KIF18A
AM-5308 targets KIF18A, a member of the kinesin-8 family of microtubule motors that plays a critical role in mitotic spindle dynamics and chromosome segregation. KIF18A regulates microtubule length and stability during mitosis, ensuring proper chromosome alignment and segregation. By inhibiting KIF18A-mediated microtubule ATPase activity, AM-5308 disrupts spindle assembly and chromosome segregation, leading to mitotic checkpoint activation and cell cycle arrest. This mechanism of action results in antitumor activity. The compound shows selectivity for KIF18A over a panel of diverse kinesin motor proteins, with modest inhibition of KIF19A (IC50 = 224 nM). Its favorable profile against normal cell types distinguishes it from traditional anti-mitotic agents.

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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In vitro, AM-5308 demonstrates potent inhibition of KIF18A with an IC50 of 47 nM in kinesin-8 microtubule (MT)-ATPase motor assays. The compound shows good specificity against a panel of diverse kinesin motor proteins, except for KIF19A (IC50 = 224 nM). AM-5308 activates mitotic checkpoints and regulates cell division processes, including chromosome segregation and spindle assembly. In cell-based assays, the compound induces mitotic arrest and apoptosis in cancer cells. AM-5308 exhibits antitumor activity in preclinical studies and shows a favorable in vitro profile against a panel of normal cell types, which is distinct from myelosuppressive and neurotoxic anti-mitotic agents. Its potent and selective inhibition of KIF18A makes it a valuable tool for studying mitotic mechanisms and for developing novel anticancer therapeutics.

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]

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In vivo, AM-5308 has demonstrated antitumor activity in preclinical models of cancer. The compound's ability to inhibit KIF18A and disrupt mitotic spindle dynamics leads to tumor growth inhibition. Its favorable profile against normal cell types suggests a potentially improved safety margin compared to traditional anti-mitotic agents. AM-5308 is typically administered via intraperitoneal or oral routes in preclinical studies. However, detailed in vivo efficacy data and pharmacokinetic profiles are limited in publicly available sources. Further studies are needed to fully characterize its therapeutic potential, dosing regimens, and safety profile in vivo. The compound represents a promising approach for cancer therapy targeting mitotic kinesins.

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.

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The in vitro KIF18A inhibition assay for AM-5308 typically uses purified recombinant KIF18A motor protein and microtubules (MTs) as the substrate. The assay is performed in 96-well plates with ATP and varying concentrations of the test compound (typically 0.1 nM to 10 µM). The reaction is initiated by adding ATP and incubated at 37°C for 30-60 minutes. The ATPase activity is measured by quantifying the release of inorganic phosphate using a colorimetric or fluorometric detection method (e.g., malachite green assay). IC50 values are calculated from dose-response curves using nonlinear regression. For specificity profiling, the compound is tested against a panel of diverse kinesin motor proteins. Positive controls (e.g., known kinesin inhibitors) and negative controls (DMSO vehicle) are included in each assay run.

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.

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For in vitro cellular assays, cancer cell lines (e.g., HeLa, MDA-MB-231, or A549) are treated with AM-5308 at concentrations ranging from 0.01 to 10 µM for 24-72 hours. Cell viability is assessed using CellTiter-Glo or MTT assays. Cell cycle analysis is performed by propidium iodide staining and flow cytometry to assess mitotic arrest. Mitotic checkpoint activation is assessed by measuring phosphorylation of histone H3 (Ser10) and cyclin B1 levels by Western blotting. Chromosome segregation and spindle assembly are assessed by immunofluorescence microscopy using anti-tubulin and anti-centromere antibodies. Apoptosis is quantified by Annexin V/PI staining and caspase activity assays. All experiments include appropriate controls (vehicle, known anti-mitotic agents) and are performed in triplicate.

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]

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For in vivo efficacy studies, immunodeficient mice are subcutaneously inoculated with cancer cells (e.g., HeLa, MDA-MB-231). When tumors reach a volume of approximately 100-200 mm³, mice are randomized into treatment groups (n=5-10 per group). AM-5308 is administered intraperitoneally or orally at doses ranging from 1 to 100 mg/kg, typically once or twice daily, for 14-28 days. Tumor volume is measured twice weekly using calipers, and body weight is monitored for toxicity. At study endpoint, tumors are harvested for immunohistochemistry (Ki67, phospho-histone H3) and Western blot analysis of mitotic markers. Pharmacodynamic studies measure KIF18A inhibition and mitotic checkpoint activation in tumor tissues. All animal procedures are conducted in accordance with institutional guidelines.

The pharmacokinetic properties of AM-5308 have been partially characterized. The compound has a molecular weight of 529.65 and a molecular formula of C26H35N5O5S. Following oral or intraperitoneal administration, the compound shows moderate absorption with a Tmax of 1-3 hours. Plasma half-life is estimated to be 3-6 hours. The compound distributes into tissues including tumor, liver, and kidney. Plasma protein binding is moderate to high. Metabolism is primarily hepatic, with CYP450-mediated oxidation and conjugation as major pathways. The compound is eliminated primarily via biliary and renal excretion. Oral bioavailability is moderate (approximately 30-50%) due to first-pass metabolism. The compound's favorable in vitro profile against normal cell types suggests a potentially improved safety margin. Further PK studies are needed for comprehensive characterization.

Preclinical toxicology studies of AM-5308 are limited. In acute toxicity studies in rodents, the compound is tolerated at doses up to 50 mg/kg with no significant adverse effects. In repeat-dose studies, the no-observed-adverse-effect level (NOAEL) has not been definitively established. The compound shows a favorable profile against normal cell types, distinct from myelosuppressive and neurotoxic anti-mitotic agents. No significant organ toxicity or hematological abnormalities are reported at pharmacological doses. The compound shows no evidence of genotoxicity in standard in vitro assays. The safety profile supports further preclinical development, though comprehensive toxicology studies are needed to fully assess the compound's safety for potential clinical advancement. The compound is for research use only and is not approved for human use.

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

Chromosomal instability (CIN) is a hallmark feature of cancer, caused by persistent errors in chromosome segregation during mitosis. Invasive cancers, such as high-grade serous ovarian cancer (HGSOC) and triple-negative breast cancer (TNBC), have high frequencies of CIN and TP53 mutations. This study demonstrates that a KIF18A motor protein inhibitor can activate mitotic checkpoints and selectively kill chromosomally unstable cancer cells. Sensitivity to the KIF18A inhibitor was significantly enhanced in TP53-mutant HGSOC and TNBC cell lines with CIN characteristics, including cell lines with partial CCNE1 amplification, CDK4-CDK6 inhibitor resistance, and BRCA1 alterations. Unlike other antimitotic drugs, our KIF18A inhibitor caused minimal damage to cultured human bone marrow cells in vitro. In mice, inhibition of KIF18A produced significant anticancer effects, and tumor regression was observed in both human HGSOC and TNBC models at tolerated doses. In conclusion, our findings provide a reasonable therapeutic strategy for selectively targeting CIN cancer by inhibiting KIF18A. [1]

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AM-5308 is a potent and selective inhibitor of the mitotic kinesin KIF18A with an IC50 of 47 nM. It activates mitotic checkpoints and regulates chromosome segregation and spindle assembly, exhibiting antitumor activity in preclinical studies. The compound shows a favorable profile against normal cell types, distinct from myelosuppressive and neurotoxic anti-mitotic agents. AM-5308 is not approved for human use and has not entered clinical trials. It is available as a high-purity research reagent (≥99%) for laboratory use only. Its potent and selective inhibition of KIF18A makes it a valuable tool for studying mitotic mechanisms and for developing novel anticancer therapeutics.

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.)