CDK17;LIMK2; VHL; PROTAC degrader

Here, we identified at least one lead-like degrader molecule, DD-03–156, which induces potent and selective degradation of CDK17 and LIMK2 (Figure 1J) [1].

ompetitive displacement assay for cellular CRBN and VHL engagement [1]
HEK293T cells stably expressing the BRD4BD2-GFP with mCherry reporter were seeded at 30 – 50% confluency in 384-well plates with 50 μL FluoroBrite DMEM media containing 10% FBS per well a day before compound treatment. Degrader titrations and 100 nM dBET6 or 250 nM AT1 were dispensed using a D300e Digital Dispenser (HP), normalized to 0.5% DMSO, and incubated with cells for 5 h. Assay plates were imaged using Acumen as described above. Experiments were performed in triplicates and the values for the concentrations that lead to a 50% increase in BRD4BD2-eGFP accumulation (EC50) were calculated using the nonlinear fit variable slope model

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CellTiter-Glo Viability Assay [1]
MM1.S was seeded in a 96-well microplate at 10,000 cells per well in RPMI-1640 media supplemented with 10% FBS and incubated with compounds (final DMSO concentration at 0.1%). Relative cell viability was measured 72 h after addition of drug using CellTiter-Glo (Promega) according to the manufacturer’s protocol. Each analysis was performed in biological triplicate.

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KiNativ Live Cell Profiling Protocol [1]
CRBN−/− MOLT-4 cells were plated in fresh media (RPMI-1640 + 10% FBS) in 15 cm plates and treated for 5 h with candidate compounds. To harvest cells, plates were harvested using detachment using CellStripper detachment solution and washed twice with cold PBS, followed by centrifugation and snap-freezing of cell pellets in liquid nitrogen. The remainder of the KiNativ profiling experiment was performed by ActivX Biosciences

[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 protein degradation. TPD is of great significance in drug development because it can target previously inaccessible targets. However, the discovery and optimization of degradative agents remain inefficient due to a lack of understanding of the relative importance of the key molecular events required to induce target degradation. In this paper, we use chemical proteomics to annotate the degradable kinase genome. Our large dataset provides chemical lead compounds for approximately 200 kinases and demonstrates that current screening methods starting with the most active binders are not an efficient way to discover active compounds. We develop multi-target degradative agents to answer fundamental questions about the ubiquitin-proteasome system and reveal that kinase degradation depends on p97. This work will not only advance the discovery of kinase degradative agents but also provide a blueprint for assessing targeted degradation across the entire gene family, thereby accelerating the understanding of TPD beyond the kinase genome. [1]

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Technological advancements often facilitate new biological discoveries (Botstein, 2010). We demonstrate that this database can serve as a rich source of small-molecule tools for studying the fundamental biology of the ubiquitin-proteasome system (UPS), achieving this goal by exploring the role of AAA⁺-ATPase p97. Our observations show that the processing of most degradable kinases depends on p97, and this dependence is independent of the recruited E3 ligases (CRBN, VHL, and IAP). Although many unknowns remain regarding the role of p97 in promoting kinase-proteasome degradation, this study demonstrates how we can use our collection of multi-target degraders to reveal the effects of UPS perturbations on the degradation of proteins from different gene families. The limitation of our approach is that it only provides TPD information for degraders developed based on reported kinase binders, common linkers, and E3 ubiquitin ligase-recruited ligands; generating all possible variants is impractical. Furthermore, these degraders were tested in the biological environment of immortalized cancer cell lines. The study found that all these variables significantly affect the degradation of specific targets; therefore, further findings are likely if the scope of the study is expanded beyond what was presented here. Therefore, we envision the degradable kinase database as a dynamic resource that expands as new results emerge. We anticipate that this large dataset will not only accelerate the development of degradative chemical probes and clinically significant lead compounds targeting the entire kinase genome, but also promote the development of informatics and molecular modeling-based methods to improve the predictive power of degradation activity and facilitate the rational design of these bifunctional entities. [1]

C53H62F3N9O8S3

1106.30509901047

1105.38355

2769753-69-5

163196237

White to off-white solid powder

7.6

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

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

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

Solubility in Formulation 1: 5 mg/mL (4.52 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 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: 5 mg/mL (4.52 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 5 mg/mL (4.52 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 50.0 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.)