DCAF1; BRD4; drug-linker conjugate

Targeted protein degradation induced by heterobifunctional compounds and molecular glues presents an exciting avenue for chemical probe and drug discovery. To date, small-molecule ligands have been discovered for only a limited number of E3 ligases, which is an important limiting factor for realizing the full potential of targeted protein degradation. We report herein the discovery by chemical proteomics of azetidine acrylamides that stereoselectively and site-specifically react with a cysteine (C1113) in the E3 ligase substrate receptor DCAF1. We demonstrate that the azetidine acrylamide ligands for DCAF1 can be developed into electrophilic proteolysis-targeting chimeras (PROTACs) that mediated targeted protein degradation in human cells. We show that this process is stereoselective and does not occur in cells expressing a C1113A mutant of DCAF1. Mechanistic studies indicate that only low fractional engagement of DCAF1 is required to support protein degradation by electrophilic PROTACs. These findings, taken together, demonstrate how the chemical proteomic analysis of stereochemically defined electrophilic compound sets can uncover ligandable sites on E3 ligases that support targeted protein degradation [1].

[1]. Targeted Protein Degradation by Electrophilic PROTACs that Stereoselectively and Site-Specifically Engage DCAF1. J Am Chem Soc. 2022 Oct 12;144(40):18688-18699.

Here, we have described the chemical proteomic discovery of a ligandable cysteine (C1113) in the E3 ligase substrate receptor protein DCAF1 and the conversion of azetidine acrylamides targeting this cysteine into electrophilic PROTACs. These findings are significant because, to our knowledge, they describe the first electrophilic PROTACs that have been shown to act in a stereo- and site-selective manner. The properties of stereoselectivity and site-specificity provide convenient ways to establish controls to verify on-target activity for electrophilic PROTACs, as we have shown herein for DCAF1 and others have demonstrated for reversibly binding PROTACs that engage VHL in a stereoselective manner. We further interpret these features to indicate the presence of a high-quality druggable pocket in proximity to DCAF1_C1113. Also supportive of this conclusion is the recent report of a noncovalent DCAF1 ligand that binds a pocket near C1113 (PDB code: 7SSE, Figure S10). While we have taken advantage of the druggability of DCAF1_C1113 to create electrophilic PROTACs, we also imagine that more advanced covalent ligands might serve as molecular glues or antagonists of the various physiological and pathological functions of DCAF1. Indeed, small-molecule antagonists of DCAF1 could counteract the pathogenesis of viruses like HIV-1 and HIV-2 that co-opt this E3 ligase substrate receptor to suppress immune cell responses. More generally, the rich dataset of stereoselective interactions reported herein between azetidine acrylamides and cysteines in the human T-cell proteome (Figure 1B and Dataset S1) should offer attractive starting points for chemical probe discovery for additional proteins from structurally and functionally diverse classes.[1]
As has been described previously, electrophilic PROTACs have the potential to maximally leverage the catalytic potential of targeted protein degradation by creating “neo”-E3 ligases that are permanently modified (until physical turnover) with a substrate-binding compound. However, key variables can impact the success of such endeavors, including the half-life and substrate compatibility of the E3 ligase, as well as the quality of the chemical probes that target it. On the latter point, we view the azetidine acrylamide reactive group found herein to stereoselectively engage DCAF1_C1113 as an encouraging starting point for probe optimization, especially in comparison to electrophilic PROTACs reported to target other E3 ligases, which mostly use high-reactivity groups such as α-chloroacetamides. Nonetheless, we have, so far, only observed targeted protein degradation for electrophilic PROTACs in cells expressing recombinant DCAF1, and it will be important in future studies to determine if this activity can be extended to endogenous DCAF1. That we observed evidence of electrophilic PROTACs promoting a ternary complex with endogenous DCAF1 and FBKP12, as well as inducing FKBP12 ubiquitination, is encouraging, even though these effects were not apparently robust enough to lead to substantial FKBP12 degradation in cells expressing endogenous DCAF1 (possibly due to counteracting cellular deubiquitinases). Further improvements in electrophilic PROTAC performance may require greater levels of cellular engagement of DCAF1, as our first-generation compounds only appear to modify ≤ 20% of recombinant DCAF1 at functional concentrations in cells. A recent study has also described an autoinhibitory oligomerization mechanism for DCAF1 that may not be shared by other CRL4 substrate receptors58. If a substantial proportion of endogenous DCAF1 is in an autoinhibited tetrameric state, then a greater quantity of total DCAF1 may need to be modified by electrophilic PROTACs to support targeted protein degradation. On the other hand, we wonder whether this autoregulatory feature of DCAF1 might also be exploited to create electrophilic PROTACs and/or molecular glues that carry out context-dependent protein degradation only in those cell types where DCAF1 is in an activated state.[1]

C27H35CLN6O4S

575.12

2098790-24-8

2241669-80-5

Typically exists as solids at room temperature

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)