DCAF1; BRD4; drug-linker conjugate

异双官能化合物和分子胶诱导的靶向蛋白质降解为化学探针和药物发现提供了一条令人兴奋的途径。迄今为止，只发现了有限数量的E3连接酶的小分子配体，这是实现靶向蛋白质降解全部潜力的重要限制因素。我们在这里报告了通过化学蛋白质组学发现氮杂环丁烷丙烯酰胺与E3连接酶底物受体DCAF1中的半胱氨酸（C1113）立体选择性和位点特异性反应。我们证明，DCAF1的氮杂环丁烷丙烯酰胺配体可以开发成亲电蛋白水解靶向嵌合体（PROTAC），介导人类细胞中靶向蛋白的降解。我们表明，这一过程是立体选择性的，在表达DCAF1 C1113A突变体的细胞中不会发生。机制研究表明，仅需要DCAF1的低分数参与来支持亲电PROTAC的蛋白质降解。这些发现共同证明了立体化学定义的亲电化合物集的化学蛋白质组学分析如何揭示E3连接酶上支持靶向蛋白质降解的可连接位点[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]

C27H36CL2N6O4S

611.58

2241669-80-5

2098790-24-8

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