MRTX1133

KRas G12D (Kd = 0.2 pM)

MRTX1133可与活化和失活的KRAS G12D突变体可逆结合并抑制其活性。它是突变型KRAS的高选择性抑制剂。MRTX1133对KRAS G12D的特异性比野生型KRAS高出1000倍以上。MRTX1133不仅在Panc 04.03异种移植物模型中表现出肿瘤消退，而且在细胞增殖测定中表现出个位数的nM效力。

MRTX1133是通过广泛的基于结构的活性改进发现的，并在KRASG12D突变异种移植物小鼠肿瘤模型中显示出有效性。在CD-1小鼠中腹膜内（IP）给予30 mg/kg的MRTX1133导致KRASG12D突变体Panc 04.03细胞系中持续的血浆暴露超过游离组分调节的pERK IC50值约8小时。受此结果的鼓舞，我们评估了以30 mg/kg BID（IP）调节Panc 04.03-异种移植物肿瘤模型中KRAS依赖性ERK磷酸化的能力，并在第二次给药后1小时和12小时分别观察到pERK信号抑制62%和74%。该模型中的一项抗肿瘤疗效研究导致MRTX1133具有剂量依赖性抗肿瘤活性，在3 mg/kg BID（IP）时观察到94%的生长抑制，在10 mg/kg BID和30 mg/kg BID时分别观察到−62%和−73%的肿瘤消退）。相反，在非KRASG12D肿瘤模型MKN1中没有观察到显著的抗肿瘤活性（数据未显示）[2]。

HTRF结合测定
将重组人KRAS 4B G12D蛋白（对应于氨基酸1-169，在大肠杆菌中表达，具有C-末端Avi生物素化标签MW＝22kDa）与缓冲液（50mM HEPES pH 7.5，5mM MgCl2，1mM DTT，约0.1%DMSO）、5nM KRASG12D、100nM示踪剂（化合物45）和0.5nM Tb SA（Cisbio）中的测试化合物一起孵育。在室温下孵育1小时后，根据制造商的说明，使用Clariostar读取器[（BMG）激发滤光片（Ex Tr）、二向色滤光片（LP TP）和发射滤光片（F 665-10和F 620-10）]测量HTRF信号。HTRF比率使用公式计算：[发射665/发射620]*10000。使用Xlfit软件（IDBS）拟合IC50，Hill方程固定为1（拟合背景+Bmax/（1+（x/IC50）^Hill））。

AGS ICW测定方案[2]
1.KRASG12D突变体AGS细胞（ATCC CRL-1739）在补充有10%胎牛血清和青霉素/链霉素的DMEM培养基中生长。将细胞以20000个细胞/孔的密度接种在经黑色透明底部组织培养物处理的96孔板中，并使其附着12-14小时。用化合物的3倍9点连续稀释液处理接种的细胞，最高终浓度为10µM。将稀释的化合物以0.5%DMSO的最终浓度加入到接种的细胞中。药物处理3小时后，通过在室温下将平板在50µl 4.0%甲醛中孵育20分钟来固定细胞。然后倾倒甲醛，加入150µL冰冷的100%甲醇10分钟，使细胞透化。倾倒甲醇，在室温下加入100µL Licor封闭缓冲液（Li-Cor Biotechnology，Lincoln NE）1小时，以抑制板中的非特异性抗体结合
2.使用对磷酸化形式的ERK特异性的抗体测定磷酸化ERK的量，并将其与GAPDH的量进行比较。用于检测的初级抗体添加如下：在Odyssey阻断缓冲液+0.05%Tween 20中稀释1:500的磷酸ERK（细胞信号传导CS-9101）和稀释1:5000的GAPDH（Millipore MAB374）。将平板在4°C下孵育过夜。将平板用150uL PBS+0.1%吐温20洗涤3次
3.用于显示一级抗体的二级抗体添加如下：在奥德赛阻断缓冲液+0.05%Tween20中以1:800稀释的山羊抗Rabbit-800（LI-COR，926-32211）和山羊抗Mouse680（LI-COR926-68070），并在室温下孵育1小时。将平板用150uL PBS+0.1%Tween20洗涤3次。在LiCOR Odyssey CLX印版读取器上对印版进行干燥成像。S192 4。通过将磷酸化ERK（Thr202/Tyr204）信号归一化为每个孔的GAPDH信号来分析板，并计算DMSO对照值的百分比。使用剂量响应曲线的4参数拟合生成IC50值
2D细胞增殖测定方案[2]
1.KRASG12D突变细胞系GP2d（Sigma-Aldrich#95090714）在补充有10%胎牛血清和青霉素/链霉素的DMEM培养基中生长。KRASWT细胞系MKN1（JCRB0252）在补充有10%胎牛血清、10mM HEPES、10mM丙酮酸钠和青霉素/链霉素的RPMI细胞中生长。将细胞以2000个细胞/孔的密度接种在白色透明底部组织培养物处理的96孔板中，并使其附着12-14小时。每个细胞系也以相同的密度在基线板中的3个孔中铺板，以在药物处理之前确定每个细胞系的发光RLU值。在用药物处理平板细胞之前，立即读取基线平板，方法是将每个细胞系的三个平板孔中的每一个与30µL CTG试剂孵育30分钟，遮光并剧烈摇晃。然后在CLARIOstar微孔板读取器上读取发光RLU值。用最高终浓度为3µM的MRTX1133的3倍9点系列稀释剂量反应处理接种的细胞。以0.5%DMSO的最终浓度加入稀释的化合物。药物治疗3天后，使用上述基线平板的条件在CLARIOstar上读取每个平板
2.通过从添加MRTX1133 3 3天后的处理板的RLU值中减去基线RLU值来分析数据。细胞增殖百分比抑制值是通过将每个处理过的孔的发光单位值除以载体处理过的孔中发光单位值的平均值并乘以100来计算的。将数据转换并传递到GraphPad Prism中，以使用剂量响应曲线的4参数拟合来获得IC50值。

Tumor Pharmacodynamic and Tumor Xenograft Studies
All mouse studies were conducted in compliance with all applicable regulations and guidelines of the Institutional Animal Care and Use Committee (IACUC) from the National Institutes of Health (NIH). Mice were maintained under pathogen-free conditions, and food and water were provided ad libitum. 6–8-weekold, female, athymic nude-Foxn1nu mice (Envigo, San Diego) were injected subcutaneously with Panc 04.03 cells in 100 l of PBS and Matrigel matrix in the right hind flank with 5.0 x 106 cells (Corning #356237; Discovery Labware, MA) 50:50 cells : Matrigel. Mouse health was monitored daily, and caliper measurements began when tumors were palpable. Tumor volume measurements were determined utilizing the formula 0.5 x L x W2 in which L refers to length and W refers to width of each tumor.  Tumor pharmacodynamic studies: When tumors reached an average tumor volume of ~400 mm3 , mice were randomized into treatment groups. Mice were treated by intraperitoneal injection with either vehicle consisting of 10% research grade Captisol (CyDex Pharmaceuticals, KS) in 50 mM citrate buffer pH 5.0 or MRTX1133 at 30mg/kg. Tumors and plasma were collected at 1 hour and 12 hours after a single dose to determine exposure levels. Tumor fragments were snap frozen in homogenization tubes (Omni, #19-628-3) with liquid nitrogen and homogenized (MPBio FastPrep-24 system) with Lysis/Binding Buffer AM11 (Active Motif, #52097) with protease and phosphatase inhibitors added fresh before use. Tumor lysates were then assayed for ERK1/2 phosphorylation. Xenograft studies: When tumors reached an average tumor volume of ~350 mm3 , mice were randomized into treatment groups. Mice were treated by intraperitoneal injection with either vehicle consisting of 10% research grade Captisol (CyDex Pharmaceuticals, KS) in 50 mM citrate buffer pH 5.0 or MRTX1133 in vehicle at 3, 10, or 30mg/kg BID.  Animals were monitored daily, tumors were measured 3 times per week, and body weights were measured 2 times per week. Data are expressed as mean +/- SEM. Statistical analysis of differences in mean tumor volume between vehicle and MRTX1133-treated cohorts was run using a two-tailed Student’s t-test with equal variance in Excel (Microsoft; Redmond, WA).
Animal/Disease Models: 6-8-week age, female/athymic nude-Foxn1nu mice (Panc 04.03 model)
Doses: 3, 10, 30 mg/kg
Route of Administration: Intraperitoneal injection/ip; twice a day for 28 days
Experimental Results: MRTX1133 dose-dependent suppressed tumor growth with a TGI of 94% observed at 3 mg/kg BID (IP) and TGIs of -62% and -73% observed at 10 and 30 mg/kg BID (IP), respectively.

[1]. Front Oncol . 2021 May 3:11:672612.

[2]. J Med Chem . 2022 Feb 24;65(4):3123-3133.

[3]. Nat Med . 2022 Oct;28(10):2171-2182.

KRAS G12D Inhibitor MRTX1133 is an orally bioavailable reversible inhibitor of the oncogenic KRAS substitution mutation G12D, with potential antineoplastic activity. Upon oral administration, KRAS G12D inhibitor MRTX1133 specifically targets and noncovalently binds to KRAS G12D. This prevents KRAS G12D-mediated signaling and activation of downstream survival pathways. This leads to an inhibition of the growth of tumor cells that overexpress KRAS G12D. KRAS, a member of the RAS family of oncogenes, serves an important role in cell signaling, division and differentiation. Mutations of KRAS may induce constitutive signal transduction leading to tumor cell proliferation, invasion, and metastasis.

C33H31F3N6O2

600.6335

600.246

C, 65.99; H, 5.20; F, 9.49; N, 13.99; O, 5.33

2621928-55-8

2621928-55-8;

156124857

Yellow to brown solid powder

5.2

86.6Ų

2

11

6

44

1100

2

C#CC1=C(C=CC2=CC(=CC(=C21)C3=NC=C4C(=C3F)N=C(N=C4N5CC6CCC(C5)N6)OC[C@@]78CCCN7C[C@@H](C8)F)O)F

SCLLZBIBSFTLIN-IFMUVJFISA-N

InChI=1S/C33H31F3N6O2/c1-2-23-26(35)7-4-18-10-22(43)11-24(27(18)23)29-28(36)30-25(13-37-29)31(41-15-20-5-6-21(16-41)38-20)40-32(39-30)44-17-33-8-3-9-42(33)14-19(34)12-33/h1,4,7,10-11,13,19-21,38,43H,3,5-6,8-9,12,14-17H2/t19-,20?,21?,33+/m1/s1

4-[4-(3,8-diazabicyclo[3.2.1]octan-3-yl)-8-fluoro-2-[[(2R,8S)-2-fluoro-1,2,3,5,6,7-hexahydropyrrolizin-8-yl]methoxy]pyrido[4,3-d]pyrimidin-7-yl]-5-ethynyl-6-fluoronaphthalen-2-ol

MRTX1133; MRTX 1133; MRTX-1133

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:50~100 mg/mL (83.3~166.5 mM)

配方 1 中的溶解度: 10 mg/mL (16.65 mM) in 10% SBE-β-CD/50 mM citrate pH 5.0 (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。

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配方 2 中的溶解度: 3.5 mg/mL (5.83 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
例如，若需制备1 mL的工作液，可将 100 μL 35.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (4.16 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 4 中的溶解度: 5%DMSO+40%PEG300+5%Tween80+50%ddH2O: 25mg/ml

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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；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。