Screening of Covalent Kinase Inhibitors Yields Hits for Cysteine Protease USP7 / HAUSP

Larissa N Ernst,1 Simon J Jaag,2 Valentin R Wydra,3 Benedikt Masberg,2 Cornelius Knappe,2 Stefan Gerstenecker,3 Ricardo AM Serafim,3 Xiaojun Julia Liang,4,5 Nico J Seidler,3,5 Michael Lämmerhofer,2 Matthias Gehringer,3– 5 Frank M Boeckler1,6 1Department of Pharmacy and Biochemistry, Eberhard Karls Universität Tübingen, Laboratory for Molecular Design and Pharmaceutical Biophysics, Institute of Pharmaceutical Sciences, Tübingen, 72076, Germany; 2Department of Pharmacy and Biochemistry, Eberhard Karls Universität, Pharmaceutical (Bio-)Analysis, Institute of Pharmaceutical Sciences, Tübingen, 72076, Germany; 3Department of Pharmacy and Biochemistry, Eberhard Karls Universität, Pharmaceutical Chemistry, Institute of Pharmaceutical Sciences, Tübingen, 72076, Germany; 4Department of Medicinal Chemistry, Eberhard Karls Universität Tübingen, Faculty of Medicine, Institute for Biomedical Engineering, Tübingen, 72076, Germany; 5Cluster of Excellence iFIT (EXC 2180) ‘Image-Guided and Functionally Instructed Tumor Therapies’, Eberhard Karls Universität Tübingen, Tübingen, 72076, Germany; 6Interfaculty Institute for Biomedical Informatics (IBMI), Eberhard Karls Universität Tübingen, Tübingen, 72076, GermanyCorrespondence: Frank M Boeckler, Lab for Molecular Design and Pharm. Biophysics, Institute for Pharmaceutical Sciences, Eberhard Karls Universität Tübingen, Auf der Morgenstelle 8, Tübingen, 72076, Germany, Tel +49 7071 29 74567, Email [email protected]: The ubiquitin-specific protease 7 (USP7), also known as herpes-associated ubiquitin-specific protease (HAUSP) is an interesting target due to its role in the tumor suppressor p53 pathway. In recent years targeted covalent inhibitors have gained significant importance in pharmaceutical research. Thus, we have investigated a small library of 129 ligands bearing different types of covalent reactive groups (“warheads”) from various kinase drug discovery projects for their reactivity towards the catalytic cysteine of USP7, as well as their influence on its melting temperature. These compounds mainly encompassed α,β-unsaturated amides specifically acrylamides, SNAr reacting compounds, aryl fluorosulfates and sulfonyl fluorides.Methods: We analyzed an array of 129 electrophilic compounds which had been designed as covalent kinase inhibitors in a DSF-based (differential scanning fluorimetry) screen against USP7. The hits were evaluated for their ability to cause similar thermal shifts for a CYS-deficient USP7 control mutant (USP7asoc), where only the catalytic Cys223 was retained. Additionally, covalent binding was evaluated by intact protein mass spectrometry (MS).Results: The DSF screen revealed that, predominantly 18 of the 129 tested compounds decreased the melting temperature of USP7 and its mutant USP7asoc. For 8 of these, the hypothesized covalent binding mode was corroborated with native and mutant USP7 by intact protein MS. Nearly all identified hits have a covalent warhead that reacts via nucleophilic aromatic substitution (SNAr).Conclusion: The screening and evaluation of the kinase library revealed several initial hits of interest. Seven SNAr warheads and one acrylamide warhead compound covalently modified the target protein (USP7) and showed clear shifts in the melting temperatures ranging from − 6.0 °C to +1.7 °C. Keywords: covalent cysteine modification, differential scanning fluorimetry (DSF), intact protein mass spectrometry, nucleophilic aromatic substitution (SNAr), repurposing of kinase inhibitors