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

Abstract

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.

Keywords: 5,5'-dithiobis-(2-nitrobenzoic acid); covalent fragment-based drug discovery; differential scanning fluorimetry; glutathione; intact protein mass spectrometry.

Graphical abstract

Figure 1

Chemical structures of the 20 fragments purchased for the CovLib. The scaffolds for the respective warhead types are highlighted in cyan for α-cyanoacrylamides/acrylates, in green for epoxides, in yellow for vinyl sulfones, and in light red for S_NAr-type electrophiles.

Figure 2

Chemical structures of the cysteine surrogates and reference compounds used for the reactivity assessment (A) Time-dependent depletion of selected compounds and the reference compound Iodoacetamide in the DTNB assay. The measurement points are shown as the mean value of the triplicate determination with the respective standard deviation as error bars. The second order fit function is shown as colored line (B) Time-dependent decrease of the relative AUC of selected compounds and the reference compound Afatinib in the GSH assay (C), and in PBS as a reference measurement (D) For GSH, the measurement points are shown as the mean of the duplicate determination, and the pseudo-first order fit function is shown as a colored line.

Figure 3

Overall structures of JNK3 (Protein Data Bank (PDB) code 8BZP) (A), USP7 (PDB code 4M5W) (B), and T-p53C (PDB code 1UOL) (C) All cysteines in the protein structures are highlighted.

Figure 4

First derivatives of the melting curves of α-cyanoacylamides/acrylates (A), epoxides (B), vinyl sulfones (C), and S_NAr (D) fragments (1 mM) with JNK3 (8 µM protein) after 30 min of incubation.

Figure 5

First derivatives of the melting curves of α-cyanoacrylamides/acrylates (A), epoxides (B), vinyl sulfones (C), and S_NAr (D) fragments (1 mM) with USP7 (8 µM protein) after 30 min of incubation.

Figure 6

First derivatives of the melting curves of α-cyanoacrylamides/acrylates (A), epoxides (B), vinyl sulfones (C), and S_NAr (D) fragments (1 mM) with T-p53C (8 µM protein) after 30 min of incubation.

Figure 7

Deconvoluted MS spectra of VS001 (A), VS004 (measured without TCEP) (C), SN001 (E) with USP7 (protein-to-compound ratio 1:100, theoretical mass of unmodified USP7: 41,145.61 Da) and VS001 (B), VS004 (measured without DTT) (D), and SN001 (F) with T-p53C (protein-to-compound ratio 1:100, theoretical mass of unmodified T-p53C: 24,756.03 Da) after 4 h of incubation at 20 °C.

Figure 8

Deconvoluted MS spectra of SN002 (measured without TCEP) (A), SN006 (C), SN007 (E) with USP7 (protein-to-compound ratio 1:100, theoretical mass of unmodified USP7: 41,145.61 Da) and SN002 (measured without DTT) (B), SN006 (D), and SN007 (F) with T-p53C (protein-to-compound ratio 1:100, theoretical mass of unmodified T-p53C: 24,756.03 Da) after 4 h of incubation at 20 °C.