AM-9022

KIF18A

在这项研究中，研究人员筛选了多种化合物的小分子文库，用于选择性抑制KIF18A mt - atp酶运动活性。在细胞中发现了两个化合物hit，它们表型复制了KIF18A KD的作用;这些hit在结构上不同于KIF18A抑制剂BTB-1)。接下来，研究人员发起了一项药物化学筛选，以优化有希望的Hit，化合物3 (AM-7710);结构-活性关系(SAR)研究产生了四个有希望的系列类似物，分别代表早期SAR导联(AM-0277, AM-1882)和晚期SAR导联(AM-5308， AM-9022  )(图(Fig.2a).2a)。与AM-7710相比，这四种化合物在kif18a抑制活性和细胞效力方面均有显著改善，并且对多种运动蛋白表现出良好的特异性，除了KIF19A运动蛋白(图1)。2b2b和扩展数据图2a-c)。[1]

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5株细胞株对AM-0277、AM-1882和AM-9022 敏感，平均半最大有效浓度(EC50)分别为0.047µM、0.021µM和0.045µM。[1]

口服候选AM-9022，剂量为10 mg / kg，在小鼠中获得高血浆暴露，并在ovcar - 3ph3有丝分裂标记物检测中显示出两位数的纳摩尔效力(扩展数据图8a)。AM-9022 对人和小鼠KIF18A的运动活性有相似的抑制作用。[1]

AM-9022使OVCAR-3肿瘤中pH3有丝分裂标志物水平升高3.4倍(P值= 0.042)，肿瘤中化合物暴露量高于血浆(图6a)。为了评估口服候选药物的疗效和耐受性，已建立OVCAR-3肿瘤的小鼠以30 mg / kg /日的剂量给予载药或AM-9022，持续18 d。令人鼓舞的是，AM-9022抑制肿瘤生长(P值= 1.24 × 10−130)，具有TR (95% TR, 10只无肿瘤小鼠中的6只)和体重减轻的证据(图(Fig.6b)。研究结束时，AM-9022的血浆AUC值为53µM·h。[1]

已建立JIMT-1肿瘤的小鼠分别给予30和100 mg / kg / d的载药或AM-9022，持续21 d。AM-9022 在30和100 mg / kg剂量下抑制肿瘤生长(P值≤5.1 × 10−13)，在耐受良好的剂量下，TR(分别为16%和94%)(图6c)。研究结束时，JIMT-1模型中30 mg / kg AM-9022的血浆AUC值比OVCAR-3模型高3.7倍。[1]

已建立PDX肿瘤的小鼠给予载药或AM-9022，剂量为60 mg / kg，每日≥28 d。超过28天的治疗时间取决于抗癌活性水平和肿瘤是否达到预定的大小临界值。我们纳入了CTG-0017和CTG-0437模型停止治疗后的观察期，以评估持久性和肿瘤再生。对AM-9022 处理的敏感性模式在TNBC PDX组中有所不同(图(Fig.6d)。值得注意的是，AM-9022在CTG-0017模型中抑制肿瘤生长(P值= 6.5 × 10−164)，具有TR (83% TR)的证据，导致第58天90%的小鼠无肿瘤。AM-9022在CTG-0437模型中抑制肿瘤生长(101% TGI, P值= 2.2 × 10−101)。AM-9022在CTG-0888模型中效果较差(63% TGI, P值= 5.1 × 10−31)，在CTG-1019模型中无抗癌作用(图(Fig.6d)。与之前一样，AM-9022对KIF18A活性的抑制小鼠耐受良好(图6d)。综上所述，这些数据表明口服候选AM-9022在耐受良好的剂量下，对六种人类乳腺和卵巢肿瘤模型中的五种具有显著的抗癌作用，导致OVCAR-3、JIMT-1、CTG-0017和CTG-0437模型出现TR或停滞。[1]

运动蛋白测定[1]
使用ADP-Glo发光法(Promega)评估运动活性，使用先前描述的分析条件。表达和纯化重组截断的马达蛋白(hKIF18A (1 - 467,4 nM)， mKIF18A (1 - 467,4 nM)， hKIF19A(1 - 463,32或100 nM)， hKIF18B (1 - 436,8 nM)， hKIFC1 (266 - 673,4 nM))或获得(hEG5, 4 nM;hCENP-E, 8 nM)。化合物用30µM ATP和30µg ml - 1 MTs和上述运动蛋白浓度进行评估;数据来自两个或四个独立的实验。KIF18A化合物用hif18a (160 nM)检测或不检测MTs(0或30µg ml−1);数据来自两个独立的实验，一式两份。KIF18A化合物用30或300µM ATP和5或80µg ml - 1 mt的hKIF18A (4 nM)进行评估;数据来自一个实验。am -7710系列模拟mt - atp酶IC50值来自Amgen的Genedata Screener数据存储(扩展数据图Fig. Fig.2a2a)。

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酶联无机磷酸盐马达测定[1]
KIF18A化合物(1µM)与一组运动蛋白(hCENP-E, hEG5, hKIFC3, hKIF3C，人染色体动力学蛋白，hMCAK, hMKLP1, hMKLP2)进行酶联无机磷酸盐测定，根据制造商的方案和前面所述。数据来自一个或两个独立的实验，有两份或三份。研究由Cytoskeleton进行[1]

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Kinome结合试验[1]
KIF18A化合物(1µM)与一组激酶(n = 96)使用上文所述的竞争结合法进行评估。数据来自一个实验。研究由Eurofins DiscoverX进行。[1]

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Tubulin-polymerization化验[1]< br > 荧光微管蛋白聚合实验根据制造商的方案(Cytoskeleton)，用DMSO、KIF18A化合物(10µM)、紫杉醇(5µM)和诺可达唑(5µM)进行。微管蛋白聚合使用SpectraMax M5平板阅读器(Molecular Devices)进行检测，检测波长为440 nm，在37°C下每分钟测量一次，持续90分钟。数据来自一个或三个独立的实验。数据以平均荧光强度与时间的关系以及相应的AUC值作图。

细胞生长测定(化合物)细胞生长测定如前所述28。细胞株(n = 11)分别用DMSO或AM-0277、AM-1882、AM-9022 和palbociclib(最大浓度6µM)或ispinesib(最大浓度0.6µM)在19点或17点浓度范围内处理96 h。数据来自两个独立的实验，一式两份。每口井从相同数量的油田收集成像数据。计数POC的计算公式为(计数POC =(化合物处理过的核计数)÷ (dmso处理过的核计数)× 100)。如果在6µM时最大响应<50%，则认为该细胞系不敏感。测定敏感细胞系组的EC50平均值。对于OVCAR-8和OVCAR-8 ADRRES细胞生长分析，细胞用AM-1882和AM-9022(最大浓度为6µM)或紫杉醇和阿霉素(最大浓度为1µM)在19点浓度范围内处理96小时，有或没有p- gp抑制剂GF120918(1µM)。在两个独立的实验中评估了使用或不使用P-gp抑制剂的OVCAR-8细胞系。将数据绘制成具有相应EC50计数值的浓度-反应曲线。[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

White to off-white solid powder

4.2

136Ų

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)

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
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