20S CP Research Articles

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.

Method of Monitoring 26S Proteasome in Cells Revealed the Crucial Role of PSMA3 C-Terminus in 26S Integrity.

Proteasomes critically regulate proteostasis via protein degradation. Proteasomes are multi-subunit complexes composed of the 20S proteolytic core particle (20S CP) that, in association with one or two 19S regulatory particles (19S RPs), generates the 26S proteasome, which is the major proteasomal complex in cells. Native gel protocols are used to investigate the 26S/20S ratio. However, a simple method for detecting these proteasome complexes in cells is missing. To this end, using CRISPR technology, we YFP-tagged the endogenous PSMB6 (β1) gene, a 20S CP subunit, and co-tagged endogenous PSMD6 (Rpn7), a 19S RP subunit, with the mScarlet fluorescent protein. We observed the colocalization of the YFP and mScarlet fluorescent proteins in the cells, with higher nuclear accumulation. Nuclear proteasomal granules are formed under osmotic stress, and all were positive for YFP and mScarlet. Previously, we have reported that PSMD1 knockdown, one of the 19 RP subunits, gives rise to a high level of "free" 20S CPs. Intriguingly, under this condition, the 20S-YFP remained nuclear, whereas the PSMD6-mScarlet was mostly in cytoplasm, demonstrating the distinct subcellular distribution of uncapped 20S CPs. Lately, we have shown that the PSMA3 (α7) C-terminus, a 20S CP subunit, binds multiple intrinsically disordered proteins (IDPs). Remarkably, the truncation of the PSMA3 C-terminus is phenotypically reminiscent of PSMD1 knockdown. These data suggest that the PSMA3 C-terminal region is critical for 26S proteasome integrity.

Discovery of Phytoalexin Camalexin and Its Derivatives as Novel Antiviral and Antiphytopathogenic-Fungus Agents

In response to the invasion of plant viruses and pathogenic fungi, higher plants produce defensive allelochemicals. Finding candidate varieties of botanical pesticides based on allelochemicals is one of the important ways to create efficient and green pesticides. Here, a series of camalexin derivatives based on a phytoalexin camalexin scaffold were designed, synthesized, and assessed for their antiviral and fungicidal activities systematically. Most of these camalexin derivatives exhibited better antiviral activities against tobacco mosaic virus (TMV) than the control antiviral agent ribavirin. Under the same test conditions, the anti-TMV activities of compounds 3d, 5a, 5d, and 10f-10h were found to be equivalent to or better than that of ningnanmycin, an agricultural cytosine nucleoside antibiotic with excellent protective effect. The antiviral mechanism research showed that compound 5a could cause 20S CP disk fusion and disintegration, thus affecting the assembly of virus particles. The results of molecular docking indicate that there were obvious hydrogen bonds between compounds 3d, 5a, and 10f and TMV CP. The binding constants of compounds 5a and 10f to TMV CP were also calculated using fluorescence titration. These camalexin derivatives also presented broad spectrum fungicidal activities, especially for Rhizoctonia solani and Physalospora piricola. In this work, the design, synthesis, structure optimization, and mode of action of camalexin derivatives were carried out progressively. This work provides a reference for using defensive chemical compounds as novel pesticide lead compounds.

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.

Toad Alkaloid for Pesticide Discovery: Dehydrobufotenine Derivatives as Novel Agents against Plant Virus and Fungi.

Plant viruses and fungi are a serious threat to food security and natural ecosystems. The efficient and environment-friendly control methods are urgently needed to help safeguard such resources. Here, we achieved the efficient synthesis of toad alkaloid dehydrobufotenine in eight steps with an overall yield of 8% from 5-methoxyindole. A series of dehydrobufotenine derivatives were designed, synthesized, and evaluated for their antiviral and fungicidal activities systematically. It was found for the first time that these compounds have good anti-plant virus activities and anti-plant pathogen activities. The antiviral activities of 21 compounds were similar to or better than those of ribavirin. Compounds 12 and 17 displayed better antiviral activities than ningnanmycin which is perhaps the most effective anti-plant virus agent. The antiviral mechanism research study of 12 revealed that it could make 20S CP disk fusion and aggregation. Further molecular docking results showed that there are hydrogen bonds between compounds 12, 17, and tobacco mosaic virus CP. The docking results are consistent with the antiviral activity. These compounds also displayed broad-spectrum fungicidal activities against 14 kinds of fungi, especially for Sclerotinia sclerotiorum. In this work, the synthesis, structure optimization, structure-activity relationship studies, and mode of action research of dehydrobufotenine alkaloids were carried out. It provides a reference for the development of the anti-plant virus agent and anti-plant pathogen agent from toad alkaloids.

Survival in Hostile Conditions: Pupylation and the Proteasome in Actinobacterial Stress Response Pathways.

Bacteria employ a multitude of strategies to cope with the challenges they face in their natural surroundings, be it as pathogens, commensals or free-living species in rapidly changing environments like soil. Mycobacteria and other Actinobacteria acquired proteasomal genes and evolved a post-translational, ubiquitin-like modification pathway called pupylation to support their survival under rapidly changing conditions and under stress. The proteasomal 20S core particle (20S CP) interacts with ring-shaped activators like the hexameric ATPase Mpa that recruits pupylated substrates. The proteasomal subunits, Mpa and pupylation enzymes are encoded in the so-called Pup-proteasome system (PPS) gene locus. Genes in this locus become vital for bacteria to survive during periods of stress. In the successful human pathogen Mycobacterium tuberculosis, the 20S CP is essential for survival in host macrophages. Other members of the PPS and proteasomal interactors are crucial for cellular homeostasis, for example during the DNA damage response, iron and copper regulation, and heat shock. The multiple pathways that the proteasome is involved in during different stress responses suggest that the PPS plays a vital role in bacterial protein quality control and adaptation to diverse challenging environments.

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.

Proteasome in action: substrate degradation by the 26S proteasome.

Ubiquitination is the major criteria for the recognition of a substrate-protein by the 26S proteasome. Additionally, a disordered segment on the substrate — either intrinsic or induced — is critical for proteasome engagement. The proteasome is geared to interact with both of these substrate features and prepare it for degradation. To facilitate substrate accessibility, resting proteasomes are characterised by a peripheral distribution of ubiquitin receptors on the 19S regulatory particle (RP) and a wide-open lateral surface on the ATPase ring. In this substrate accepting state, the internal channel through the ATPase ring is discontinuous, thereby obstructing translocation of potential substrates. The binding of the conjugated ubiquitin to the ubiquitin receptors leads to contraction of the 19S RP. Next, the ATPases engage the substrate at a disordered segment, energetically unravel the polypeptide and translocate it towards the 20S catalytic core (CP). In this substrate engaged state, Rpn11 is repositioned at the pore of the ATPase channel to remove remaining ubiquitin modifications and accelerate translocation. C-termini of five of the six ATPases insert into corresponding lysine-pockets on the 20S α-ring to complete 20S CP gate opening. In the resulting substrate processing state, the ATPase channel is fully contiguous with the translocation channel into the 20S CP, where the substrate is proteolyzed. Complete degradation of a typical ubiquitin-conjugate takes place over a few tens of seconds while hydrolysing tens of ATP molecules in the process (50 kDa/∼50 s/∼80ATP). This article reviews recent insight into biochemical and structural features that underlie substrate recognition and processing by the 26S proteasome.

The Proteasome Activators Blm10/PA200 Enhance the Proteasomal Degradation of N-Terminal Huntingtin.

The Blm10/PA200 family of proteasome activators modulates the peptidase activity of the core particle (20S CP). They participate in opening the 20S CP gate, thus facilitating the degradation of unstructured proteins such as tau and Dnm1 in a ubiquitin- and ATP-independent manner. Furthermore, PA200 also participates in the degradation of acetylated histones. In our study, we use a combination of yeast and human cell systems to investigate the role of Blm10/PA200 in the degradation of N-terminal Huntingtin fragments (N-Htt). We demonstrate that the human PA200 binds to N-Htt. The loss of Blm10 in yeast or PA200 in human cells results in increased mutant N-Htt aggregate formation and elevated cellular toxicity. Furthermore, Blm10 in vitro accelerates the proteasomal degradation of soluble N-Htt. Collectively, our data suggest N-Htt as a new substrate for Blm10/PA200-proteasomes and point to new approaches in Huntington’s disease (HD) research.

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.

Drug Development Targeting the Ubiquitin-Proteasome System (UPS) for the Treatment of Human Cancers.

Cancer cells are characterized by a higher rate of protein turnover and greater demand for protein homeostasis compared to normal cells. In this scenario, the ubiquitin–proteasome system (UPS), which is responsible for the degradation of over 80% of cellular proteins within mammalian cells, becomes vital to cancer cells, making the UPS a critical target for the discovery of novel cancer therapeutics. This review systematically categorizes all current reported small molecule inhibitors of the various essential components of the UPS, including ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), ubiquitin ligases (E3s), the 20S proteasome catalytic core particle (20S CP) and the 19S proteasome regulatory particles (19S RP), as well as their mechanism/s of action and limitations. We also discuss the immunoproteasome which is considered as a prospective therapeutic target of the next generation of proteasome inhibitors in cancer therapies.

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. The proteasome is composed of two main subunits. The first is the core particle (20S CP), and within this core particle are three types of threonine proteases. The second is the regulatory complex (19S RP), which has a myriad of activities including recognizing proteins marked for degradation and shuttling the protein into the 20S CP to be degraded. Small-molecule inhibitors of the 20S CP have been developed and are exceptional treatments for multiple myeloma (MM). 20S CP inhibitors disrupt the protein balance, leading to cellular stress and eventually to cell death. Unfortunately, the 20S CP inhibitors currently available have dose-limiting off-target effects and resistance can be acquired rapidly. Herein, we discuss small molecules that have been discovered to interact with the 19S RP subunit or with a protein closely associated with 19S RP activity. These molecules still elicit their toxicity by preventing the proteasome from degrading proteins, but do so through different mechanisms of action.

Monitoring the Immunoproteasome in Live Cells Using an Activity-Based Peptide-Peptoid Hybrid Probe.

Activity-based probes have greatly improved our understanding of the intrinsic roles and expression levels of various proteins within cells. To be useful in live cells, probes must be cell permeable and provide a read-out that can be measured without disrupting the cells or the activity of the target. Unfortunately, probes for the various forms of the proteasome that can be utilized in intact cells are limited; commercially available probes are most effectively used with purified protein or cell lysate. The proteasome, both the 26S and various isoforms of the 20S CP, is an important target with reported roles in cancer, autoimmune disorders, and neurodegenerative diseases. Here, we present the development of a selective probe for the immunoproteasome, a specialized isoform of the 20S proteasome, that becomes expressed in cells that encounter an inflammatory signal. Using a one-bead, one-compound library of small peptides, we discovered a trimer sequence efficiently cleaved by the immunoproteasome with significant selectivity over the standard proteasome. Upon conjugating this sequence to rhodamine 110 and a peptoid, we generated a probe with a considerable improvement in sensitivity compared to that of current aminomethylcoumarin-based proteasome probes. Importantly, our probe was capable of labeling immunoproteasome-expressing cells while maintaining its selectivity over other cellular proteases in live cell cultures. We anticipate this probe to find wide utility for those that wish to study the immunoproteasome's activity in a variety of cell lines and to be used as a reporter to discover small molecules that can perturb the activity of this proteasome isoform.

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. Several small molecule stimulators of the 20S CP have since been discovered, including AM-404, an arachidonic acid derivative. AM-404 has previously been shown to inhibit fatty acid amide hydrolase activity. We wished to evaluate what structural components of AM-404 are required to stimulate the 20S CP with the long-term goal of using this information to design a stimulator with better drug-like qualities. We synthesized numerous derivatives of AM-404, varying the chain length, substitutions, and degree of unsaturation. Through this endeavor, we obtained several molecules capable of stimulating the 20S CP to various degrees. We discovered that though chain length is important, the presence of a cis-alkene in a specific location in the aliphatic chain has the greatest impact on the ability to stimulate the 20S CP. Two of the derivatives maintain modest stimulatory activity, and have improved toxicity over AM-404.

A Sensitive High-Throughput Screening Method for Identifying Small Molecule Stimulators of the Core Particle of the Proteasome.

Fluorescence resonance energy transfer (FRET) technology is a useful tool to monitor protein interactions as well as protease activity. We have recently reported a biochemical assay utilizing a FRET reporter peptide to monitor the activity of the 20S catalytic particle (20S CP) of the proteasome. This assay is designed specifically to have increased sensitivity to identify stimulators of the 20S CP, which may hold therapeutic potential to treat protein-accumulation diseases. The protocol described here details the necessary steps in synthesizing the FRET reporter peptide and performing the FRET assay with the 20S CP. © 2018 by John Wiley & Sons, Inc.

Development and Application of a Sensitive Peptide Reporter to Discover 20S Proteasome Stimulators.

To attenuate an overabundance of cellular protein, it has been hypothesized that the 20S core particle (20S CP) of the proteasome can be chemically stimulated to degrade proteins into nontoxic peptides more quickly. Screening for small molecule 20S CP stimulators is typically performed with a reporter peptide composed of four amino acids and a coumarin group that is released upon proteasome-mediated hydrolysis to generate a fluorescent signal. Screening with this small reporter can lead to false negatives because the reporter peptide is rapidly turned-over without stimulation. To improve the screening for 20S CP stimulators, we have developed a peptide FRET reporter nearly four times more sensitive to stimulation but still amenable for high throughput screening. Through application of our FRET reporter, we have discovered two 20S CP gate-opening stimulators and also a molecule that elicits its mechanism of action through an interaction with a 20S CP active site.

Probing the cooperativity of Thermoplasma acidophilum proteasome core particle gating by NMR spectroscopy

The 20S proteasome core particle (20S CP) plays an integral role in cellular homeostasis by degrading proteins no longer required for function. The process is, in part, controlled via gating residues localized to the ends of the heptameric barrel-like CP structure that occlude substrate entry pores, preventing unregulated degradation of substrates that might otherwise enter the proteasome. Previously, we showed that the N-terminal residues of the α-subunits of the CP from the archaeon Thermoplasma acidophilum are arranged such that, on average, two of the seven termini are localized inside the lumen of the proteasome, thereby plugging the entry pore and functioning as a gate. However, the mechanism of gating remains unclear. Using solution NMR and a labeling procedure in which a series of mixed proteasome rings are prepared such that the percentage of gate-containing subunits is varied, we address the energetics of gating and establish whether gating is a cooperative process involving the concerted action of residues from more than a single protomer. Our results establish that the intrinsic probability of a gate entering the lumen favors the in state by close to 20-fold, that entry of each gate is noncooperative, with the number of gates that can be accommodated inside the lumen a function of the substrate entry pore size and the bulkiness of the gating residues. Insight into the origin of the high affinity for the in state is obtained from spin-relaxation experiments. More generally, our approach provides an avenue for dissecting interactions of individual protomers in homo-oligomeric complexes.

Identification of 4-arylidene curcumin analogues as novel proteasome inhibitors for potential anticancer agents targeting 19S regulatory particle associated deubiquitinase

The proteasomal 19S regulatory particle (RP) associated deubiquitinases (DUBs) have attracted much attention owing to their potential as a therapeutic target for cancer therapy. Identification of new entities against 19S RP associated DUBs and illustration of the underlying mechanisms is crucial for discovery of novel proteasome blockers. In this study, a series of 4-arylidene curcumin analogues were identified as potent proteasome inhibitor by preferentially blocking deubiquitinase function of proteasomal 19S RP with moderate 20S CP inhibition. The most active compound 33 exhibited a major inhibitory effect on 19S RP-associated ubiquitin-specific proteases 14, along with a minor effect on ubiquitin C-terminal hydrolase 5, which resulted in dysfunction of proteasome, and subsequently accumulated ubiquitinated proteins (such as IκB) in several cancer cells. Remarkably, though both 19S RP and 20S CP inhibition induced significantly endoplasmic reticulum stress and triggered caspase-12/9 pathway activation to promote cancer cell apoptosis, the 19S RP inhibition by 33 avoided slow onset time, Bcl-2 overexpression, and PERK-phosphorylation, which contribute to the deficiencies of clinical drug Bortezomib. These systematic studies provided insights in the development of novel proteasome inhibitors for cancer treatment.

Abstract 1736: The anticancer activity of the DUB inhibitor b-AP15 is associated with accumulation of proteasome bound ubiquitin and oxidative stress

Abstract Background: The ubiquitin proteasome system (UPS) is the major cellular degradation system for damaged or short-lived proteins. Tumor cells are dependent on a functioning UPS, making it an ideal target for the development of anti-cancer therapies. Bortezomib, an inhibitor of the proteolytic activities of the proteasome core particle (20SCP), is approved for treatment of multiple myeloma and mantle cell lymphoma; however several problems have emerged including dose-limiting toxicity and tumor relapse. We previously identified b-AP15 as a novel inhibitor of the proteasome associated deubiquitinases (DUBs) USP14 and UCH37/UCHL5. Treatment with b-AP15 induced cell death in several in vitro and in vivo cancer models. The aim of this study was to investigate how the different modes of UPS inhibition affect the structure of the 26S proteasome and cellular response as well as the potential for proteasome DUB inhibitors as a therapeutic option for cancer. Methods: HCT116 colon carcinoma cells were cultured in McCoy's 5A modified medium/10% fetal calf serum. For protein expression analysis cell extracts were resolved by Tris-Acetate gel and transferred to nitrocellulose membrane for western blotting. Proteasome integrity and cellular distribution of polyubiquitin chains were assessed in cell extracts subjected to density gradient centrifugation, conducted in 4-24% linear glycerol gradients, followed by western blot analysis. Apoptotic response was assessed with M30-CytoDeath ELISA, measuring level of caspase-cleavage of the endogenous cellular substrate keratin-18. Results: We found that inhibitors of proteasome function do not alter proteasome assembly or structure; however treatment induced the accumulation of polyubiquitinated proteins associated with the 26S proteasome. Comparison of 20S CP and 19S DUB inhibition showed that b-AP15 induced more rapid and sustained apoptotic response in comparison to bortezomib. Proteasome inhibition has been previously associated with endoplasmic reticulum and oxidative stress, however our findings show that reactive oxygen species (ROS) are main mediators of b-AP15 induced apoptosis. b-AP15 strongly induces several genes associated with oxidative stress response; hemoxygenase-1 (HMOX1), p38-MAPK, c-JUN and Jun N-terminal kinase (JNK). Additionally, treatment with a pharmacological inhibitor of JNK and several ROS scavengers decreased the levels of apoptosis in response to b-AP15 treatment. Conclusions: Cellular response to b-AP15 mediated proteasome inhibition is distinct from clinically used bortezomib; therefore b-AP15 has a potential clinical use, in particular for treatment of malignancies resistant to other proteasome inhibitors. Citation Format: Magdalena Mazurkiewicz, Slavica Brnjic, Mårten Fryknäs, Chao Sun, Xiaonan Zhang, Rolf Larsson, Pádraig D'Arcy, Stig Linder. The anticancer activity of the DUB inhibitor b-AP15 is associated with accumulation of proteasome bound ubiquitin and oxidative stress. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1736. doi:10.1158/1538-7445.AM2015-1736

Synthesis and evaluation of derivatives of the proteasome deubiquitinase inhibitor b-AP15.

The ubiquitin-proteasome system (UPS) is increasingly recognized as a therapeutic target for the development of anticancer therapies. The success of the 20S proteasome core particle (20S CP) inhibitor bortezomib in the clinical management of multiple myeloma has raised the possibility of identifying other UPS components for therapeutic intervention. We previously identified the small molecule b-AP15 as an inhibitor of 19S proteasome deubiquitinase (DUB) activity. Building upon our previous data, we performed a structure-activity relationship (SAR) study on b-AP15 and identified VLX1570 as an analog with promising properties, including enhanced potency and improved solubility in aqueous solution. In silico modeling was consistent with interaction of VLX1570 with key cysteine residues located at the active sites of the proteasome DUBs USP14 and UCHL5. VLX1570 was found to inhibit proteasome deubiquitinase activity invitro in a manner consistent with competitive inhibition. Furthermore, using active-site-directed probes, VLX1570 also inhibited proteasome DUB activity in exposed cells. Importantly, VLX1570 did not show inhibitory activity on a panel of recombinant non-proteasome DUBs, on recombinant kinases, or on caspase-3 activity, suggesting that VLX1570 is not an overtly reactive general enzyme inhibitor. Taken together, our data shows the chemical and biological properties of VLX1570 as an optimized proteasome DUB inhibitor.