Protein-protein interactions: developing small-molecule inhibitors/stabilizers through covalent strategies

Abstract

The development of small-molecule inhibitors or stabilizers of selected protein-protein interactions (PPIs) of interest holds considerable promise for the development of research tools as well as candidate therapeutics. In this context, the covalent modification of selected residues within the target protein has emerged as a promising mechanism of action to obtain small-molecule modulators of PPIs with appropriate selectivity and duration of action. Different covalent labeling strategies are now available that can potentially allow for a rational, ground-up discovery and optimization of ligands as PPI inhibitors or stabilizers. This review article provides a synopsis of recent developments and applications of such tactics, with a particular focus on site-directed fragment tethering and proximity-enabled approaches.

Keywords: covalent PROTAC; fragment-based drug discovery; imine; protein–protein interaction; targeted covalent ligand; tethering.

Figure 1.. Site-directed approaches.

(A) Outline of the disulfide tethering approach utilized to screen for stabilizers of the 14-3-3σ/ERα protein–protein interaction (PPI). A library of disulfide fragments was screened and tethering was determined via mass spectrometry. Fragments were classified as neutral (no preference for either the PPI or the apo form), cooperative (preference for the PPI complex), or competitive (only binding to the apo-14-3-3). (B) Chemical structures of 14-3-3σ/ERα PPI stabilizers 1 and 2. The co-crystal structure of 1 bound to the 14-3-3σ/ERα complex provided insight into the compound’s mechanism of PPI stabilization by revealing both the formation of a disulfide bond with the 14-3-3σ Cys⁴² and hydrophobic contacts between the para-phenyl of 1 and the C-terminal Val⁵⁹⁵ of ERα [Protein Data Bank (PDB): 6HMT]. (C) Outline of the imine-based tethering approach. (D) Imine-based tethering in the 14-3-3 hub protein utilizes the native 14-3-3 Lys¹²², which is located near several known 14-3-3 binding partners (Complex 1). Screening efforts with aldehyde libraries identified aromatic aldehydes capable of forming aldimines with 14-3-3 Lys¹²² and cooperatively bind with the protein partner (ERα and Pin1; Complex 2). Ligands that cooperatively bind the PPI and induce conformational change increase PPI stability and selectivity (Complex 3). Co-crystal structures of PPI stabilizers, 3 (E) PDB: 6YQ2, 4 (F) PDB: 7NJ9, and 5 (G) PDB: 7BIW, bound to the 14-3-3 (pink surface)/ERα (green) PPI. Co-crystal structures of 14-3-3/Pin1 (orange) PPI stabilizers, 6 (H) PDB: 7AXN and 7 (I) PDB: 7BFW. (J) Co-crystal structure of the HEG1-KRIT1 (yellow surface) inhibitor, 8, bound to KRIT1 (PDB: 6UZK). Figure (A, C, D) created with BioRender.com.

Figure 2.. Ligand-directed chemistry approach.

(A) General mechanism of protein tagging by the ligand-directed chemistry approach. (B) Covalent ligands utilizing electrophilic cleavable linkers and their binding affinity and kinetics in Escherichia coli dihydrofolate reductase (eDHFR) or FK506-binding protein (FKBP12). Binding affinity and kinetics were determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) [46]. (C) An irreversible covalent inhibitor of Hsp90 was developed utilizing electrophilic cleavable linker (ECL). Figure (A, C) created with BioRender.com. Abbreviation: POI, protein of interest.

Figure 3.. Other examples of proximity-enabled covalent strategies.

(A) Affinity enhancement of locked nucleic acid (LNA) ligands via imine formation with a lysine residue in the vicinity of the ligand binding site, the structures of known ligands for the target proteins, and the binding affinities of aldehyde-based LNAs to their respective targets. Binding affinities were determined by fluorescence polarization (FP) assay. (B) Urokinase-type plasminogen activator (uPa) inhibition activity of benzamidine derivatives as determined by enzymatic inhibition assay. (C) Summary of imine chemistries, including the stabilization by iminoboronate and diazaborine. (D) Imine-forming Mcl-1 inhibitors, including aldehyde 23 and iminoboronate-forming 24 and 25. TR-FRET IC₅₀ values are in cell-free conditions. Mcl-1-dependent multiple myeloma (MOLP-8) EC₅₀ values represent the caspase activity in MOLP-8 cells. (E) Crystal structure of Staphylococcus aureus SrtA (Protein Data Bank: 1T2W) indicates the presence of several lysine residues (blue) near the binding site. Srt A is inhibited by cyclic peptide 26 and appending an iminoboronate (27) or a diazaborine (28) forming warhead resulted in improved inhibition. The IC₅₀ values shown are from cell-free conditions. Figure (A) created with BioRender.com. Abbreviations: CAII, bovine carbonic anhydrase II; HAS, human serum albumin; IL2, human interleukin-2.

Figure 4.. Covalent proteolysis targeting chimeras (PROTACs).

(A) Examples of covalent PROTACs, including a first unsuccessful attempt (29) and an irreversible PROTAC (30). (B) A reversible covalent PROTAC (31), including its mechanism of action that involves the formation of a thioether between 31 and Bruton’s tyrosine kinase (BTK) Cys⁴⁸¹. PROTAC structure: BTK ligand (blue), the known cereblon (CRBN) ligand pomalidomide (pink), and the pomalidomide with linker (POM). The noncovalent analog, 32, resulted in loss of activity. MOLM-14 half-maximal degradation concentration (DC₅₀) values represent BTK degradation in MOLM-14 cells. Figure (B) created with BioRender.com.

Figure I.. General mechanism of proteolysis targeting chimeras (PROTACs). Figure created with BioRender.com.

Abbreviation: POI, protein of interest.