Covalent Fragment Screening and Optimization Identifies the Chloroacetohydrazide Scaffold as Inhibitors for Ubiquitin C-terminal Hydrolase L1.

Dysregulation of the ubiquitin-proteasome systems is a hallmark of various disease states including neurodegenerative diseases and cancer. Ubiquitin C-terminal hydrolase L1 (UCHL1), a deubiquitinating enzyme, is expressed primarily in the central nervous system under normal physiological conditions, however, is considered an oncogene in various cancers, including melanoma, lung, breast, and lymphoma. Thus, UCHL1 inhibitors could serve as a viable treatment strategy against these aggressive cancers.

High-throughput assay exploiting disorder-to-order conformational switches: application to the proteasomal Rpn10:E6AP complex.

Conformational switching is pervasively driven by protein interactions, particularly for intrinsically disordered binding partners. We developed a dually orthogonal fluorescence-based assay to monitor such events, exploiting environmentally sensitive fluorophores. This assay is applied to E3 ligase E6AP, as its AZUL domain induces a disorder-to-order switch in an intrinsically disordered region of the proteasome, the so-named Rpn10 AZUL-binding domain (RAZUL).

Discovery and Development of Cyclic Peptide Proteasome Stimulators.

The proteasome degrades proteins, which is essential for cellular homeostasis. Ubiquitin independent proteolysis degrades highly disordered and misfolded proteins. A decline of proteasomal activity has been associated with multiple neurodegenerative diseases due to the accumulation of misfolded proteins.

Discovery of a non-covalent ligand for Rpn-13, a therapeutic target for hematological cancers.

The ubiquitin-proteasome system serves as the major proteolytic degradation pathway in eukaryotic cells. Many inhibitors that covalently bind to the proteasome's active sites have been developed for hematological cancers, but resistance can arise in patients. To overcome limitations of active-site proteasome inhibitors, we and others have focused on developing ligands that target subunits on the 19S regulatory particle (19S RP).

20S proteasome hydrolysis of LLVY substrates to determine preferences for moieties in its primed substrate channel.

The proteasome is an essential multi-catalytic enzyme in cells that is responsible for degrading proteins with a ubiquitin-dependent or -independent mechanism. Many activity-based probes, inhibitors, and stimulators have been developed to study or modulate the activity of the proteasome. The development of these proteasome probes or inhibitors have been based on their interaction with the amino acids of the β5 substrate channel proceeding the catalytically active threonine residue.

Targeting KRAS Mutant Cancers via Combination Treatment: Discovery of a Pyridopyridazinone pan-RAF Kinase Inhibitor.

Structure-based optimization of a set of aryl urea RAF inhibitors has led to the identification of Type II pan-RAF inhibitor GNE-9815 (), which features a unique pyrido[2,3-]pyridazin-8(7)-one hinge-binding motif. With minimal polar hinge contacts, the pyridopyridazinone hinge binder moiety affords exquisite kinase selectivity in a lipophilic efficient manner. The improved physicochemical properties of GNE-9815 provided a path for oral dosing without enabling formulations.

Targeting KRAS Mutant Cancers via Combination Treatment: Discovery of a 5-Fluoro-4-(3)-quinazolinone Aryl Urea pan-RAF Kinase Inhibitor.

Optimization of a series of aryl urea RAF inhibitors led to the identification of type II pan-RAF inhibitor GNE-0749 (), which features a fluoroquinazolinone hinge-binding motif. By minimizing reliance on common polar hinge contacts, this hinge binder allows for a greater contribution of RAF-specific residue interactions, resulting in exquisite kinase selectivity. Strategic substitution of fluorine at the C5 position efficiently masked the adjacent polar NH functionality and increased solubility by impeding a solid-state conformation associated with stronger crystal packing of the molecule.

Optimization and Anti-Cancer Properties of Fluoromethylketones as Covalent Inhibitors for Ubiquitin C-Terminal Hydrolase L1.

The deubiquitinating enzyme (DUB) UCHL1 is implicated in various disease states including neurodegenerative disease and cancer. However, there is a lack of quality probe molecules to gain a better understanding on UCHL1 biology. To this end a study was carried out to fully characterize and optimize the irreversible covalent UCHL1 inhibitor VAEFMK.

N-Terminal Targeting of Regulator of G Protein Signaling Protein 2 for F-Box Only Protein 44-Mediated Proteasomal Degradation.

Regulator of G protein signaling (RGS) proteins are negative modulators of G protein signaling that have emerged as promising drug targets to improve specificity and reduce side effects of G protein-coupled receptor-related therapies. Several small molecule RGS protein inhibitors have been identified; however, enhancing RGS protein function is often more clinically desirable but presents a challenge. Low protein levels of RGS2 are associated with various pathologies, including hypertension and heart failure.

Small-Molecule Inhibitors of the Proteasome's Regulatory Particle.

Cells need to synthesize and degrade proteins consistently. Maintaining a balanced level of protein in the cell requires a carefully controlled system and significant energy. Degradation of unwanted or damaged proteins into smaller peptide units can be accomplished by the proteasome.

Analysis of chain length, substitution patterns, and unsaturation of AM-404 derivatives as 20S proteasome stimulators.

Proteasome-mediated degradation of proteins is a vital cellular process and is performed by the ubiquitin-dependent proteasome system (UPS) and the ubiquitin-independent proteasome system (UIPS). While both systems are necessary to maintain healthy cell function, many disease states are characterized by reduced activity of the UPS, and the UIPS cannot by itself maintain proper protein levels. It has been suggested that the 20S core particle (20S CP), the isoform of the proteasome in the UIPS that can degrade proteins without a ubiquitin tag, can be stimulated with a small molecule to assist the 20S CP to accept and hydrolyze substrates more rapidly.