Small Molecule Inhibitors Targeting the Ubiquitin-Proteasome System

The ubiquitin-proteasome system (UPS) is an important mechanism for intracellular protein quality control, which maintains intracellular protein homeostasis by labeling proteins and sending them to the proteasome for degradation. In recent years, research on small molecule inhibitors targeting the ubiquitin-proteasome system has attracted much attention. These inhibitors can modulate specific proteasome subsystems, thereby affecting physiological processes and disease development within the cell.

Ubiquitin-Proteasome System (UPS)

The ubiquitin-proteasome system (UPS) is one of the vital protein degradation pathways in human cells, mainly comprising two major processes: substrate protein ubiquitination and degradation of ubiquitinated proteins by the proteasome. Ubiquitin-tagged proteins can be recognized and degraded by the 26S proteasome, or degraded via the autophagy-lysosome pathway, or have their ubiquitin tags removed by deubiquitinating enzymes (DUBs), thereby regulating downstream related signals. The 26S proteasome can effectively degrade short-lived, soluble, unfolded, or misfolded proteins and peptides, while the autophagy-lysosome pathway is responsible for the degradation of long-lived proteins, insoluble protein aggregates, and dysfunctional organelles (such as damaged mitochondria), with ubiquitination modification being the core of both pathways.

Increasing evidence suggests that dysfunction in the UPS can affect processes such as cell cycle regulation, cell growth, proliferation, apoptosis, DNA repair, and other cellular signaling, and is closely associated with the development of malignant tumors, cardiovascular diseases, neurodegenerative diseases, and other disorders. Therefore, the UPS has become one of the targets for the treatment of related diseases. Inhibiting the ubiquitin-proteasome system, regulating the degradation of relevant substrate proteins, and achieving disease treatment are important strategies for drug development.

Small Molecule Inhibitors Targeting the Ubiquitin-Proteasome System

Currently, research on small molecule inhibitors targeting the ubiquitin-proteasome system mainly includes inhibitors of ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, ubiquitin ligase E3, and proteasome inhibitors.

Small Molecule Inhibitors Targeting Ubiquitin-Activating Enzyme E1

Currently, only two ubiquitin-activating enzymes E1(UBA1, and UBA6) have been identified in mammalian cells. The classical E1 enzymes have an adenylation domain (AD), catalytic cysteine domain (CCD), and ubiquitin-fold domain (UFD). First, in the presence of ATP and Mg2+, the E1 enzyme catalyzes the formation of a Ub-AMP adduct of Ub with ATP and binds to the AD structural domain of the E1 enzyme while releasing the phosphate group PPi, the cysteine sulfhydryl nucleophilic attack of the CCD structural domain on the Ub-AMP bond to form the Ub-E1 thioester bond and release AMP, and the other Ub noncovalently binds to the AAD structural domain to form the E1 enzyme carrying two Ub molecules of E1 enzyme. The E1 enzyme carrying two molecules of Ub then binds the E2 enzyme and transfers the Ub to the E2 enzyme via the UFD to form the Ub-E2 complex, which then participates in the activation of the next Ub. Therefore, inhibition of E1 enzyme activity can block the activation of ubiquitin molecules and inhibit the ubiquitination process, which is a potential anti-tumor target.

Small Molecule Inhibitors Targeting Ubiquitin-Conjugating Enzyme E2

In mammalian cells, over 40 different E2 enzymes have been identified, which are involved in ubiquitination and ubiquitin-like processes. Ubiquitin molecules activated by E1 enzymes can form E2-Ub complexes with E2 enzymes through thioester bonds. These E2-Ub complexes then bind to various E3 ligases. Under the catalysis of E3 ligases, ubiquitin molecules are transferred to substrate proteins. As the central enzymes in ubiquitination, E2 enzymes determine the topology of ubiquitination and are responsible for recruiting different E3 ligases. Most E2 enzymes contain a ubiquitin-conjugating region (UBC), which allows them to bind to activated ubiquitin or ubiquitin-like molecules. Since the number of ubiquitin-activating E1 enzymes is relatively low, inhibiting E1 enzymes would suppress most ubiquitination processes with poor selectivity. Therefore, targeting E2 enzymes for the development of anticancer drugs can provide specificity that E1 enzyme inhibitors cannot achieve. Currently reported E2 inhibitors can target active sites, allosteric sites, and protein-protein interaction sites.

Small Molecule Inhibitors Targeting Ubiquitin Ligase E3

The ubiquitin E3 ligase specifically recognizes substrate proteins and catalyzes the transfer of the E2-Ub complex to the substrate protein, determining the specificity of ubiquitination. Currently, the human genome encodes over 600 types of E3 ubiquitin ligases. According to their structures and modes of ubiquitin transfer, they can be mainly classified into three types: RING E3 ligases, HECT E3 ligases, and RBR E3 ligases. Among them, the RING E3 ligases constitute the largest superfamily of ubiquitin E3 ligases. They contain an interesting new gene (RING) or U-box domain, with similar folding patterns, but the former binds to zinc ions, whereas the latter does not. RING E3 ligases act as bridges to transfer activated ubiquitin from E2 to the substrate protein, without binding to the ubiquitin molecule. HECT E3 ligases contain the homologous to the E6AP carboxyl terminus (HECT) domain, where a conserved Cys residue can form a thioester bond with the ubiquitin molecule carried by E2, and then transfer the ubiquitin to the substrate protein. RBR E3 ligases contain the RING-between-RING-RING (RBR) domain, namely the RING1 and RING2 domains, separated by the in-between-RING domain (IBR). RING1 recruits E2-Ub, while RING2 contains a catalytic Cys residue that can form a thioester bond with the ubiquitin molecule, transferring the ubiquitin molecule to the substrate protein.

Abnormal expression of ubiquitin E3 ligases is closely related to the occurrence and development of various tumors and poor prognosis, making them an important target for anti-tumor drugs. Numerous small molecule inhibitors have been developed targeting different types of ubiquitin E3 ligases, with the most extensive and in-depth research conducted on MDM2 inhibitors.

Proteasome Inhibitors

Proteasomes are the protein degradation machinery of the ubiquitin-proteasome system. The 26S proteasome is an ATP-dependent proteolytic complex composed of a 20S catalytic core and two 19S regulatory particles. The 20S core consists of two α outer rings and two β inner rings, forming a cylindrical structure of αββα rings, which serves as the catalytic center for protein degradation, conferring proteasomes with proteolytic activity. The 19S regulatory particles are located at both ends of the 20S core, capable of recognizing substrates tagged with ubiquitin, unfolding substrate proteins, and transporting them to the 20S catalytic core for degradation.

Proteasomes in eukaryotic cells are mainly found in the cytoplasm and nucleus. They can degrade unnecessary or misfolded proteins into amino acids, which are then reused for protein synthesis, thereby maintaining protein homeostasis within the cell. Changes in proteasome activity can affect the physiological functions of cells. Compared to normal cells, the life activities of tumor cells are more frequent, making them more sensitive to changes in proteasome activity. Therefore, inhibiting proteasome activity can affect cell function, and kill, or inhibit the growth of tumor cells, making it an important target in the research of anti-tumor drugs. Additionally, altering proteasome activity by inhibiting proteolytic cleavage sites has also become a hot topic in research related to immunology, inflammation, and other fields. Currently, many proteasome inhibitors have been approved and reported, demonstrating effective tumor-suppressive effects.

References

1. LaPlante G.; Zhang W. Targeting the ubiquitin-proteasome system for cancer therapeutics by small-molecule inhibitors. Cancers. 2021, 13(12): 3079.
2. Wu H. Q.; et al. Small molecules that target the ubiquitin system. Biochemical Society Transactions. 2020, 48(2): 479-497.