Mps1 (IC50 = 1 nM)

Empesertib (BAY1161909) is a very potent Mps-1 inhibitor, showing an IC50 lower than 400 nM in a HeLa cell proliferation assay and an IC50 lower than or equal to 1 nM (more potent than 1 nM) in an Mps-1 kinase assay with a concentration of 1 μM/2 mM ATP.[1]

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BAY 1161909 and BAY 1217389 inhibited Mps1 kinase activity with IC50 values below 10 nmol/L while showing an excellent selectivity profile. In cellular mechanistic assays, both Mps1 inhibitors abrogated nocodazole-induced SAC activity and induced premature exit from mitosis (“mitotic breakthrough”), resulting in multinuclearity and tumor cell death. Both compounds efficiently inhibited tumor cell proliferation in vitro (IC50 nmol/L range).[2]

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Only the corresponding R-isomers 39–41/Empesertib (BAY1161909) (Table 6) had good potency; S-isomer 42 and larger α-substituents (43, 44) resulted in reduced potency (Table 7). Compared to the fluoroazetidine-derived amide 39 and oxazolidinone 40, methylsulfone 41/Empesertib (BAY1161909) showed the best overall profile, further improved kinase selectivity compared to the early lead compound 9 (41/Empesertib (BAY1161909) only inhibits two kinases, JNK2 and JNK3, more than 50% at a concentration of 1 μmol/L and no other kinase at 100 nmol/L in the Millipore kinase panel of 230 kinases), and was selected as our first clinical candidate. Indeed, it was the first MPS1 inhibitor to enter a phase I clinical trial, in May 2014 (NCT02138812) [4].

Empesertib (BAY1161909) is an extremely potent Mps-1 inhibitor. Its maximum oral bioavailability (Fmax) in rats is greater than 70%, as measured by rat liver microsomes; similarly, its maximum oral bioavailability (Fmax) in dogs is greater than 50%, as measured by dog liver microsomes; and its maximum oral bioavailability in humans is greater than 60%, as measured by human liver microsomes.[1]

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In vivo, Empesertib (BAY1161909) and BAY 1217389 achieved moderate efficacy in monotherapy in tumor xenograft studies. However, in line with its unique mode of action, when combined with paclitaxel, low doses of Mps1 inhibitor reduced paclitaxel-induced mitotic arrest by the weakening of SAC activity. As a result, combination therapy strongly improved efficacy over paclitaxel or Mps1 inhibitor monotreatment at the respective MTDs in a broad range of xenograft models, including those showing acquired or intrinsic paclitaxel resistance. Both Mps1 inhibitors showed good tolerability without adding toxicity to paclitaxel monotherapy. These preclinical findings validate the innovative concept of SAC abrogation for cancer therapy and justify clinical proof-of-concept studies evaluating the Mps1 inhibitors BAY 1161909 and BAY 1217389 in combination with antimitotic cancer drugs to enhance their efficacy and potentially overcome resistance [2].

Kinase assay [2]
The inhibition of recombinant human Mps1 by Empesertib (BAY1161909) or BAY 1217389 was assessed in TR-FRET–based in vitro kinase assays via phosphorylation of a biotinylated peptide (biotin-Ahx-PWDPDDADITEILG-NH2). Kinase and test compound were preincubated for 15 minutes before enzyme reaction was started by the addition of substrate and ATP upon 10 μmol/L. For further details, see Supplementary Materials and Methods.

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Kinase selectivity profiling [2]
Empesertib (BAY1161909) and BAY 1217389 were counterscreened against a panel of kinases using the Millipore Kinase or DiscoveRx profiler screen. Empesertib (BAY1161909) was initially tested at 1 μmol/L in the DiscoveRx kinase panel, followed by KD determination for 11 kinases (Supplementary Table S1). BAY 1161909 was tested at 10 μmol/L in the Millipore kinase panel, followed by retesting at 1 and 0.1 μmol/L and IC50 determination for JNK1alpha, JNK2alpha, and JNK3 (Supplementary Table S1). BAY 1217389 was initially tested at 1, 0.1, and 0.01 μmol/L in the DiscoveRx kinase panel (Supplementary Table S2).

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Biochemical assays [3]
The inhibition of the kinase activity of biochemically purified full-length TTK was determined in a IMAP® fluorescence polarization-based assay, as previously described. Briefly, kinase inhibitors and TTK were diluted in IMAP reaction buffer, which consists of 10 mM Tris–HCl (pH 7.5), 10 mM MgCl2, 0.01% Tween-20, 0.1% NaN3, and 1 mM DTT. After preincubation of 1 h in the dark at room temperature, fluorescein-labeled MBP-derived substrate peptide was added, followed by ATP to start the reaction. Final enzyme concentration was 3.9 nM, final substrate concentration was 50 nM, and final ATP concentration was 5 μM. The reaction was allowed to proceed for 2 h at room temperature in the dark. Fluorescein polarization was measured on an Envision multimode reader. Data were fit using a four parameter logistics curve in XLfit™5. Experiments were performed at least three times in duplicate (Supplementary Table S2). Broad kinase selectivity profiling was carried out at Carna Biosciences.

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Purification of TTK kinase domain [3]
TTK was expressed and purified according to a protocol developed at the Structural Genomics Consortium. Plasmid TTKA-c013/SGC2-B08 was used. TTK was expressed in the Escherichia coli Rosetta™ strain in LB medium with 50 μg/L kanamycin and 35 μg/L chloramphenicol, to OD600 of 0.6–0.8 at 37 °C in a shaker incubator. After this, 1 mM IPTG was added, followed by further incubation for 4 h at room temperature. Bacterial pellets were frozen at − 20 °C. For purification, cells were resuspended in binding buffer [50 mM Hepes (pH 7.5), 500 mM NaCl, and 5% (vol/vol) glycerol]. We added 10 mM imidazole and EDTA-free protease inhibitors (1 tablet per 50 mL), and the cells were lysed using an Avastin liquid homogenizer, followed by centrifugation. The supernatant was incubated for 1 h at 4 °C with NiNTA Superflow-sepharose beads, after which the beads were isolated and washed with binding buffer with 50 mM imidazole. Protein was eluted with binding buffer and 250 mM imidazole. The next day, the protein-containing fractions were further purified by size-exclusion chromatography on a Superdex 75 column running in 50 mM Hepes (pH 7.5), 150 mM NaCl, and 5 mM DTT. Peak fractions were pooled and subsequently flash-frozen in dry ice/ethanol in 50 μl aliquots at − 80 °C. Specifically for protein crystallography, peak fractions were concentrated at 4 °C on a Spin-X UF 30-kDa filter to 8–10 mg/ml, before flash-freezing and storage.

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Thermal shift assays [3]
An aliquot of purified TTK kinase domain was thawed and diluted to 4.8 μM in IMAP buffer [10 mM Tris (pH 7.5), 10 mM MgCl2, 0.01% Tween-20, and 0.1% NaN3]. Subsequently, 10 μl of the protein was mixed with 5 μl of 40 μM compound of interest in IMAP buffer, in a Greiner 96-well PCR plate. After 30-min incubation on ice, 5 μl of 1250 times diluted SyproOrange in IMAP buffer was added. Final concentrations were 2.4 μM TTK and 10 μM compound. SyproOrange was 5000 times diluted. Immediately after incubation, the plate was sealed with TopSeal A-plus and transferred to a Biorad CFX96, where the temperature was increased from 20 °C to 90 °C in 0.5 °C increment per 5 s. Reported values were measured in quadruplicate, in three independent experiments (Supplementary Table S2). Melting temperatures were determined by taking the minimum of the first derivative of the melting curve.

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SPR [3]
Binding kinetics were determined by SPR using a Biacore T200. Because initial tests with full-length TTK did not yield stable surfaces, we used the His-tagged kinase domain of TTK, as described previously. Briefly, TTK kinase domain was immobilized on a Ni-NTA sensor chip with His-tag capturing and amine-coupling to a level of 4000–6000 RU. Inhibitors were diluted in binding buffer to a final concentration of DMSO of 1% (vol/vol). Compound binding was measured in binding buffer (10 mM Tris, 10 mM MgCl2, 0.01% Tween-20, and 1 mM DTT at pH 6.8) with 1% (vol/vol) DMSO using single cycle kinetics by injecting an increasing concentration range of 1 – 3.16 – 10 – 31.6 – 100 nM. Flow rate was 30 μl/min, and association time per injection was 100 s. Dissociation following the last injection was monitored for at least 30 min. No regeneration was carried out. From the compound signal, we subtracted the buffer injection and the reference channel signals (double referencing). Resulting data were fit using the Biacore Evaluation software to the simple 1:1 Langmuir binding model. All kinetic constants were within the working range of Biacore T200. To determine the reliability of the curve fit, we applied standard Biacore checks. The uniqueness of a fit (U value), the standard error of the ka and kd, and the mass transfer constant were monitored as outlined before.

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Biochemical MPS1 Inhibition Assays.The inhibition of human MPS1 was assessed in time-resolved fluorescence resonance energy transfer (TR-FRET) based in vitrokinase assays measuring the phosphorylation of a biotinylated substrate peptide with a phosphospecific antibody.Compounds were tested on the same microtiter plate at 11 different concentrations in the range 20μM to 0.1 nM, in duplicates for each concentration. The dilution series was prepared separately prior to the assay as 100-fold concentrated stock solutions in DMSO. For the assay, 50nL of each stock solution of the test compound in DMSO was pipetted into a black, low volume, 384-well microtiter plate. Two microliters of a solution of human recombinant MPS1 in aqueous assay buffer [25 mM HEPES pH 7.7, 10 mM MgCl2, 2mM DTT, 0.05% BSA (w/v), 0.1 mM activated sodium orthovanadate, 0.001% Pluronic F-127] was added, and the mixture was incubated for 15 min at 22°C to allow pre-binding of the test compound to the enzyme. Then, the kinasereaction was started by the addition of 3μL of a solution of ATP (16.7μM for the low ATP assay and 3.3mM for the high ATP assay; final concentration in the 5μL assay volume=10μM and 2mM, respectively) and the biotinylated peptide biotin-Ahx-PWDPDDADITEILG (C-terminus in amide form) (1.67μM; final concentration=1μM) in assay buffer, and the resulting mixture was incubated for 60min at 22°C. The enzyme concentration was adjusted depending on the activity of the enzyme lot and was chosen appropriately to have the assay in the linear range (typical concentrations were in the S4range 0.25–0.5nM). The reaction was stopped by the addition of 5μL of a solution of TR-FRET detection reagents [140 nM streptavidin-XLent and 1.5nM anti-phospho(Ser/Thr)-europium antibody in an aqueous EDTA solution (40mM EDTA, 0.1% (w/v) BSA in 100mM HEPES pH 7.4)].The resulting mixture was incubated for 1 h at 22°C to allow formation of the complex between the phosphorylated biotinylated peptide and the detection reagents. Subsequently, the amount of phosphorylated substrate was evaluated by measurement of the resonance energy transfer from the europium chelate to the streptavidin-XLent. For this, the fluorescence emissions at 620nm and at 665nm after excitation at 350nm were measured using a TR-FRET reader [e.g., PHERAstar]. The ratio of the emissions at 665nm and at 620nm was taken as the measure for the amount of phosphorylated substrate. Data were normalized (enzyme reaction without inhibitor=0% inhibition, all other assay components but no enzyme=100% inhibition), and IC50values were calculated using a four-parameter fit.Binding Kinetics Studies.Surface plasmon resonance (SPR) spectroscopy was performed in a Biacore T100 instrument. The recombinant kinase domain of hMPS1 (N515–T806) was expressed in E. colias N-terminal GST-fusion protein containing a thrombin cleavage site, purified via glutathione affinity chromatography followed by thrombin cleavage and subsequent size exclusion chromatography as described below for the protein production for the crystallization experiments. The protein was immobilized on Series S CM5 sensor chips via amine coupling at densities of 1000 and 5000 RU. Upon pre-equilibration of the immobilized protein in assay buffer [25mM HEPES pH7.7, 10mM MgCl2, 2mM DTT, 0.001% (v/v) Surfactant P20, 2% DMSO], S5serial dilutions of test compounds were applied in either multiple injection cycles (16: concentrations of 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, 500, and 1000 nM) or single injection cycles (41: concentrations of 2, 7.8, 31.3, 125, and 500 nM). The resulting sensorgrams were fit to a simple Langmuir 1:1 interaction model including a standard term for mass transport, from which the kinetic parameters [kon, koff, (=1/koff)] were obtained. [4]

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In Vitro Metabolic Stability in Rat Hepatocytes. [4]
Hepatocytes from male Wistar rats were isolated via a two-step perfusion method. After perfusion, the liver was carefully removed from the rat, the liver capsule was opened, and the hepatocytes were gently S6shaken out into a Petri dish with ice-cold Williams’ medium E (WME). The resulting cell suspension was filtered through sterile gauze into 50mL Falcon tubes and centrifuged at 50×gfor 3 min at rt. The cell pellet was resuspended in 30mL of WME and centrifuged through a Percoll gradient two times at 100×g. The hepatocytes were washed again with WME and resuspended in medium containing 5% FCS. Cell viability was determined by trypan blue exclusion. For the metabolic stability assay, liver cells were distributed in WME containing 5% FCS to glass vials at a density of 1.0 × 106vital cells/mL. The test compound was added to a final concentration of 1μM. Organic solvent in the incubations was limited to ≤0.01% DMSO and ≤1% acetonitrile. During incubation, the hepatocyte suspensions were continuously shaken at 580 rpm and aliquots were taken at 2, 8, 16, 30, 45, and 90 min, to which an equal volume of cold acetonitrile was immediately added. Samples were frozen at –20°C overnight, and subsequently centrifuged at 3000rpm for 15 min. The supernatant was analyzed with an Agilent 1200 HPLC system with MS/MS detection. The half-life of a test compound was determined from the concentration–time plot. From the half-life, the intrinsic and the in vitropredicted blood clearances were calculated, as well as the hepatic extraction ratio with EH=(CLb/LBF)·100%, according to the ‘well-stirred’ liver model.3In combination with the standardized liver blood flow (LBF) and amount of liver cells in vivoand in vitro, the in vitroblood clearance (CLb, in vitro) and the hepatic extraction ratio (EH, in vitro) were calculated. The following parameter values were used: liver blood flow: 4.2, 2.1, and 1.32L/h/kg for rat, dog, and human, respectively; specific liver weight: 32, 39, and 21 g/kg body weight for rat, dog, and human, respectively; liver cells in vivo, 1.1×108cells/g liver; liver cells in vitro, 1.0×106/mL.

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In Vitro Metabolic Stability in Liver Microsomes. [4]
The in vitrometabolic stability of test compounds was determined by incubation at 1μM in a suspension of liver microsomes in 100mM phosphate buffer pH7.4 (NaH2PO4·H2O + Na2HPO4·2H2O) and at a protein concentration of 0.5 mg/mL at 37°C. The microsomes were activated by adding a cofactor mix containing 8 mM glucose-6-phosphate, 0.5 mM NADP, and 1 IU/mL glucose-6-phosphate dehydrogenase in phosphate buffer pH 7.4. The metabolic assay was started shortly afterwards by adding the test compound to the incubation at a final volume of 1mL. During incubation, the microsomal suspensions were continuously shaken at 580 rpm and aliquots were taken at 2, 8, 16, 30, 45, and 60 min. Further handling and analysis as per the hepatocyte method described above.

On a 96-well multititer plate, cultured tumor cells are plated at densities of 5000 cells/well (MCF7, DU145, HeLa-MaTu-ADR), 3000 cells/well (NCI-H460, HeLa-MaTu, HeLa), or 1000 cells/well (B16F10) in 200 μl of each of their five growth media supplementsed with 10% fetal calf serum." The test substances are added to the fresh culture medium (200 μl) in the other plates, which has been replaced with varying concentrations (0 μM, as well as in the range of 0.01-30 μM). The cells are incubated for 4 days in the presence of the test substances. After 24 hours, the cells on one plate (the zero-point plate) are stained with crystal violet. Cell proliferation is assessed by staining the cells with crystal violet. To fix the cells, add 20 μl/measuring point of an 11% glutaric aldehyde solution and let it sit at room temperature for 15 minutes. The fixed cells 15 are washed three times with water, and then the plates are dried at room temperature. Staining the cells involves adding 100 μl/measuring point of a 0.1% crystal violet solution (pH 3.0). The plates are dried at room temperature following three rounds of water washing for the stained cells. To dissolve the dye, add 100 μl/measuring point of a 10% acetic acid solution.

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Multinuclearity assay. [2]
U-2 OS (osteosarcoma ATCC: HTB-96) cells were plated at a density of 2,500 cells per well in a 384-well microtiter plate in 20 μL cell culture medium and incubated overnight at 37°C. Empesertib (BAY1161909) or BAY 1217389 was added at a final concentration of 100 nmol/L in triplicates. Cells were treated for 0, 24, 48, and 72 hours at 37°C in the presence of test compounds. Thereafter, cells were fixed with 4% (v/v) PFA, permeabilized with 0.5% (v/v) Triton X-100, and blocked with 0.5% (v/v) BSA in PBS. α-Tubulin structures were detected by antibody labeling. After blocking with goat IgG, secondary antibodies were applied in the blocking solution. Cells were washed with PBS, and nuclei were marked with Hoechst 33342. Finally, cells were washed and covered with PBS and stored at 4°C until image acquisition. Images were acquired with a PerkinElmer Opera High-Content Analysis reader.

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Cellular Assays.[4]
Human tumor cell lines were obtained from the ATCC or from the German Collection of Microorganisms and Cell Cultures. Authentication of the cell lines was performed at the German Collection of Microorganisms and Cell Cultures. Cells were propagated under the suggested growth conditions in a humidified 37°C incubator. HeLa and HT29 cell proliferation assays were performed as previously described.

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Spindle Assembly Checkpoint (SAC) Assay (Cell-Based Mechanistic Assay).[4]
The SAC assay was carried out according to our published protocol.1Live-Cell Imaging for Co-treatment Study.For fluorescence live-cell imaging, the experiments were carried out according to our published protocol

rat, dog and humans
1-2 mg/mL
Oral gavage, IV

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Pharmacokinetic investigations [2]
Pharmacokinetic studies were performed in male Wistar rats and female CD1 or NMRI nu/nu mice. For i.v. studies in rats and mice, Empesertib (BAY1161909) was solubilized in 1% DMSO, 99% plasma; for p.o. studies in rats in 50% polyethylene glycol (PEG) 400, 10% ethanol, 40% water, and for p.o. studies in mice in 75% PEG 400, 5% ethanol, and 25% solutol. BAY 1217389 was solubilized in 50% PEG 400, 10% ethanol, and 40% water for intravenous and p.o. dosing in rats and mice. In pharmacokinetic studies, plasma samples were collected after 2, 5, 15, 30, 45 minutes, 1, 2, 4, 6, 8, and 24 hours after intravenous application and after 8, 15, 30, 45 minutes, 1, 2, 4, 6, 8, and 24 hours after p.o. administration and precipitated with ice-cold acetonitrile (1:5). Supernatants were analyzed via LC/MS-MS. Pharmacokinetic parameters were estimated from the plasma concentration data, e.g., using the log-linear trapezoidal rule for AUC estimation. Maximal plasma concentrations (Cmax) and time thereof (Tmax) were taken directly from the concentration time profiles.

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Animal efficacy studies [2]
For tumor xenograft studies, female athymic NMRI nu/nu mice (Taconic), 50 days old, average body weight 20 to 22 g, were used after an acclimatization period of 14 days. Feeding and drinking was ad libitum 24 hours per day. Human tumor cells derived from exponentially growing cell cultures were resuspended for A2780cis, NCI-H1299, and SUM-149 models in 100% Matrigel to a final concentration of 2 × 107, 3 × 107, or 5 × 107 cells/mL, respectively. Subcutaneous implants of 0.1 mL of 2 × 106 A2780cis, 3 × 106 NCI-H1299, or 5 × 106 SUM-149 cells were inoculated into the inguinal region of athymic mice. Tumor fragments of patient tumor explants MAXF 1384 or LU384, obtained from serial passage in nude mice, were cut into fragments of 4 to 5 mm diameter and transplanted subcutaneously into the flank of athymic mice. Tumor area (product of the longest diameter and its perpendicular), measured with a caliper, and body weight were determined two to three times a week. Tumor growth inhibition is presented as treatment/control ratio (T/C) calculated with tumor areas at the end of the study. Animal body weight was used as a measure for treatment-related toxicity. Body weight loss > 20% was dedicated as toxic. When tumors reached a size of approximately 20 to 40 mm², depending on growth of the tumor model, animals were randomized to treatment and control groups (8–10 mice/group) and treated p.o. with vehicle (70% PEG 400, 5 % ethanol, and 25% Solutol), Empesertib (BAY1161909), BAY 1217389, and/or paclitaxel, as indicated in Tables and Figure legends. In combination treatment groups, Mps1 inhibitor and paclitaxel were applied at the same day within a time frame of 1 hour. The treatment of each animal was based on individual body weight.

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In vivo mode of action studies [2]
For analysis of polyploidy and multinuclearity induction in vivo, A2780cis tumor–bearing female NMRI nude mice (see above) were treated with paclitaxel (intravenously once with 24 mg/kg), Empesertib (BAY1161909) (p.o. twice daily for 2 days with 2.5 mg/kg), and in combination with paclitaxel (i.v. once 24 mg/kg) and Empesertib (BAY1161909) (p.o. twice daily for 2 days 1 mg/kg). Treatment for all groups started at a tumor size of 60 mm² at day 14 after tumor cell inoculation. Tumor samples were prepared 4 and 8 hours after first Empesertib (BAY1161909) application at treatment day 1, as well as 4, 8, and 24 hours after first application of BAY 1161909 on treatment day 2. At each time point, 3 animals per treatment group were analyzed. Tumors were used for histologic examination after paraffin embedding and hematoxylin and eosin staining.

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In Vivo Pharmacokinetics in Rats and Dogs. [4]
All animal experiments were conducted in accordance with the German Animal Welfare Law and were approved by local authorities.For in vivopharmacokinetic experiments, test compounds were administered to male Wistar rats or female Beagle dogs intravenously at doses of 0.1 to 0.5 mg/kg and intragastrically at doses of 0.2 to 1.0 mg/kg formulated as solutions using solubilizers such as PEG400in well-tolerated amounts. Concerning iv administration, test compounds were given as bolus (rat) or 15 min infusion (dog). Blood samples were taken at various time points after dosing, usually up to 24 h. Depending on the expected half-life, additional samples were taken at later time points (e.g., 48h, 7h). Blood was collected into lithium heparin tubes and centrifuged at 3000rpm for 15min. An aliquot of 100μL from the supernatant (plasma) was taken and precipitated by the addition of 400μL of cold acetonitrile. Samples were frozen at –20°C overnight, and subsequently thawed and centrifuged at 3000rpm, 4°C for 20 S8min. Aliquots of the supernatants were analyzed using an Agilent 1200 HPLC system with MS/MS detection. PK parameters such as AUC and CL were calculated by non-compartmental analysis using PK calculation software.

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Animal [4]
For tumor xenograft studies, female athymic NMRI nu/nu mice (Taconic), 50 days old, average body weight 20–22 g, were used after an acclimatization period of 14 days. Feeding and drinking was ad libitum 24 hours per day. Human NCI-H1299 tumor cells derived from exponentially growing cell cultures were resuspended in 100% Matrigel to a final concentration of 3×107cells/mL and implanted subcutaneously in 0.1mL into the inguinal region of athymic NMRI nude mice. Tumor fragments of patient tumor explants MAXF 1384, obtained from serial passage in nude mice, were cut into fragments of 4–5mm diameter and transplanted subcutaneously into the flank of athymic NMRI nude mice. Tumor area (product of the longest diameter and its perpendicular), measured with a caliper, and body weight were determined 2–3 times per week. Tumor growth inhibition is presented as the treatment/control ratio (T/C), calculated with tumor areas at the end of the study. Animal body weight was used as a measure of treatment-related toxicity. Body weight loss >20% was considered toxic. When tumors reached a size of approximately 20–40 mm2, animals were randomized into treatment and control groups (8–10 mice/group), S9and treated twice daily for 2 days on and 5 days off orally (po) with vehicle (70% PEG 400, 5% EtOH, 25% Solutol), 41(Empesertib (BAY1161909)), 79(BAY 1217389), and/or once per week intravenously (iv) with paclitaxel, as indicated in figure 8. In combination treatment groups, MPS1 inhibitor and paclitaxel were applied on the same day within a time frame of 1 h. The treatment of each animal was based on individual body weight. Animals were euthanized according to the German Animal Welfare Guidelines. Data are expressed as tumor area means ± SD. Statistical analysis included one-way ANOVA and differences were compared versus the control group by a pairwise comparison procedure using SigmaStat software

In vivo pharmacokinetic parameters [2]
Pharmacokinetic parameters were determined in mouse and rat. Following intravenous administration of BAY 1161909 as bolus of 0.5 mg/kg to male CD1 mouse and 0.5 mg/kg to male Wistar rat, the compound exhibited low blood clearance. The volume of distribution (Vss) was high in both species and terminal half-lives were long. After oral administration of 1 mg/kg to female NMRI mouse and 0.5 mg/kg to male Wistar rat, peak plasma levels were reached after 4 hours. The oral bioavailability was moderate in mouse and rat (Table 1). BAY 1217389 was administered intravenously as bolus of 1.0 and 0.5 mg/kg to female CD1 mouse and male Wistar rat, respectively. Blood clearance was found to be low in the tested species. Vss was high and terminal half-lives were long. BAY 1217389 was administered orally to female NMRI mouse (1 mg/kg) and male Wistar rat (0.5 mg/kg). Peak plasma concentrations were observed between 1.5 and 7 hours. Oral bioavailability was high in rat and moderate in mouse (Table 1). Our data demonstrate that we have identified two novel Mps1 kinase inhibitors with a favorable pharmacokinetic profile, supporting further development for clinical application.

[1]. PRODRUG DERIVATIVES OF SUBSTITUTED TRIAZOLOPYRIDINES. WO2014198647A2.

[2]. Novel Mps1 Kinase Inhibitors with Potent Antitumor Activity. Mol Cancer Ther. 2016 Apr;15(4):583-92.

[3]. Target Residence Time-Guided Optimization on TTK Kinase Results in Inhibitors with Potent Anti-Proliferative Activity. J Mol Biol. 2017 Jul 7;429(14):2211-2230.

[4]. Treating Cancer by Spindle Assembly Checkpoint Abrogation: Discovery of Two Clinical Candidates, BAY 1161909 and BAY 1217389, Targeting MPS1 Kinase. J Med Chem. 2020 Aug 13;63(15):8025-8042.

Empesertib is an orally bioavailable, selective inhibitor of the serine/threonine monopolar spindle 1 (Mps1) kinase, with potential antineoplastic activity. Upon administration, empesertib binds to and inhibits the activity of Mps1. This causes inactivation of the spindle assembly checkpoint (SAC), accelerated mitosis, chromosomal misalignment, chromosomal missegregation, mitotic checkpoint complex destabilization, and increased aneuploidy. This leads to the induction of cell death in cancer cells overexpressing Mps1. Mps1, a kinase expressed in proliferating normal tissues and aberrantly overexpressed in a wide range of human tumors, is activated during mitosis and is essential for SAC functioning and controls chromosome alignment.

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Monopolar spindle 1 (Mps1) has been shown to function as the key kinase that activates the spindle assembly checkpoint (SAC) to secure proper distribution of chromosomes to daughter cells. Here, we report the structure and functional characterization of two novel selective Mps1 inhibitors, BAY 1161909 and BAY 1217389, derived from structurally distinct chemical classes. BAY 1161909 and BAY 1217389 inhibited Mps1 kinase activity with IC50 values below 10 nmol/L while showing an excellent selectivity profile. In cellular mechanistic assays, both Mps1 inhibitors abrogated nocodazole-induced SAC activity and induced premature exit from mitosis ("mitotic breakthrough"), resulting in multinuclearity and tumor cell death. Both compounds efficiently inhibited tumor cell proliferation in vitro (IC50 nmol/L range). In vivo, BAY 1161909 and BAY 1217389 achieved moderate efficacy in monotherapy in tumor xenograft studies. However, in line with its unique mode of action, when combined with paclitaxel, low doses of Mps1 inhibitor reduced paclitaxel-induced mitotic arrest by the weakening of SAC activity. As a result, combination therapy strongly improved efficacy over paclitaxel or Mps1 inhibitor monotreatment at the respective MTDs in a broad range of xenograft models, including those showing acquired or intrinsic paclitaxel resistance. Both Mps1 inhibitors showed good tolerability without adding toxicity to paclitaxel monotherapy. These preclinical findings validate the innovative concept of SAC abrogation for cancer therapy and justify clinical proof-of-concept studies evaluating the Mps1 inhibitors BAY 1161909 and BAY 1217389 in combination with antimitotic cancer drugs to enhance their efficacy and potentially overcome resistance. [2]

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The protein kinase threonine tyrosine kinase (TTK; also known as Mps1) is a critical component of the spindle assembly checkpoint and a promising drug target for the treatment of aggressive cancers, such as triple negative breast cancer. While the first TTK inhibitors have entered clinical trials, little is known about how the inhibition of TTK with small-molecule compounds affects cellular activity. We studied the selective TTK inhibitor NTRC 0066-0, which was developed in our own laboratory, together with 11 TTK inhibitors developed by other companies, including Mps-BAY2b, BAY 1161909, BAY 1217389 (Bayer), TC-Mps1-12 (Shionogi), and MPI-0479605 (Myrexis). Parallel testing shows that the cellular activity of these TTK inhibitors correlates with their binding affinity to TTK and, more strongly, with target residence time. TTK inhibitors are therefore an example where target residence time determines activity in in vitro cellular assays. X-ray structures and thermal stability experiments reveal that the most potent compounds induce a shift of the glycine-rich loop as a result of binding to the catalytic lysine at position 553. This "lysine trap" disrupts the catalytic machinery. Based on these insights, we developed TTK inhibitors, based on a (5,6-dihydro)pyrimido[4,5-e]indolizine scaffold, with longer target residence times, which further exploit an allosteric pocket surrounding Lys553. Their binding mode is new for kinase inhibitors and can be classified as hybrid Type I/Type III. These inhibitors have very potent anti-proliferative activity that rivals classic cytotoxic therapy. Our findings will open up new avenues for more applications for TTK inhibitors in cancer treatment. [3]

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Inhibition of monopolar spindle 1 (MPS1) kinase represents a novel approach to cancer treatment: instead of arresting the cell cycle in tumor cells, cells are driven into mitosis irrespective of DNA damage and unattached/misattached chromosomes, resulting in aneuploidy and cell death. Starting points for our optimization efforts with the goal to identify MPS1 inhibitors were two HTS hits from the distinct chemical series "triazolopyridines" and "imidazopyrazines". The major initial issue of the triazolopyridine series was the moderate potency of the HTS hits. The imidazopyrazine series displayed more than 10-fold higher potencies; however, in the early project phase, this series suffered from poor metabolic stability. Here, we outline the evolution of the two hit series to clinical candidates BAY 1161909 and BAY 1217389 and reveal how both clinical candidates bind to the ATP site of MPS1 kinase, while addressing different pockets utilizing different binding interactions, along with their synthesis and preclinical characterization in selected in vivo efficacy models.[4]

C29H26FN5O4S

559.61

559.168

C, 62.24; H, 4.68; F, 3.39; N, 12.51; O, 11.44; S, 5.73

1443763-60-7

1443763-60-7;2170218-39-8;2170218-40-1 (tosylate hydrate);

71599640

White to off-white solid powder

1.4±0.1 g/cm3

1.658

4.44

2

8

8

40

951

1

C1(F)=CC=C(C=C1)[C@@H](C)C(NC1C=CC(C2C=CC3N(C=2)N=C(NC2C=CC(S(C)(=O)=O)=CC=2OC)N=3)=CC=1)=O

NRJKIOCCERLIDG-GOSISDBHSA-N

InChI=1S/C29H26FN5O4S/c1-18(19-4-9-22(30)10-5-19)28(36)31-23-11-6-20(7-12-23)21-8-15-27-33-29(34-35(27)17-21)32-25-14-13-24(40(3,37)38)16-26(25)39-2/h4-18H,1-3H3,(H,31,36)(H,32,34)/t18-/m1/s1

(2R)-2-(4-fluorophenyl)-N-[4-[2-(2-methoxy-4-methylsulfonylanilino)-[1,2,4]triazolo[1,5-a]pyridin-6-yl]phenyl]propanamide

Empesertib; Mps1-IN-5; BAY1161909; 1443763-60-7; Mps1-IN5; BAY 1161,909; BAY-1161,909; BAY1161,909; Empesertib [INN]; Empesertib [WHO-DD]; BAY-1161909; BAY 1161909

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: ≥ 35 mg/mL (~62.5 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.47 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 2: 2.5 mg/mL (4.47 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 25.0 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: ≥ 2.5 mg/mL (4.47 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.)