DD-03-156 ((S,R,S)-AHPC-Me-PEG2-dabrafenib)

CDK17;LIMK2; VHL; PROTAC degrader

在这里，我们发现了至少一种Lead降解分子DD-03-156，它能诱导CDK17和LIMK2的有效和选择性降解（图1J）[1]。

细胞CRBN和VHL参与的竞争性置换分析[1]
在化合物处理前一天，将用mCherry报告基因稳定表达BRD4BD2-GFP的HEK293T细胞以30-50%的融合率接种在384孔板中，每孔含有10%FBS的50μL FluoroBrite DMEM培养基。使用D300e数字分配器（HP）分配降解剂滴定和100 nM dBET6或250 nM AT1，标准化为0.5%DMSO，并与细胞孵育5小时。如上所述，使用Acumen对测定板进行成像。实验进行了三次，使用非线性拟合变斜率模型计算了导致BRD4BD2 eGFP累积（EC50）增加50%的浓度值

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CellTiter肾小球活力测定[1]
将MM1.S以每孔10000个细胞的速度接种在96孔微孔板中，在补充有10%FBS的RPMI-1640培养基中，并与化合物一起孵育（最终DMSO浓度为0.1%）。根据制造商的方案，在添加药物后72小时使用CellTiter Glo（Promega）测量相对细胞存活率。每项分析均以生物三份进行。

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KiNativ活细胞分析方案[1]
CRBN−/−MOLT-4细胞被置于15 cm的新鲜培养基（RPMI-1640+10%FBS）中，并用候选化合物处理5小时。为了收获细胞，使用CellStripper分离溶液通过分离收获平板，用冷PBS洗涤两次，然后离心并在液氮中快速冷冻细胞颗粒。KiNativ分析实验的其余部分由ActivX Biosciences进行

[1]. Mapping the Degradable Kinome Provides a Resource for Expedited Degrader Development. Cell. 2020;183(6):1714-1731.e10.

Targeted protein degradation (TPD) refers to the use of small molecules to induce ubiquitin-dependent degradation of proteins. TPD is of interest in drug development, as it can address previously inaccessible targets. However, degrader discovery and optimization remains an inefficient process due to a lack of understanding of the relative importance of the key molecular events required to induce target degradation. Here, we use chemo-proteomics to annotate the degradable kinome. Our expansive dataset provides chemical leads for ∼200 kinases and demonstrates that the current practice of starting from the highest potency binder is an ineffective method for discovering active compounds. We develop multitargeted degraders to answer fundamental questions about the ubiquitin proteasome system, uncovering that kinase degradation is p97 dependent. This work will not only fuel kinase degrader discovery, but also provides a blueprint for evaluating targeted degradation across entire gene families to accelerate understanding of TPD beyond the kinome. [1]

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Technological advances often facilitate new biological discoveries (Botstein, 2010). We demonstrate that this database can serve as a rich source of small molecule tools with which to study the basic biology of the ubiquitin proteasome system (UPS), by interrogating the role of the AAA+-ATPase p97. Our observations suggest that the majority of the degradable kinome is processed in a p97-dependent fashion, and that this dependence occurs irrespective of the E3 ligase recruited (CRBN, VHL and IAP). Although much still remains to be understood about the role of p97 in facilitating the proteasomal degradation of kinases, this study demonstrates how our collection of multitargeted degraders can be harnessed to reveal effects of perturbations to the UPS on protein degradation across gene families. A limitation of our approach is that it informs on TPD in the context of degraders developed from reported kinase binders and commonly employed linkers and E3-recruiting ligands, of which it is implausible to generate all possible variants. In addition, these degraders are tested in the biological setting of immortalized cancer cell lines. These variables were all found to dramatically influence the degradation of specific targets, and it is probable that there are more discoveries to be made by expanding beyond the scope of this study. Hence, we envision the degradable kinome database as a living resource that will continue to expand as new results become available. We anticipate this large dataset will accelerate development not only of degrader chemical probes and clinically relevant lead compounds across the kinome, but also of informatics and molecular modeling-based approaches that may lead to improved prediction of degradation activity and rational design of these bifunctional entities.[1]

C53H62F3N9O8S3

1106.30509901047

1105.38355

2769753-69-5

163196237

White to off-white solid powder

7.6

292 Ų

5

19

23

76

1990

4

S1C=NC(C)=C1C1C=CC(=CC=1)[C@H](C)NC([C@@H]1C[C@H](CN1C([C@H](C(C)(C)C)NC(CCOCCOCCNC1=NC=CC(C2=C(C3C=CC=C(C=3F)NS(C3C(=CC=CC=3F)F)(=O)=O)N=C(C(C)(C)C)S2)=N1)=O)=O)O)=O

BMLDGVCSNFURAJ-QUQZIOAQSA-N

InChI=1S/C53H62F3N9O8S3/c1-30(32-15-17-33(18-16-32)44-31(2)59-29-74-44)60-48(68)40-27-34(66)28-65(40)49(69)47(52(3,4)5)62-41(67)20-23-72-25-26-73-24-22-58-51-57-21-19-39(61-51)45-43(63-50(75-45)53(6,7)8)35-11-9-14-38(42(35)56)64-76(70,71)46-36(54)12-10-13-37(46)55/h9-19,21,29-30,34,40,47,64,66H,20,22-28H2,1-8H3,(H,60,68)(H,62,67)(H,57,58,61)/t30-,34+,40-,47+/m0/s1

(2S,4R)-1-[(2S)-2-[3-[2-[2-[[4-[2-tert-butyl-4-[3-[(2,6-difluorophenyl)sulfonylamino]-2-fluorophenyl]-1,3-thiazol-5-yl]pyrimidin-2-yl]amino]ethoxy]ethoxy]propanoylamino]-3,3-dimethylbutanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methyl-1,3-thiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

DD-03-156; 2769753-69-5; DD 03-156;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 请将本产品存放在密封且受保护的环境中（例如氮气保护），避免吸湿/受潮。

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

DMSO : 200 mg/mL (180.78 mM)
H2O : < 0.1 mg/mL

配方 1 中的溶解度: 5 mg/mL (4.52 mM) 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), 悬浮液；超声助溶。
例如，若需制备1 mL的工作液，可将100 μL 50.0 mg/mL澄清DMSO储备液加入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 2 中的溶解度: 5 mg/mL (4.52 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 50.0 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: ≥ 5 mg/mL (4.52 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (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 50.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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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。