Evaluation of a Covalent Library of Diverse Warheads

• Published in: Drug design, development and therapy Vol. 18; p. 2653
• Main Authors: Schwer, Martin, Engelhardt, Marc U, Boeckler, Frank M, Klett, Theresa, Ernst, Larissa N, Jaag, Simon J, Lammerhofer, Michael, Gehringer, Matthias, Masberg, Benedikt, Knappe, Cornelius
• Format: Journal Article
• Language: English
• Published: Dove Medical Press Limited 31.07.2024
• Subjects: Cysteine; Drug discovery; Mass spectrometry; Organosulfur compounds; Proteases; Protein binding; Tumor proteins; Germany
• ISSN: 1177-8881; 1177-8881

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 a-cyanoacacrylamides/acrylates (CA), three epoxides (EO), four vinyl sulfones (VS), and eight electron-deficient heteroarenes with a leaving group ([S.sub.N]Ar/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 [alpha]-cyanoacrylamides/acrylates and all structurally confirmed epoxides turned out to be less reactive compounds, possibly due to steric hindrance and reversibility (for a-cyanoacrylamides/acrylates). The [S.sub.N]Ar 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.sub.N]Ar-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 [degrees]C and -9.1 [degrees]C. Keywords: covalent fragment-based drug discovery, differential scanning fluorimetry, 5,5'-dithiobis-(2-nitrobenzoic acid), intact protein mass spectrometry, glutathione