quenching of fluorescein). The IC50 values were calculated by fitting the titration curves to a standard logistic regression model. Refer to Supplementary Table 2 for the HCA-I and HCA-II data.

Normal keratinocytes, such as recessive dystrophic epidermolysis bullosa keratinocytes (RDEBK) and normal human keratinocytes (NHK), as well as cSCC cell lines, are seeded into 96-well plates. The CellTox Green Cytotoxicity Assay is used to analyze live cell number and cell death using an IncuCyte ZOOM real-time imager. One tool used to calculate relative EC50 values is GraphPad Prism. In clonogenic assays, six-well plates are seeded with cells. After adding inhibitors (such as TAK-243; 0.01, 0.1, 1, and 10 μM) for the specified durations, cells are kept in drug-free medium for a maximum of two weeks to enable colony formation. Colonies are stained with crystal violet after being fixed in 10% methanol and 10% acetic acid[1].

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Western blot analysis.[1]
HCT-116 and WSU-DLCL2 cells were maintained in log-phase growth in McCoy's 5A modified or RPMI-1460 medium, respectively supplemented with 10% fetal bovine serum at 37 °C in a 5% CO2 incubator. Cells were grown in 6-well cell culture dishes and treated with DMSO (0.1%) or with 0.01, 0.10 or 1.00 μM TAK-243 for the times indicated. Whole-cell extracts were prepared using RIPA buffer. Immunoblotting (all antibodies were used at 1:1,000 dilution unless otherwise noted) after SDS–PAGE conducted under nonreducing (E2-thioester) or reducing conditions was done as previously described3; 30 μg total protein was fractionated by SDS–PAGE and immunoblotted with the following primary antibodies to the following proteins.

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Cellular thioester assays.[1]
HCT-116 cells grown in 6-well cell culture dishes were treated with DMSO (0.1%) or increasing concentrations of TAK-243 for 4 h, and whole-cell extracts were prepared using RIPA buffer. Cellular lysates for E2–UBL thioester detection were fractionated by SDS–PAGE under nonreducing conditions and immunoblotted with primary antibodies to UBCH10, USE1, UBC12, UBC9 and ATG7. Mouse- or rabbit-specific Alexa-Fluor-680-conjugated secondary antibodies (for information on all antibodies used, see Supplementary Table 3) were used, and blots were imaged using the Li-Cor Odyssey Infrared Imaging system. To analyze E2 thioesters involved in DNA damage repair, HCT-116 cells were cultured with DMSO or TAK-243 for 8 h. The following antibodies were used to detect E2–ubiquitin thioesters on immunoblots: UBE2A and UBE2B, ubiquitin-conjugating enzyme E2 T (UBE2T), proliferating cell nuclear antigen, Fanconi anemia complementation group D2 (FANCD2) and UBE1. All primary and secondary antibodies were used at 1:1,000 dilution. Refer to Supplementary Table 3 for a full antibody list and to Supplementary Figure 15 for uncropped western blot images.

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Cell cycle analysis.[1]
Logarithmically growing HCT-116 cells (plated at 6 ×105) were incubated with DMSO, pevonedistat (0.25 μM) or TAK-243 (0.05 μM or 1 μM) for 16 h. The cells were collected, fixed with 70% ethanol and stored overnight at 4 °C. Fixed cells were centrifuged and washed with PBS to remove the ethanol. The pellets were resuspended in propidium iodide and RNAse A in PBS for 1 h on ice, protected from light. Cell-cycle distribution was determined using flow cytometry, and the analysis was completed using BD FACSDiva software version 6.1.1. The gating strategy was defined by forward (FSC) or side (SSC) scatter of untreated control cells. Gating on live, single PI-positive cells were used for cell cycle analysis (Supplementary Fig. 16). 10,000 total events and 5,000 gated events were recorded for each sample analyzed.

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Lys48 immunofluorescence assay.[1]
HCT-116 cells were plated (1.5 × 104 cells/well) in 96-well cell culture plates and incubated for 24 h at 37 °C and 5% CO2 for attachment. Increasing concentrations of TAK-243 were added to the plates (with a starting concentration of 20 μM), and the plates were incubated for 3.5 h at 37 °C and 5% CO2. Cells were fixed in 2% paraformaldehyde for 10 min followed by 10 min of permeabilization in 0.5% Triton X-100 at room temperature. Cells were blocked for 1 h at room temperature with Roche Blocking Buffer. A primary antibody specific for Lys48 of ubiquitin was incubated with the cells for 1 h at room temperature. An Alexa-Fluor-488-conjugated goat anti-rabbit-IgG (catalog no. A-11034, Invitrogen; 1:500) was used as the secondary antibody and incubated with the cells for 1 h at room temperature. Images were analyzed using an Opera confocal high-content screening imaging system.

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CellLights microscopy for endoplasmic reticulum (ER) expansion.[1]
HCT-116 cells were transduced with CellLight ER-RFP, BacMam 2.0 at a multiplicity of infection (MOI) of 20 and treated 24 h later with DMSO or 0.1 μM TAK-243 for the times indicated in Supplementary Figure 6. After treatment with the compound, the cells were added to a humidified chamber kept at 37 °C with 5% CO2 and attached to an Eclipse TE2000-U microscope (Nikon Instruments) equipped with an automated xyz stage, a filter wheel and an Orca-ER camera controlled by MetaMorph software.

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Comet assay.[1]
Calu-6 cells were plated into 6-well dishes at a density of 0.2 × 106 cells/well. Cells were treated with either DMSO or 1 μM TAK-243 for 1 h before exposure to UV treatment (20 J/m2). Following UV treatment, cells were harvested at time t = 0 min or incubated for 6 h in the presence or absence of 1 μM TAK-243. Preparation and execution of the alkaline comet assay were performed according to the Trevigen CometAssay protocol. Electrophoresis was conducted with alkaline buffer (pH 13 solution containing 200 mM NaOH and 1 mM EDTA) at a constant voltage of 21 V over a 30-min period. Following neutralization in water and fixation with 70% Ethanol, the slides were allowed to dry, and finally they were stained using a 1:5,000 dilution of SYBR green in 1× PBS for 5 min at 4 °C. After a quick rehydration step in distilled water, the slides were imaged on an imageXpress MICRO using a 4× pan-fluorescence objective lens. Comet tails were measured using Comet Assay IV software.

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Cell viability assays.[1]
Cell lines were maintained under logarithmic growth in the appropriate cell culture medium for each cell line according to the manufacturer's instructions. Cells were plated in appropriate complete medium in triplicate in 384-well culture plates in the presence of increasing concentrations of TAK-243 and incubated at 37 °C for 72 h. Cell viability was assessed using the Cell Titer Glo assay kit according to the manufacturer's protocol.

Mice[2]
TAK-243's preclinical effectiveness and toxicity are evaluated using AML mouse models. SCID mice are given subcutaneous (sc) injections of OCI-AML2 cells, and TAK-243 (20 mg/kg sc twice weekly) is administered to the mice once tumors are palpable. In an extra model, NOD-SCID mice are given injections of primary AML cells from two patients into their femurs. Mice are administered TAK-243 (20 mg/kg sc twice weekly) two weeks post-injection. Following three weeks of therapy, the mice are killed, and flow cytometry is used to quantify the amount of AML engraftment in the non-injected femur[2].

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Generation of in vivo xenograft models of human tumors and efficacy studies.[1]
Eight- to 12-week-old mice were inoculated subcutaneously in the flanks with either tumor fragments or a tumor cell suspension in serum-free medium. Female CB-17 SCID mice were used for the following tumor models: WSU-DLCL2 (4.0 × 106 cells/mouse), MM1.S (5.0 × 106 cells/mouse), (PHTX-132Lu (primary NSCLC, 2 × 3 mm tumor fragments), THP-1 (4.0 × 106 cells/mouse), PHTX-24c (primary colon, 2 × 3 mm tumor fragments), and THP-1 UBA3 A171T (previously described). Male CB-17 SCID mice were used for the CWR22 model (2 × 3 mm tumor fragments). Female CB-17 SCID mice (Taconic) were used for the PHTX-55B model (primary breast, 2 × 3 mm tumor fragments). Immunodeficient, female NOD SCID mice were used for the PHTX-235O model (primary ovarian, 2 × 3 mm tumor fragments). Female nude mice (Taconic) were used for the HCT-116 and Calu-6 models (5.0 × 106 cells/animal). CrownBio performed the experiment using the HNM626 model, in which female nude mice were implanted in the flank with 3 × 3 × 3 mm tumor fragments. Medicilon performed the experiment using the HCC-70 model, in which female nude mice were implanted in the flank with 5.0 × 106 cells/mouse.

Tumor growth was monitored with vernier calipers. The mean tumor volume was calculated using the formula: volume (V) = W2 × L/2, where W and L are the width and length of the tumor, respectively. When the mean tumor volume reached approximately 200 mm3, the animals were randomized into groups of n = 6–10 animals (depending on the model). Our randomization approach used a technique called minimization to reduce imbalances in the baseline characteristics of the mice across the groups. In particular, our approach reduced imbalances in the average tumor volume and the initial average animal weight. First, we generated a large number of candidate designs using simple randomization, in which every possible design with a fixed number of animals per group was equally likely. The number of candidate designs was not fixed; rather, new candidate designs were added until the total computation time reached 3 s. For each candidate design, the overall imbalance was calculated. The overall imbalance was defined as the sum of two terms in which the first term was the variance of the means of the tumor volumes for each group divided by the square of the overall volume mean, and the second term was the variance of the means of the animal weights for each group divided by the square of the overall weight mean. The candidate design with the lowest overall imbalance was selected as the final design. The number of mice per group was used to enable calculation of statistical significance. Mice were dosed with 20% 2-hydroxypropyl-β-cyclodextrin (HPbCD) or TAK-243 over a 21-d period on a BIW schedule; no blinding was used. Tumor growth and body weights were measured twice per week. The percentage TGI (mean tumor volume (MTV) of the control group – MTV of a treated group)/MTV of the control group) was calculated within 5 d of the last dose, and the statistical analysis was as described previously43.

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TAK-243 and radiation combination efficacy experiments.[1]
In experiments were performed at WUXI (China), 6- to 9-week-old female BALB/c nude mice were implanted subcutaneously with ∼30-mm3 tumor slices in the right flank, using either LU-01-0030 tumor tissue (a patient-derived xenograft (PDX) model of human NSCLC) or BR-05-0026 tumor tissue (a PDX model of breast cancer). When the mean tumor volume reached approximately 159 mm3 (LU-01-0030) or 177 mm3 (BR-05-0026), animals were randomized into four treatment groups. TAK-243 was administered intravenously on a BIW schedule for 3 weeks on the indicated days (Supplementary Fig. 8). Beam-focused radiation was delivered using 2 Gy on the indicated days. A Rad Source RS-2000 X-ray irradiator was used for therapy. Before being irradiated, the mice were anesthetized by intraperitoneal injection using 1.0% sodium pentobarbital at 80 mg/kg. Beam-focused radiation was applied to each mouse at 1 Gy/min. Tumors were measured twice a week using vernier calipers. Tumor volumes were calculated using the following formula: 0.5 × (length × width2). Tumor size and body weight were measured approximately twice a week for the duration of the study. Mice were euthanized when their tumor volume reached approximately 1,000 mm3. Synergistic antitumor analysis was evaluated on days 0 to 21. All tumor volumes had a value of 1 added to them before log10 transformation. For each mouse, the log(tumor volume) at day 0 was subtracted from the log(tumor volume) on the subsequent days. This difference versus time was used to calculate an area-under-the-curve (AUC) value for each animal using the trapezoid rule. The synergy score for the combination of agents A and B was defined as

Where AUCAB, AUCA, AUCB and AUCctl are the AUC values for animals in the combination group, the A group, the B group, and the control group, respectively. The standard error of the synergy score was computed based on the variation in the AUC values among the animals. A two sided t-test was used to determine whether the synergy score was significantly different from zero. If the P value was below 0.05, and the synergy score was less than zero, then the combination was considered to be synergistic.

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Pharmacokinetic (PK) analysis.[1]
SCID mice bearing WSU-DLCL2 NHL xenograft tumors were administered a single intravenous dose of TAK-243 in 20% hydroxypropyl β-cyclodextrin at 12.5, 18.75 and 25 mg/kg, and the tumor and plasma were harvested over a 72-h period. TAK-243 exposures were determined using mass spectrometry (LC/MS/MS) methods with a lower limit of quantification (LLOQ) of 1 nM for plasma and 5 nM for tumors.

Pharmacokinetic and pharmacodynamic analysis of TAK-243 in tumor bearing mice [1]
We characterized the pharmacokinetic (PK) parameters of TAK-243 in immunocompromised CB-17 SCID mice that bore a subcutaneous diffuse, large B cell lymphoma (WSU-DLCL2) tumor following an acute, intravenously administered dose of TAK-243. Both plasma and WSU-DLCL2 tumor tissues from these mice were analyzed for exposure to TAK-243. In plasma, the TAK-243 parent compound was characterized by a high clearance (CL) rate (3.99 to 4.99 liter per h per kg body weight (liter/h/kg)) and a short terminal half-life (t1/2) (0.2–0.4 h) (Supplementary Fig. 10a). TAK-243 rapidly forms a TAK-243–ubiquitin adduct, and our PK measurements did not account for TAK-243 that was sequestered as a ubiquitin adduct. Despite the high plasma clearance rate observed for TAK-243, the drug showed a high volume of distribution at steady state (Vss) (1.13–1.74 liter/h/kg) with a 5.8- to 8-fold higher area-under-the-curve (AUC) drug exposure in tumor tissue relative to that in plasma, and a prolonged tumor t1/2 of 16.9–20.6 h (Supplementary Fig. 10b).[1]

[1]. A small-molecule inhibitor of the ubiquitin activating enzyme for cancer treatment. Nat Med. 2018 Feb;24(2):186-193.

[2]. TAK-243, a small molecule inhibitor of ubiquitin-activating enzyme (UAE), induces ER stress and apoptosis in diffuse large B-cell lymphoma (DLBCL) cells. Blood 2017 130:1533.

[3]. TAK-243 Is a Selective UBA1 Inhibitor That Displays Preclinical Activity in Acute Myeloid Leukemia (AML). Blood 2017, 130:814.

TAK-243 is under investigation in clinical trial NCT03816319 (TAK-243 in Treating Patients With Relapsed or Refractory Acute Myeloid Leukemia or Refractory Myelodysplastic Syndrome or Chronic Myelomonocytic Leukemia).
UAE Inhibitor TAK-243 is a small molecule inhibitor of ubiquitin-activating enzyme (UAE), with potential antineoplastic activity. UAE inhibitor TAK-243 binds to and inhibits UAE, which prevents both protein ubiquitination and subsequent protein degradation by the proteasome. This results in an excess of proteins in the cells and may lead to endoplasmic reticulum (ER) stress-mediated apoptosis. This inhibits tumor cell proliferation and survival. UAE, also called ubiquitin E1 enzyme (UBA1; E1), is more active in cancer cells than in normal, healthy cells.

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The ubiquitin-proteasome system (UPS) comprises a network of enzymes that is responsible for maintaining cellular protein homeostasis. The therapeutic potential of this pathway has been validated by the clinical successes of a number of UPS modulators, including proteasome inhibitors and immunomodulatory imide drugs (IMiDs). Here we identified TAK-243 (formerly known as MLN7243) as a potent, mechanism-based small-molecule inhibitor of the ubiquitin activating enzyme (UAE), the primary mammalian E1 enzyme that regulates the ubiquitin conjugation cascade. TAK-243 treatment caused depletion of cellular ubiquitin conjugates, resulting in disruption of signaling events, induction of proteotoxic stress, and impairment of cell cycle progression and DNA damage repair pathways. TAK-243 treatment caused death of cancer cells and, in primary human xenograft studies, demonstrated antitumor activity at tolerated doses. Due to its specificity and potency, TAK-243 allows for interrogation of ubiquitin biology and for assessment of UAE inhibition as a new approach for cancer treatment.[1]

C19H20F3N5O5S2

519.517811775208

519.085

C, 43.93; H, 3.88; F, 10.97; N, 13.48; O, 15.40; S, 12.34

1450833-55-2

71715374

White to off-white crystalline solid

1.8±0.1 g/cm3

1.712

1.49

4

13

7

34

804

4

S(=O)(=O)(OC[C@@H]1[C@@H](O)[C@@H](O)[C@H](NC2=CC=NC3N2N=C(C=3)C2C=C(SC(F)(F)F)C=CC=2)C1)N

PUMGWJMQPGYZFC-IDCJVQTKSA-N

InChI=1S/C19H20F3N5O5S2/c1-32-34(30,31)26-14-8-13(17(28)18(14)29)24-15-5-6-23-16-9-12(25-27(15)16)10-3-2-4-11(7-10)33-19(20,21)22/h2-7,9,13-14,17-18,24,26,28-29H,8H2,1H3/t13-,14+,17+,18-/m1/s1

methyl ((1S,2R,3S,4R)-2,3-dihydroxy-4-((2-(3-((trifluoromethyl)thio)phenyl)pyrazolo[1,5-a]pyrimidin-7-yl)amino)cyclopentyl)sulfamate

TAK-243; TAK 243; TAK243; MLN7243; 1450833-55-2; Uae inhibitor Sulfamic acid, [(1R,2R,3S,4R)-2,3-dihydroxy-4-[[2-[3-[(trifluoromethyl)thio]phenyl]pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]methyl ester; TAK 243; UNII-V9GGV0YCDI; MLN-7243; MLN 7243; AOB87172; AOB-87172; AOB 87172

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 : 50~100 mg/mL ( 96.24~192.48 mM )
Ethanol : ~25 mg/mL
Water : ~1 mg/mL (~1.92 mM )

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.00 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 20.8 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: ≥ 2.08 mg/mL (4.00 mM) (saturation unknown) 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.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 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: ≥ 2.08 mg/mL (4.00 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 5% DMSO + 40% PEG300 + 5% Tween80 + 50% ddH2O: 5mg/ml

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