KIF18A

In this study, researchers screened a small-molecule library of diverse compounds for selective inhibitors of KIF18A MT-ATPase motor activity. Two compound hits were discovered that phenocopied the effects of KIF18A KD in cells; these hits are structurally distinct from the KIF18A inhibitor BTB-1 (refs. 27,28). Next, researchers initiated a medicinal chemistry campaign to optimize the promising hit, compound 3 (AM-7710)28; structure–activity relationship (SAR) efforts led to a quartet of promising series analogs representing early SAR leads (AM-0277, AM-1882) and late SAR leads (AM-5308 , AM-9022 ) (Fig. ​(Fig.2a).2a). All four compounds showed a significant improvement in KIF18A-inhibitory activity and cell potency relative to AM-7710 and exhibited good specificity against a panel of diverse kinesin motor proteins, except for the KIF19A motor (Fig. ​(Fig.2b2b and Extended Data Fig. 2a–c). [1]

To examine the effects of KIF18A inhibition in vivo, researchers selected AM-1882 and AM-5308  based on their acceptable plasma exposures achieved by intraperitoneal (i.p.) dosing in rodents and their double-digit nanomolar potency in the OVCAR-3 pH3 mitotic marker assay (Extended Data Fig. ​Fig.7a).7a). Researchers confirmed that AM-1882 and AM-5308  have similar inhibitory effects on the motor activity of human and mouse KIF18A, which share 90% amino acid identity in their motor domains (Extended Data Fig. 7b,c). This allows us to evaluate KIF18A-inhibitor tolerability in mice.[1]

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To investigate the pharmacodynamic (PD) effects of our KIF18A inhibitors, mice with established OVCAR-3 cell line-derived xenograft (CDX) tumors were administered vehicle, AM-1882 at 100 mg per kg or AM-5308  at 50 mg per kg. Tumor and blood samples were collected 24 h after treatment for pH3 PD and pharmacokinetic (PK) analysis. AM-1882 and AM-5308 increased pH3 mitotic marker levels in OVCAR-3 tumors by 5.9-fold (P value = 0.0068) and 7.1-fold (P value = 0.0022), respectively (Fig. ​(Fig.5a).5a).AM-5308  exposure was higher in the tumor relative to plasma, whereas AM-1882 exposure was similar in both. Next, Researchers selected AM-5308 for tumor PD assessment by imaging. Mice with established OVCAR-3 tumors were administered vehicle or AM-5308 at 25 mg per kg for 2 d. AM-5308 increased pH3 mitotic marker counts in OVCAR-3 tumors by 12.7-fold (P value = 0.037), with evidence of abnormal mitotic cell features. [1]

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To establish whether the robust PD effects observed with our KIF18A inhibitors would result in efficacy, mice with established OVCAR-3 tumors were administered vehicle, AM-1882 at 100 mg per kg or AM-5308  at 25 mg per kg daily for 18 d. As a positive control, mice were administered docetaxel at 20 mg per kg once weekly. AM-1882 and AM-5308 inhibited tumor growth (P values ≤ 1.3 × 10−89) with evidence of tumor regression (TR; 73% and 46%, respectively) (Fig. ​(Fig.5c).5c). Treatment with docetaxel resulted in 74% tumor growth inhibition (TGI) (P value = 8.8 × 10−41). Our KIF18A inhibitors were well tolerated by the mice with no changes in body weight or blood counts (Fig. ​(Fig.5c5c and Extended Data Fig. ​Fig.7d).7d). By contrast, docetaxel decreased neutrophil counts (P value = 5.0 × 10−4). At the end of the study, plasma AUC values for AM-1882 and AM-5308 were 123 and 45 µM·h, respectively.[1]

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To further examine the in vivo activity of our KIF18A inhibitors, we evaluated the OVCAR-8 CDX tumor model. Mice with established tumors were administered vehicle, AM-1882 at 50 or 100 mg per kg or AM-5308  at 25 or 50 mg per kg daily for 18 d. AM-1882 and AM-5308 inhibited tumor growth (P values ≤ 1.7 × 10−61) with evidence of TR (16% or 73% TR and 19% or 75% TR, respectively) (Fig. ​(Fig.5e).5e). As before, inhibition of KIF18A was well tolerated by the mice (Fig. ​(Fig.5e).5e). The plasma AUC value for AM-5308 at 25 mg per kg was 2.6-fold higher in the OVCAR-8 study, while AM-1882 showed similar exposures across CDX studies (Extended Data Fig. ​Fig.7e).7e). After treatment cessation, researchers monitored the mice to determine the durability of treatment and the timing of tumor regrowth. The OVCAR-8 tumors that regressed on KIF18A-inhibitor treatment showed a delayed resumption in growth, except for in one animal in the AM-5308 group.[1]

Kinesin motor assays[1]
ADP-Glo motor assays Motor activity was assessed with the ADP-Glo luminescence assay (Promega) using assay conditions as described previously. Recombinant truncated motor proteins were expressed and purified (hKIF18A (1–467, 4 nM), mKIF18A (1–467, 4 nM), hKIF19A (1–463, 32 or 100 nM), hKIF18B (1–436, 8 nM), hKIFC1 (266–673, 4 nM)) or procured (hEG5, 4 nM; hCENP-E, 8 nM). Compounds were assessed with 30 µM ATP and 30 µg ml−1 MTs and the motor protein concentrations indicated above; data are from two or four independent experiments. KIF18A compounds were assessed with or without MTs (0 or 30 µg ml−1) with hKIF18A (160 nM); data are from two independent experiments in duplicate. KIF18A compounds were assessed with 30 or 300 µM ATP and 5 or 80 µg ml−1 MTs for hKIF18A (4 nM); data are from one experiment. AM-7710-series analog MT-ATPase IC50 values were obtained from the Genedata Screener datastore at Amgen (Extended Data Fig. ​Fig.2a2a).

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Enzyme-linked inorganic phosphate motor assays[1]
KIF18A compounds (1 µM) were assessed against a panel of motor proteins (hCENP-E, hEG5, hKIFC3, hKIF3C, human chromokinesin, hMCAK, hMKLP1, hMKLP2) using an enzyme-linked inorganic phosphate assay according to the manufacturer’s protocol and as described previously. Data are from one or two independent experiments in duplicate or triplicate. Studies were conducted by Cytoskeleton.[1]

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Kinome binding assay[1]
KIF18A compounds (1 µM) were assessed against a panel of kinases (n = 96) using a competition binding assay as described previously. Data are from one experiment. Studies were conducted by Eurofins DiscoverX.[1]

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Tubulin-polymerization assays[1]
The fluorescence-based tubulin-polymerization assay was performed according to the manufacturer’s protocol (Cytoskeleton) with DMSO, KIF18A compounds (10 µM), paclitaxel (5 µM) and nocodazole (5 µM). Tubulin polymerization was measured using the SpectraMax M5 plate reader (Molecular Devices) set to detect at 440 nm with one measurement per minute for 90 min at 37 °C. Data are from one or three independent experiments. Data are graphed as mean fluorescence intensity versus time with corresponding AUC values.

Pericentrin and α-tubulin staining (compound) [1]
MDA-MB-157 cells were seeded in two-well glass chamber slides and cultured for 2 d. Cells were treated for 24 h with DMSO, AM-1882 (0.2 µM) or AM-5308  (0.5 µM). Fixation, staining and imaging were performed as described previously28. Data are from one experiment. Representative images were captured for each treatment condition.[1]

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Cell cycle and cell growth assays (compound) [1]
Human bone marrow mononuclear cells from four normal donors were expanded for 8 d as described previously57. Cells were seeded in 24-well plates and treated with DMSO or KIF18A compounds (1 µM), ispinesib (0.05 µM), paclitaxel (0.1 µM) and palbociclib (1 µM). AM-5308 and AM-9022 were assessed in two donors; the other test compounds were assessed in four donors in independent experiments. Separate plates were collected at 48 h (cell cycle) or 96 h (cell growth). The first set of plates was pulsed with BrdU for 2 h and processed as described previously57. Cells were analyzed with a BD LSRFortessa flow cytometer running FACSDiva software, and post-acquisition analysis was performed with FSC Express software. Cells were collected and counted from the second set of plates with the Vi-CELL XR Analyzer. Data were graphed for cell cycle (BrdU, sub-G1) and cell growth (count). Statistical significance was determined for each group relative to the DMSO control by one-way ANOVA at a significance level of 0.05, followed by Dunnett’s multiplicity adjustment.

OVCAR-3 tumor PD (pH3 immunoassay)[1]
Tumor PD assays were performed as described previously28. Animals were randomized into treatment groups (n = 3 mice per group) based on similar tumor size and dosed with vehicle (i.p. or p.o.), AM-1882 (100 mg per kg, i.p.), AM-5308  (50 mg per kg, i.p.) or AM-9022 (30 mg per kg, p.o.). Tumor and blood plasma were collected 24 h after treatment and processed for PD (pH3) or PK (plasma, tumor) analysis. Data were graphed for tumor PD, plasma PK and tumor PK. Statistical significance was determined for AM-1882 and AM-5308  relative to vehicle by one-way ANOVA at a significance level of 0.05 with Dunnett’s multiplicity adjustment and for AM-9022 relative to vehicle by two-tailed t-test at a significance level of 0.05 with Welch’s correction.[1]

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OVCAR-3 tumor PD (pH3 imaging) [1]
Mice were injected with OVCAR-3 cells (5.0 × 106) subcutaneously in the right flank. Animals were randomized into treatment groups (n = 3 mice per group) based on similar tumor size and dosed with vehicle or AM-5308  (25 mg per kg, i.p.) for 2 consecutive days. Tumors were collected 24 h after treatment and processed for tumor imaging analysis. Formalin-fixed paraffin-embedded tumors were sectioned onto glass slides and deparaffinized, rehydrated and treated with citrate buffer and heat for antigen retrieval (Reveal Decloaker, Biocare Medical). Slides were blocked and stained in wash buffer with anti-α-tubulin (T6199, Sigma) and anti-pH3 (06-570, Millipore) antibodies overnight at 4 °C. Slides were washed twice and stained with secondary antibodies (anti-mouse IgG Alexa Fluor 488 (A11029, Invitrogen), anti-rabbit IgG Alexa Fluor 647 (A-21244, Invitrogen)) for 2 h at room temperature. Slides were washed twice and counterstained with DAPI. ProLong antifade was added before mounting the coverslips. Slides were imaged with a confocal UltraVIEW VoX fluorescence microscope running Volocity software (PerkinElmer). A primary scan was performed with a ×20 objective to select three regions of interest per tumor followed by enumerating pH3+ counts per area for each region. Representative maximum projection images were captured with a ×60 objective for DNA, α-tubulin and pH3 channels. Data were graphed for tumor PD. Statistical significance was determined for AM-5308  relative to the vehicle by unpaired two-tailed t-test at a significance level of 0.05 with Welch’s correction.[1]

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Cell line-derived xenograft tumor model efficacy (intraperitoneal dosing) [1]
Mice were injected with OVCAR-3 cells (5.0 × 106) subcutaneously in the right flank. Animals were randomized into treatment groups (n = 10 mice per group) based on equivalent tumor size and dosed i.p. with vehicle, AM-1882 (100 mg per kg) or AM-5308  (25 mg per kg) daily for 18 consecutive days or weekly with docetaxel (20 mg per kg). Plasma PK analysis was performed at 2, 4, 8, 16 and 24 h (n = 2 mice per time point). After the final dose on day 42, samples were collected for mouse blood count analysis (n = 6 mice per treatment group) by IDEXX BioResearch; platelet counts were not reported due to technical processing issues. Data were graphed for mouse blood counts (neutrophils, reticulocytes, red blood cells, lymphocytes and white blood cells). Statistical significance was determined for treatment groups relative to the vehicle by one-way ANOVA at a significance level of 0.05 with Dunnett’s multiplicity adjustment. Mice were injected with CAL-51 cells (5.0 × 106) subcutaneously in the right flank. Animals were randomized into treatment groups (n = 10 mice per group) based on equivalent tumor size and dosed i.p. with vehicle, AM-1882 (100 mg per kg) or AM-5308  (25 mg per kg) daily for 18 consecutive days or twice weekly with gemcitabine (120 mg per kg). After the final dose on day 36, plasma PK analysis was performed as described above. Mice were injected with OVCAR-8 cells (5.0 × 106) subcutaneously in the right flank. Animals were randomized into treatment groups (n = 10 mice per group) based on equivalent tumor size and dosed i.p. with vehicle, AM-1882 (50 or 100 mg per kg) or AM-5308  (25 or 50 mg per kg) daily for 18 consecutive days. After the final dose on day 46, blood was obtained by the retro-orbital method, and plasma PK analysis was performed as described above. After treatment cessation, tumor volumes and body weights were recorded until day 81. Mice with no measurable tumor on day 81 were classified as tumor free.[1]

[1]. Kif18a inhibitors for treatment of neoplastic diseases. WO2021211549A1.

Chromosomal instability (CIN) is a hallmark of cancer, caused by persistent errors in chromosome segregation during mitosis. Aggressive cancers like high-grade serous ovarian cancer (HGSOC) and triple-negative breast cancer (TNBC) have a high frequency of CIN and TP53 mutations. Here, we show that inhibitors of the KIF18A motor protein activate the mitotic checkpoint and selectively kill chromosomally unstable cancer cells. Sensitivity to KIF18A inhibition is enriched in TP53-mutant HGSOC and TNBC cell lines with CIN features, including in a subset of CCNE1-amplified, CDK4-CDK6-inhibitor-resistant and BRCA1-altered cell line models. Our KIF18A inhibitors have minimal detrimental effects on human bone marrow cells in culture, distinct from other anti-mitotic agents. In mice, inhibition of KIF18A leads to robust anti-cancer effects with tumor regression observed in human HGSOC and TNBC models at well-tolerated doses. Collectively, our results provide a rational therapeutic strategy for selective targeting of CIN cancers via KIF18A inhibition.[1]

C26H35N5O5S

529.65

529.2358

2410796-89-1

153625074

White to light yellow solid powder

3

3

9

8

37

884

1

C(NC1=NC(N2CCO[C@H](C)C2)=CC=C1)(=O)C1=CC=C(NS(CCO)(=O)=O)C=C1N1CCC2(CC2)CC1

IUDCFCQNVQWCCI-LJQANCHMSA-N

InChI=1S/C26H35N5O5S/c1-19-18-31(13-15-36-19)24-4-2-3-23(27-24)28-25(33)21-6-5-20(29-37(34,35)16-14-32)17-22(21)30-11-9-26(7-8-26)10-12-30/h2-6,17,19,29,32H,7-16,18H2,1H3,(H,27,28,33)/t19-/m1/s1

2-(6-azaspiro[2.5]octan-6-yl)-4-(2-hydroxyethylsulfonylamino)-N-[6-[(2R)-2-methylmorpholin-4-yl]pyridin-2-yl]benzamide

AM-5308; 2410796-89-1; CHEMBL5085449; SCHEMBL22152106; (R)-AM-5308;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~188.80 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.72 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 (4.72 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.)