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 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

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.)