AM404 Analogs as Activators of the 20S Isoform of the Human Proteasome.

The proteasome is a multisubunit protease system responsible for the majority of the protein turnover in eukaryotic organisms. Dysregulation of this enzymatic complex leads to protein accumulation, subsequent aggregation, and ultimately diseased states; for that reason, positive modulation of its activity has been recently investigated as a therapeutic strategy for neurodegenerative and age-related diseases. The small molecule AM404 was recently identified as an activator of the 20S isoform of the proteasome and further exploration of the scaffold revealed the importance of the polyunsaturated fatty acid chain to elicit activity. Herein, we report the investigation of the aromatic region of the scaffold and the evaluation of the small molecules in a variety of proteasome activity and protein degradation assays. We found that derivatives A22 and A23, compared to AM404, exhibit enhanced proteasome activity in biochemical and cellular proteasome assays and more favorable cellular viability profiles. Additionally, these compounds demonstrate the ability to degrade intrinsically disordered proteins, regardless of their molecular weight, and the ability to restore the proteasome activity in the presence of toxic oligomeric α-Syn species in a biochemical setting.

ByeTAC: Bypassing an E3 Ligase for Targeted Protein Degradation.

Targeted protein degradation utilizing a bifunctional molecule to initiate ubiquitination and subsequent degradation by the 26S proteasome has been shown to be a powerful therapeutic intervention. Many bifunctional molecules, including covalent and non-covalent ligands to proteins of interest, have been developed. The traditional target protein degradation methodology targets the protein of interest in both healthy and diseased cell populations, and a therapeutic window is obtained based on the overexpression of the targeted protein. We report here a series of bifunctional degraders that do not rely on interacting with an E3 ligase, but rather a 26S proteasome subunit, which we have named ByeTACs: Bypassing E3 Targeting Chimeras. Rpn-13 is a non-essential ubiquitin receptor for the 26S proteasome. Cells under significant stress or require significant ubiquitin-dependent degradation of proteins for survival, incorporate Rpn-13 in the 26S to increase protein degradation rates. The targeted protein degraders reported here are bifunctional molecules that include a ligand to Rpn-13 and BRD4, the protein of interest we wish to degrade. We synthesized a suite of degraders with varying PEG chain lengths and showed that bifunctional molecules that incorporate a Rpn-13 binder (TCL1) and a BRD4 binder (JQ1) with a PEG linker of 3 or 4 units are the most effective to induce BRD4 degradation. We also demonstrate that our new targeted protein degraders are dependent upon proteasome activity and Rpn-13 expression levels. This establishes a new mechanism of action for our ByeTACs that can be employed for the targeted degradation of a wide variety of protein substrates.

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. In this work, cyclic peptide proteasome stimulators (CyPPSs) that enhance the clearance of misfolded proteins were discovered. In the initial screen of predicted natural products (pNPs), several cyclic peptides were found to stimulate the 20S core particle (20S CP). Development of a robust structural activity relationship led to the identification of potent, cell permeable CyPPSs. In vitro assays revealed that CyPPSs stimulate degradation of highly disordered and misfolded proteins without affecting ordered proteins. Furthermore, using a novel flow-based assay for proteasome activity, several CyPPSs were found to stimulate the 20S CP in cellulo. Overall, this work describes the development of CyPPSs as chemical tools capable of stimulating the proteasome and provides strong support for proteasome stimulation as a therapeutic strategy for neurodegenerative diseases.

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). One such 19S RP subunit, Rpn-13, is a ubiquitin receptor required for hematological cancers to rapidly degrade proteins to avoid apoptosis. Reported Rpn-13 inhibitors covalently bind to the Rpn-13's Pru domain and have been effective anti-hematological cancer agents. Here, we describe the discovery of TCL-1, a non-covalent binder to the Pru domain. Optimization of TCL-1's carboxylate group to an ester increases its cytotoxicity in hematological cancer cell lines. Altogether, our data provides a new scaffold for future medicinal chemistry optimization to target Rpn-13 therapeutically.

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 ß5 substrate channel proceeding the catalytically active threonine residue. There is potential for positive interactions with a substrate to increase selectivity or cleavage rate with the ß5 substrate channel after the catalytic threonine as evidenced by the proteasome inhibitor belactosin. To study what moieties the proteasome could accept in its primed substrate channel, we developed a liquid chromatography- mass spectrometry (LC-MS) method to quantitate the cleavage of substrates by purified human proteasome. This method allowed us to rapidly evaluate proteasome substrates that contain a moiety that could interact with the S1' site of the ß5 proteasome channel. We were able to determine a preference for a polar moiety at the S1' substrate position. We believe this information can be used in the design of future inhibitors or activity-based probes for the proteasome.

Oleic amide derivatives as small molecule stimulators of the human proteasome's core particle.

A series of oleic acid amide derivatives were synthesized based on our previous and continuing endeavors towards stimulation of the 20S core particle of the proteasome (20S CP) with the goal of increasing the protein degradation rate via the ubiquitin-independent pathway. The designed compounds were tested in a variety of biochemical and cell-based assays to assess their ability to increase the rate of hydrolysis of the 20S CP, and compared to a known fatty acid amide stimulator of the 20S CP, AM-404. AM-404 was previously described to stimulate the activity of the 20S CP, however, it does negatively affect viability of cells after prolonged dosing. Here we report the development of several small molecules with a similar ability to enhance the activity of the 20S CP as AM-404. While one molecule (17) was just as potent as AM-404, it still caused significant unwanted cytotoxicity. Molecules such as these are compatible with biochemical assays and short-term cell-based proteasome activity assays, but their unwanted toxicity limits their use in prolonged cell assays or in vivo studies.

Synthesis and Application of a Clickable Epoxomicin-Based Probe for Proteasome Activity Analysis.

The proteasome is a multisubunit protein complex responsible for the degradation of proteins, making it essential in myriad cellular processes. Several reversible and irreversible peptide substrates inspired by known proteasome inhibitors have been developed to visualize it and monitor its activity; however, they have limited commercial availability or possess fluorophores that overlap with other known chemical probes, limiting their simultaneous use. The protocols presented here describe the synthesis of a clickable epoxomicin-based probe followed by the copper-catalyzed installment of an azide-containing fluorophore, and the application of the synthesized peptide in proteasome activity assays by SDS-PAGE and flow cytometry. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Solid-phase synthesis of clickable peptide fragment (2) Basic Protocol 2: In-solution coupling of epoxy-ketone moiety to fragment (2) Basic Protocol 3: Copper-catalyzed click reaction of (3) with fluorophore of choice Basic Protocol 4: Monitoring proteasome activity by SDS-PAGE in HEK-293T cells Alternate Protocol: Monitoring proteasome activity by flow cytometry in HEK-293T cells.

Solid-Phase Synthesis and Application of a Clickable Version of Epoxomicin for Proteasome Activity Analysis.

Degradation of proteins by the proteasome is an essential cellular process and one that many wish to study in a variety of disease types. There are commercially available probes that can monitor proteasome activity in cells, but they typically contain common fluorophores that limit their simultaneous use with other activity-based probes. In order to exchange the fluorophore or incorporate an enrichment tag, the proteasome probe likely has to be synthesized which can be cumbersome. Here, we describe a simple synthetic procedure that only requires one purification step to generate epoxomicin, a selective proteasome inhibitor, with a terminal alkyne. Through a copper-catalyzed cycloaddition, any moiety containing an azide can be incorporated into the probe. Many fluorophores are commercially available that contain an azide that can be "clicked", allowing this proteasome activity probe to be included into already established assays to monitor both proteasome activity and other cellular activities of interest.

Hyperexcitability and Pharmacological Responsiveness of Cortical Neurons Derived from Human iPSCs Carrying Epilepsy-Associated Sodium Channel Nav1.2-L1342P Genetic Variant.

With the wide adoption of genomic sequencing in children having seizures, an increasing number of SCN2A genetic variants have been revealed as genetic causes of epilepsy. Voltage-gated sodium channel Nav1.2, encoded by gene SCN2A, is predominantly expressed in the pyramidal excitatory neurons and supports action potential (AP) firing. One recurrent SCN2A genetic variant is L1342P, which was identified in multiple patients with epileptic encephalopathy and intractable seizures. However, the mechanism underlying L1342P-mediated seizures and the pharmacogenetics of this variant in human neurons remain unknown. To understand the core phenotypes of the L1342P variant in human neurons, we took advantage of a reference human-induced pluripotent stem cell (hiPSC) line from a male donor, in which L1342P was introduced by CRISPR/Cas9-mediated genome editing. Using patch-clamping and microelectrode array (MEA) recordings, we revealed that cortical neurons derived from hiPSCs carrying heterozygous L1342P variant have significantly increased intrinsic excitability, higher sodium current density, and enhanced bursting and synchronous network firing, suggesting hyperexcitability phenotypes. Interestingly, L1342P neuronal culture displayed a degree of resistance to the anticonvulsant medication phenytoin, which recapitulated aspects of clinical observation of patients carrying the L1342P variant. In contrast, phrixotoxin-3 (PTx3), a Nav1.2 isoform-specific blocker, can potently alleviate spontaneous and chemically-induced hyperexcitability of neurons carrying the L1342P variant. Our results reveal a possible pathogenic underpinning of Nav1.2-L1342P mediated epileptic seizures and demonstrate the utility of genome-edited hiPSCs as an in vitro platform to advance personalized phenotyping and drug discovery.SIGNIFICANCE STATEMENT A mounting number of SCN2A genetic variants have been identified from patients with epilepsy, but how SCN2A variants affect the function of human neurons contributing to seizures is still elusive. This study investigated the functional consequences of a recurring SCN2A variant (L1342P) using human iPSC-derived neurons and revealed both intrinsic and network hyperexcitability of neurons carrying a mutant Nav1.2 channel. Importantly, this study recapitulated elements of clinical observations of drug-resistant features of the L1342P variant, and provided a platform for in vitro drug testing. Our study sheds light on cellular mechanism of seizures resulting from a recurring Nav1.2 variant, and helps to advance personalized drug discovery to treat patients carrying pathogenic SCN2A variant.

Protein degradation profile reveals dynamic nature of 20S proteasome small molecule stimulation.

Small molecules have been discovered to stimulate the 20S core particle (CP) of the proteasome to degrade proteins. However, the impact a 20S CP stimulator can have on the regulation of protein levels has not been fully characterized. Previous studies have focused on using one kind of stimulator to enhance the degradation of specific 20S CP substrates. We present here a study that utilizes several 20S CP stimulators to determine how each can affect the degradation of proteins in a biochemical assay with purified proteins and of an overexpressed GFP-fusion protein in cells. We also evaluate the effects of two stimulators on the whole cellular proteome in HEK-293T cells using label-free quantitative proteomic analysis for a broader understanding on their impact. Our studies demonstrate that 20S CP stimulation is likely to promote the degradation of significantly disordered proteins; however, the specific effect on the regulation of protein levels appears to be dependent on the mechanism of action of each stimulator due to the dynamic nature of the 20S CP. Our results reveal the potential of tailoring small molecule stimulators to influence the degradation of certain protein types and 20S CP substrates.

A Yeast Chronological Lifespan Assay to Assess Activity of Proteasome Stimulators.

Aging is characterized by changes in several cellular processes, including dysregulation of proteostasis. Current research has shown long-lived rodents display elevated proteasome activity throughout life and proteasome dysfunction is linked to shorter lifespans in a transgenic mouse model. The ubiquitin proteasome system (UPS) is one of the main pathways leading to cellular protein clearance and quality maintenance. Reduction in proteasome activity is associated with aging and its related pathologies. Small molecule stimulators of the proteasome have been proposed to help alleviate cellular stress related to unwanted protein accumulation. Here we have described the development of techniques to monitor the impact of proteasome stimulation in wild-type yeast and a strain that has impaired proteasome expression. We validated our chronological lifespan assay using both types of yeast with a variety of small molecule stimulators at different concentrations. By modifying the media conditions for the yeast, molecules can be evaluated for their potential to increase chronological lifespan in five days. Additionally, our assay conditions can be used to monitor the activity of proteasome stimulators in modulating the degradation of a YFP-α-synuclein fusion protein produced by yeast. We anticipate these methods to be valuable for those wishing to study the impact of increasing proteasome-mediated degradation of proteins in a eukaryotic model organism.

Approaches to Evaluate the Impact of a Small-Molecule Binder to a Noncatalytic Site of the Proteasome.

Proteasome activity is crucial for cell survival and proliferation. In recent years, small molecules have been discovered that can affect the catalytic activity of the proteasome. Rather than targeting the active sites of the proteasome, it might be possible to affect ubiquitin-dependent degradation of proteins by limiting the association of the 19S regulatory particle (19S RP) with the 20S core particle (20S CP) of the proteasome. We recently described the discovery of TXS-8, a peptoid that binds to Rpn-6. Rpn-6 is a proteasome-associated protein that makes critical contacts with the 19S RP and the 20S CP. Herein, we present a general workflow to evaluate the impact of a small-molecule binder on proteasome activity by using TXS-8 as an example. This workflow contains three steps in which specific probes or overexpressed proteins in cells are used to determine whether the hydrolysis activity of the proteasome is affected. Although, in our case, TXS-8 did not affect proteasome activity, our workflow is highly amenable to studying a variety of small-molecule-proteasome subunit interactions.

Identification of a covalent binder to the oncoprotein gankyrin using a NIR-Based OBOC screening method.

Despite huge advancements in the process of synthesizing small molecules as part of one-bead-one-compound (OBOC) libraries, progress lags in the ability to screen these libraries against proteins of interest. Recently, we developed a method to screen OBOC libraries in which a target protein is labeled with a near-infrared (NIR) range fluorophore. The labeled protein incubates with beads of a library in a 96-well plate, then the plate is imaged for fluorescence. Fluorescence intensities produced by the labeled protein binding the bead can be quantitated and provide a basis to rank hits. Here, we present an application of this technique by screening the oncoprotein gankyrin against a 343-member peptoid library. The library was composed of four positions occupied by one of seven amines. In the third position, an amine that facilitates covalent binding via a sulfonyl fluoride moiety was incorporated. After screening for gankyrin binders twice, ten structures showed overlap in the types of amines present at each position. These initial hits were validated with an in-gel fluorescence assay in which the labeled ligands covalently interacted with purified gankyrin. Excitingly, one peptoid was validated from this analysis. This hit was also shown to bind gankyrin in the presence of HEK 293T lysate. Results from this study demonstrate successful use of our screening method to quickly identify quality binders to a target protein of interest.

Methods for the discovery of small molecules to monitor and perturb the activity of the human proteasome.

Regulating protein production and degradation is critical to maintaining cellular homeostasis. The proteasome is a key player in keeping proteins at the proper levels. However, proteasome activity can be altered in certain disease states, such as blood cancers and neurodegenerative diseases. Cancers often exhibit enhanced proteasomal activity, as protein synthesis is increased in these cells compared with normal cells. Conversely, neurodegenerative diseases are characterized by protein accumulation, leading to reduced proteasome activity. As a result, the proteasome has emerged as a target for therapeutic intervention. The potential of the proteasome as a therapeutic target has come from studies involving chemical stimulators and inhibitors, and the development of a suite of assays and probes that can be used to monitor proteasome activity with purified enzyme and in live cells.

Fluorescent Probes with Unnatural Amino Acids to Monitor Proteasome Activity in Real-Time.

The proteasome is an essential protein complex that, when dysregulated, can result in various diseases in eukaryotic cells. As such, understanding the enzymatic activity of the proteasome and what can alter it is crucial to elucidating its roles in these diseases. This can be done effectively by using activity-based fluorescent substrate probes, of which there are many commercially available that target the individual protease-like subunits in the 20S CP of the proteasome. Unfortunately, these probes have not displayed appropriate characteristics for their use in live cell-based assays. In the work presented here, we have developed a set of probes which have shown improved fluorescence properties and selectivity toward the proteasome compared to other cellular proteases. By including unnatural amino acids, we have found probes which can be utilized in various applications, including monitoring the effects of small molecule stimulators of the proteasome in live cells and comparing the relative proteasome activity across different cancer cell types. In future studies, we expect the fluorescent probes presented here will serve as tools to support the discovery and characterization of small molecule modulators of proteasome activity.

Designing Chimeric Molecules for Drug Discovery by Leveraging Chemical Biology.

After the first seed concept introduced in the 18th century, different disciplines have attributed different names to dual-functional molecules depending on their application, including bioconjugates, bifunctional compounds, multitargeting molecules, chimeras, hybrids, engineered compounds. However, these engineered constructs share a general structure: a first component that targets a specific cell and a second component that exerts the pharmacological activity. A stable or cleavable linker connects the two modules of a chimera. Herein, we discuss the recent advances in the rapidly expanding field of chimeric molecules leveraging chemical biology concepts. This Perspective is focused on bifunctional compounds in which one component is a lead compound or a drug. In detail, we discuss chemical features of chimeric molecules and their use for targeted delivery and for target engagement studies.

Discovery of a Small Molecule Probe of Rpn-6, an Essential Subunit of the 26S Proteasome.

A considerable number of essential cellular proteins have no catalytic activity and serve instead as structural components to aid in assembling protein complexes. For example, the assembly and function of the 26S proteasome, the major enzymatic complex necessary for ubiquitin-dependent protein degradation, require a number of essential protein contacts to associate the 19S regulatory particle with the 20S core particle. Previously, small molecule inhibitors of the active sites of the 20S core particle have been developed, but the activity of the 26S proteasome could also be altered via the disruption of its assembly. We were interested in discovering a small molecule binder of Rpn-6, as it is among several essential proteins that facilitate 26S assembly, which could be used to further our understanding of the association of the 19S regulatory particle with the 20S core particle. Additionally, we were interested in whether a small molecule-Rpn-6 interaction could potentially be cytotoxic to cancer cells that rely heavily on proteasome activity for survival. A workflow for utilizing a one-bead, one-compound library and a thermal shift assay was developed to discover such a molecule. TXS-8, our lead hit, was discovered to have a low micromolar binding affinity for Rpn-6 as well as very limited binding to other proteins. The cytotoxicity of TXS-8 was evaluated in several cell lines, revealing increased cytotoxicity to hematological cancers. Discovery of this peptoid binder of Rpn-6 provides the initial evidence that Rpn-6 could be a druggable target to affect protein degradation and serves as a primary scaffold from which to design more potent binders. We suspect that Rpn-6 could have additional essential roles beyond that of a molecular clamp of the proteasome to help hematological cancer cells survive and that TXS-8 can serve as a useful tool for further elucidating its roles.

The Immunoproteasome: An Emerging Target in Cancer and Autoimmune and Neurological Disorders.

The immunoproteasome (iCP) is an isoform of the 20S proteasome that is expressed when cells are stressed or receive an inflammatory signal. The primary role of the iCP is to hydrolyze proteins into peptides that are compatible with being loaded into a MHC-I complex. When the activity of the iCP is dysregulated or highly expressed, it can lead to unwanted cell death. Some cancer types express the iCP rather than the standard proteasome, and selective inhibitors have been developed to exploit this difference. Here, we describe diseases known to be influenced by iCP activity and the current status for targeting the iCP to elicit a therapeutic response. We also describe a variety of chemical tools that have been developed to monitor the activity of the iCP in cells. Finally, we present the future outlook for targeting the iCP in a variety of disease types and with mechanisms besides inhibition.

Synthesis and Application of an Activity-Based Peptide-Peptoid Hybrid Probe for the Immunoproteasome.

The immunoproteasome (iCP), a specific isoform of the proteasome's catalytic particle, is becoming an important protein complex of interest in various diseases. However, there is still much left to be learned about its activity in cells and how this can be altered by various endogenous conditions or with treatment with small molecules. Current strategies to investigate the iCP lack in their ability to be used in live, intact cells, limiting them to use in endpoint experiments. The iCP-selective probe presented here has been shown to be compatible with various live-cell assays, including monitoring iCP activity kinetically in a plate reader-based assay and observing single cells with confocal microscopy. A well-studied iCP-selective inhibitor, ONX-0914, has also been demonstrated to decrease the fluorescence signal of the iCP probe in both of these assays, showing its potential function in investigating small-molecule modulators of the iCP. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Synthesis of an immunoproteasome-selective peptide-peptoid hybrid probe Basic Protocol 2: Expression of the immunoproteasome in A549 cells Basic Protocol 3: Using the immunoproteasome probe to monitor activity in live cells with a fluorescence plate reader Basic Protocol 4: Using the immunoproteasome probe to monitor activity in live cells with confocal microscopy.

Methods to Discover and Evaluate Proteasome Small Molecule Stimulators.

Protein accumulation has been identified as a characteristic of many degenerative conditions, such as neurodegenerative diseases and aging. In most cases, these conditions also present with diminished protein degradation. The ubiquitin-proteasome system (UPS) is responsible for the degradation of the majority of proteins in cells; however, the activity of the proteasome is reduced in these disease states, contributing to the accumulation of toxic protein. It has been hypothesized that proteasome activity, both ubiquitin-dependent and -independent, can be chemically stimulated to reduce the load of protein in diseased cells. Several methods exist to identify and characterize stimulators of proteasome activity. In this review, we detail the ways in which protease activity can be enhanced and analyze the biochemical and cellular methods of identifying stimulators of both the ubiquitin-dependent and -independent proteasome activities.