作者: Lämmerhofer, Michael ; Knappe, Cornelius ; Jaag, Simon J ; Klett, Theresa ; Schwer, Martin ; Stahlecker, Jason ; Boeckler, Frank M ; Masberg, Benedikt ; Stehle, Thilo

Purpose:

The tumor suppressor p53 is most commonly mutated in human cancer. The structural mutant in the β-sandwich of the protein, p53-Y220C, is the ninth most common p53 mutant. The p53-Y220C mutant has a solvent-accessible hydrophobic pocket, leading to thermal destabilization of the protein. Screening of our covalent fragment library (CovLib) revealed the highly reactive pyrazine derivatives SN006 and SN007, which arylate among other cysteines in p53, the mutation-generated Cys220. Herein, comprehensive structure-activity relationship (SAR) studies of these intrinsically reactive CovLib hits were performed, aiming to identify improved stabilizers for p53-Y220C, with a more balanced reactivity profile, diverse binding modes and a better potential for chemical optimization.

Methods:

The compounds were screened for enhanced stabilization of p53 wild type and its mutants using differential scanning fluorimetry (DSF). To confirm covalent modification, intact mass spectrometry was performed. Thiol reactivity profiles were determined using a standardized Glutathione-modifying (GSH) assay. The binding modes of the identified hits and covalent modification of Cys220 were elucidated by X-ray crystallography. Moreover, the influence of the hits on the DNA-binding affinity of full-length p53 was investigated employing a fluorescence polarization assay (FPA).

Results and Conclusion:

The promising pyrazine derivatives SN006/7-3, SN006/7-8, and SN006/7-9 were identified, occupying different subsites of the Y220C binding pocket. The compound SN006/7-8 substantially stabilized the thermosensitive cancer mutant Y220C by up to 5.0 °C, representing a strong enhancement over SN006 (1.8 °C) and SN007 (2.0 °C).

Drug Design Development and Therapy

Evaluation of a Covalent Library of Diverse Warheads (CovLib) Binding to JNK3, USP7, or p53

Article

作者: Klett, Theresa ; Schwer, Martin ; Jaag, Simon ; Lämmerhofer, Michael ; Jaag, Simon J ; Engelhardt, Marc U ; Masberg, Benedikt ; Gehringer, Matthias ; Boeckler, Frank ; Knappe, Cornelius ; Ernst, Larissa N ; Boeckler, Frank M ; Ernst, Larissa ; Engelhardt, Marc

Purpose:

Over the last few years, covalent fragment-based drug discovery has gained significant importance. Thus, striving for more warhead diversity, we conceived a library consisting of 20 covalently reacting compounds. Our covalent fragment library (CovLib) contains four different warhead classes, including five α-cyanoacacrylamides/acrylates (CA), three epoxides (EO), four vinyl sulfones (VS), and eight electron-deficient heteroarenes with a leaving group (S_NAr/SN).

Methods:

After predicting the theoretical solubility of the fragments by LogP and LogS during the selection process, we determined their experimental solubility using a turbidimetric solubility assay. The reactivities of the different compounds were measured in a high-throughput 5,5'-dithiobis-(2-nitrobenzoic acid) DTNB assay, followed by a (glutathione) GSH stability assay. We employed the CovLib in a (differential scanning fluorimetry) DSF-based screening against different targets: c-Jun N-terminal kinase 3 (JNK3), ubiquitin-specific protease 7 (USP7), and the tumor suppressor p53. Finally, the covalent binding was confirmed by intact protein mass spectrometry (MS).

Results:

In general, the purchased fragments turned out to be sufficiently soluble. Additionally, they covered a broad spectrum of reactivity. All investigated α-cyanoacrylamides/acrylates and all structurally confirmed epoxides turned out to be less reactive compounds, possibly due to steric hindrance and reversibility (for α-cyanoacrylamides/acrylates). The S_NAr and vinyl sulfone fragments are either highly reactive or stable. DSF measurements with the different targets JNK3, USP7, and p53 identified reactive fragment hits causing a shift in the melting temperatures of the proteins. MS confirmed the covalent binding mode of all these fragments to USP7 and p53, while additionally identifying the S_NAr-type electrophile SN002 as a mildly reactive covalent hit for p53.

Conclusion:

The screening and target evaluation of the CovLib revealed first interesting hits. The highly cysteine-reactive fragments VS004, SN001, SN006, and SN007 covalently modify several target proteins and showed distinct shifts in the melting temperatures up to +5.1 °C and -9.1 °C.

100 项与 SN007 相关的药物交易

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