Identification and optimization of molecular glue compounds that inhibit a noncovalent E2 enzyme–ubiquitin complex

Abstract

Pharmacological control of the ubiquitin-proteasome system (UPS) is of intense interest in drug discovery. Here, we report the development of chemical inhibitors of the ubiquitin-conjugating (E2) enzyme CDC34A (also known as UBE2R1), which donates activated ubiquitin to the cullin-RING ligase (CRL) family of ubiquitin ligase (E3) enzymes. A FRET-based interaction assay was used to screen for novel compounds that stabilize the noncovalent complex between CDC34A and ubiquitin, and thereby inhibit the CDC34A catalytic cycle. An isonipecotamide hit compound was elaborated into analogs with ~1000-fold increased potency in stabilizing the CDC34A-ubiquitin complex. These analogs specifically inhibited CDC34A-dependent ubiquitination in vitro and stabilized an E2~ubiquitin thioester reaction intermediate in cells. The x-ray crystal structure of a CDC34A-ubiquitin-inhibitor complex uncovered the basis for analog structure-activity relationships. The development of chemical stabilizers of the CDC34A-ubiquitin complex illustrates a general strategy for de novo discovery of molecular glue compounds that stabilize weak protein interactions.

Molecular Glues: Capable Protein-Binding Small Molecules That Can Change Protein–Protein Interactions and Interactomes for the Potential Treatment of Human Cancer and Neurodegenerative Diseases

“…Based on this CC0651 prototype molecular glue (MG), a time-resolved Forster resonance energy transfer (TR-FRET)-based assay ( Figure 9 B) was developed and used to screen a total of 995 structurally diverse chemical compounds. These compounds were chosen from an Asinex protein–protein interaction (PPI) library of 10,496 compounds through 2D fingerprinting clustering (221), computer docking (626), and structure uniqueness-based selection (148) in parallel with CC065 as positive controls and DMSO as negative controls [ 37 ].…”

“…One hit compound (BDC22455743) shared chemical structure similarity to CC0651 that could stabilize the noncovalent complex between CDC34A and ubiquitin to inhibit ubiquitin transfer to the substrate ( Figure 9 A,C). Through a series of medicinal chemistry synthetic strategy to generate BDC22455743-based analogs in parallel with TR-FRET assay testing, these authors found that a CC0651 and BDC22455743 hybrid core compound ( Figure 9 C)-based derivatives exhibits a stronger ubiquitin (Ub) IC 50 (0.03–0.2 µM) and TR-FRET EC 50 (0.44–3.6 µM) for compounds 2ab, 2cb, 2db, 2gb, and 2aη versus Ub IC 50 2.4 µM for CC0651 and TR-FRET EC 50 36 µM for CC0651 [ 37 ] ( Figure 9 C,D). These findings lay a foundation for further studies of the antitumor activity and efficacy in vitro and in vivo for the lead compounds.…”

“…EC 50 values represent the mean +/− SD, n = 3. ( B , D ) are adapted from an original paper by St-Cyr et al (some information is also from their provided supplemental materials) [ 37 ].…”

Investigation of the In Vivo, In Vitro, and In Silico Wound Healing Potential of Pinctada martensii Purified Peptides

“…Molecular docking is an effective and reliable computational technique for predicting possible binding modes and studying the ligand binding mechanism between small molecules and proteins. Molecular docking is widely used in structural molecular biology and drug discovery [ 19 ], which is a useful technology to identify the binding mode or force of ligand protein complexes. Binding energy is also an important criterion for considering the interaction between proteins and ligands, and the lowest binding energy is considered to be more stable.…”

Molecular glues: The adhesive connecting targeted protein degradation to the clinic

“…46 These compounds are currently in Phase I/II clinical trials for treatment of multiple myeloma, non-Hodgkin lymphoma, and systemic lupus erythematosus. 70,[85][86][87] CFT7455 (Table 3) is a next-generation IKZF1/3 degrader binding to CRBN E3 ligase.…”