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]

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Five cell lines were sensitive to AM-0277, AM-1882 and AM-9022  with mean half-maximum effective concentration (EC50) values of 0.047 µM, 0.021 µM and 0.045 µM, respectively. [1]

Oral candidate AM-9022 dosed at 10 mg per kg achieved high plasma exposure in mice and showed double-digit nanomolar potency in the OVCAR-3 pH3 mitotic marker assay (Extended Data Fig. ​Fig.8a).8a). AM-9022  had similar inhibitory effects on the motor activity of human and mouse KIF18A。[1]

AM-9022 increased pH3 mitotic marker levels in OVCAR-3 tumors by 3.4-fold (P value = 0.042), with higher compound exposure in tumors relative to plasma (Fig. ​(Fig.6a).6a). To evaluate the efficacy and tolerability of our oral candidate, mice with established OVCAR-3 tumors were administered vehicle or AM-9022 at 30 mg per kg daily for 18 d. Promisingly, AM-9022 inhibited tumor growth (P values = 1.24 × 10−130), with evidence of TR (95% TR, six of ten tumor-free mice) and no body weight loss (Fig. ​(Fig.6b).6b). At the end of the study, the plasma AUC value for AM-9022 was 53 µM ·h.[1]

Mice with established JIMT-1 tumors were administered vehicle or AM-9022 at 30 and 100 mg per kg daily for 21 d. AM-9022  administered at 30 and 100 mg per kg inhibited tumor growth (P values ≤ 5.1 × 10−13) with evidence of TR (16% and 94% TR, respectively) at well-tolerated doses (Fig. ​(Fig.6c).6c). At the end of the study, the plasma AUC value for AM-9022 at 30 mg per kg was 3.7-fold higher in the JIMT-1 model relative to the OVCAR-3 model.[1]

Mice with established PDX tumors were administered vehicle or AM-9022 at 60 mg per kg daily for ≥28 d. The duration of treatment beyond 28 d depended on the level of anti-cancer activity and whether the tumors reached a predetermined size cutoff. We included an observation phase after treatment cessation for CTG-0017 and CTG-0437 models to assess durability and tumor regrowth. The pattern of sensitivity to AM-9022  treatment varied across the TNBC PDX panel (Fig. ​(Fig.6d).6d). Notably, AM-9022 inhibited tumor growth in the CTG-0017 model (P value = 6.5 × 10−164) with evidence of TR (83% TR), resulting in 90% tumor-free mice by day 58. AM-9022 inhibited tumor growth in the CTG-0437 model (101% TGI, P value = 2.2 × 10−101). AM-9022 was less effective in the CTG-0888 model (63% TGI, P value = 5.1 × 10−31) and had no anti-cancer effects in the CTG-1019 model (Fig. ​(Fig.6d).6d). As before, inhibition of KIF18A activity with AM-9022 was well tolerated by the mice (Fig. ​(Fig.6d).6d). Together, these data show that the oral candidate AM-9022 has significant anti-cancer effects in five of six human breast and ovarian tumor models at well-tolerated doses, resulting in TR or stasis in OVCAR-3, JIMT-1, CTG-0017 and CTG-0437 models.[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.

Cell growth assays (compound) Cell growth assays were performed as described previously28. Cell lines (n = 11) were treated for 96 h with DMSO or AM-0277, AM-1882, AM-9022  and palbociclib (maximum concentration of 6 µM) or ispinesib (maximum concentration of 0.6 µM) over a 19-point or 17-point concentration range. Data are from two independent experiments in duplicate. Imaging data were collected from the same number of fields per well. The count POC value was computed using the formula (count POC = (compound-treated nuclear count) ÷ (DMSO-treated nuclear count) × 100). If the maximal response was <50% at 6 µM, the cell line was considered insensitive. A mean count EC50 value was determined for the sensitive cell line group. For OVCAR-8 and OVCAR-8 ADRRES cell growth analysis, cells were treated for 96 h with AM-1882 and AM-9022 (maximum concentration of 6 µM) or paclitaxel and doxorubicin (maximum concentration of 1 µM) over a 19-point concentration range, with or without P-gp-inhibitor GF120918 (1 µM). OVCAR-8 cell lines were evaluated with or without P-gp inhibitor in two independent experiments. Data were graphed as a concentration–response profile with corresponding count EC50 values.[1]

OVCAR-3 tumor PD (pH3 immunoassay) 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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Cell line-derived xenograft tumor model efficacy (p.o. dosing) 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 p.o. with vehicle or AM-9022  at 30 mg per kg daily for 18 consecutive days. After the final dose on day 45, plasma PK analysis was performed as described above. Mice with no measurable tumor on day 45 were classified as tumor free. Mice were injected with JIMT-1 cells (1.0 × 107) subcutaneously in the right flank. Animals were randomized into treatment groups (n = 10 mice per group) based on equivalent tumor size and dosed p.o. with vehicle or AM-9022 (30 or 100 mg per kg) daily for 21 consecutive days. After the final dose on day 40, plasma PK analysis was performed as described above. Mice with no measurable tumor on day 40 were classified as tumor free.[1]

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Patient-derived xenograft tumor model efficacy (p.o. dosing) Mice were implanted with low-passage PDX tumor fragments from each TNBC model (CTG-0017, CTG-0437, CTG-0888 and CTG-1019; details are in Supplementary Table 6). After tumor size reached 1,000–1,500 mm3, tumors were collected and tumor fragments were implanted subcutaneously in the left flank. Animals were assigned into treatment groups (n = 10 mice per group) based on equivalent tumor size and dosed p.o. with vehicle or AM-9022  (60 mg per kg) daily for ≥27 consecutive days. Study termination was set on a mean tumor size of 1,500 mm3 for the control group; dosing continued beyond day 28 if the tumor size threshold was not reached. CTG-0017 study dosing terminated on day 27, followed by a drug-free observation phase to day 58. CTG-0437 study dosing terminated on day 27, followed by a drug-free observation phase to day 34. Dosing on CTG-0888 and CTG-1019 terminated on day 43 (last measurement on day 41) and on day 52 (last measurement on day 51), respectively. Mice with no measurable tumor were classified as tumor free.[1]

[1].Small-molecule inhibition of kinesin KIF18A reveals a mitotic vulnerability enriched in chromosomally unstable cancers. Nat Cancer. 2024 Jan;5(1):66-84.

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]

C27H36F2N6O4S

578.67

578.24868

2446872-46-2

148785380

Typically exists as solid at room temperature

4.2

OLCRYQGHHMGOJZ-LJQANCHMSA-N

InChI=1S/C27H36F2N6O4S/c1-18-15-23(32-25(30-18)35-13-9-27(28,29)10-14-35)31-24(37)21-4-3-20(33-40(38,39)19(2)17-36)16-22(21)34-11-7-26(5-6-26)8-12-34/h3-4,15-16,19,33,36H,5-14,17H2,1-2H3,(H,30,31,32,37)/t19-/m1/s1

2-(6-azaspiro[2.5]octan-6-yl)-N-[2-(4,4-difluoropiperidin-1-yl)-6-methylpyrimidin-4-yl]-4-[[(2R)-1-hydroxypropan-2-yl]sulfonylamino]benzamide

AM-9022; CHEMBL5071728; SCHEMBL22119488; OLCRYQGHHMGOJZ-LJQANCHMSA-N; BDBM535533; EX-A8980; US11236069, Example 5-1;

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)

Typically soluble in DMSO (e.g. 10 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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