Kimberly Cornish Carmony

Proteasome inhibitors with pyrazole scaffolds from structure-based virtual screening.

We performed a virtual screen of ∼340 000 small molecules against the active site of proteasomes followed by in vitro assays and subsequent optimization, yielding a proteasome inhibitor with pyrazole scaffold. The pyrazole-scaffold compound displayed excellent metabolic stability and was highly effective in suppressing solid tumor growth in vivo. Furthermore, the effectiveness of this compound was not negatively impacted by resistance to bortezomib or carfilzomib.

A bright approach to the immunoproteasome: Development of LMP2/β1i-specific imaging probes

While the constitutive, 26S proteasome plays an important role in regulating many important cellular processes, a variant form known as the immunoproteasome is thought to primarily function in adaptive immune responses. However, recent studies indicate an association of immunoproteasomes with many physiological disorders such as cancer, neurodegenerative, and inflammatory diseases. Despite this, the detailed functions of the immunoproteasome remain poorly understood. Immunoproteasome-specific probes are essential to gain insight into immunoproteasome function. Here, we describe for the first time the development of cell-permeable activity-based fluorescent probes, UK101-Fluor and UK101-B660, which selectively target the catalytically active LMP2/β1i subunit of the immunoproteasome. These probes facilitate rapid detection of the cellular localization of catalytically active immunoproteasomes in living cells, providing a valuable tool to analyze immunoproteasome functions. Additionally, as LMP2/β1i may serve as a potential tumor biomarker, an LMP2/β1i-targeting fluorescent imaging probe may be applicable to a rapid readout assay to determine tumor LMP2/β1i levels.

Activity-based imaging probes of the proteasome.

Over the years, the proteasome has been extensively investigated due to its crucial roles in many important signaling pathways and its implications in diseases. Two proteasome inhibitors—bortezomib and carfilzomib—have received FDA approval for the treatment of multiple myeloma, thereby validating the proteasome as a chemotherapeutic target. As a result, further research efforts have been focused on dissecting the complex biology of the proteasome to gain the insight required for developing next-generation proteasome inhibitors. It is clear that chemical probes have made significant contributions to these efforts, mostly by functioning as inhibitors that selectively block the catalytic activity of proteasomes. Analogues of these inhibitors are now providing additional tools for visualization of catalytically active proteasome subunits, several of which allow real-time monitoring of proteasome activity in living cells as well as in in vivo settings. These imaging probes will provide powerful tools for assessing the efficacy of proteasome inhibitors in clinical settings. In this review, we will focus on the recent efforts towards developing imaging probes of proteasomes, including the latest developments in immunoproteasome-selective imaging probes.

PROTAC-induced proteolytic targeting.

Small-molecule modulators of protein activity are increasingly being utilized as tools to examine the functional roles of proteins. Operating at the post-translational level, these molecules provide enhanced temporal and spatial control and mitigate the potential for compensatory responses in comparison with classical genetic approaches. Proteolysis targeting chimeric molecules, or PROTACs, are small molecules that inhibit the function of their target proteins by targeting them for degradation by the ubiquitin proteasome system. This chapter summarizes strategies for PROTAC preparation and characterization.

Elucidating the Catalytic Subunit Composition of Distinct Proteasome Subtypes: A Crosslinking Approach Employing Bifunctional Activity-Based Probes

In addition to two well‐recognized proteasome subtypes—constitutive proteasomes and immunoproteasomes—mounting evidence also suggests the existence of intermediate proteasome subtypes containing unconventional mixtures of catalytic subunits. Although they appear to play unique biological roles, the lack of practical methods for detecting distinct proteasome subtypes has limited functional investigations. Here, we report the development of activity‐based probes that crosslink two catalytic subunits within intact proteasome complexes. Identification of the crosslinked subunit pairs provides direct evidence of the catalytic subunit composition of proteasomes. Using these probes, we found that U266 multiple myeloma cells contain intermediate proteasomes comprising both β1i and β2, but not β1 and β2i, consistent with previous findings with other cell types. Our bifunctional probes can be utilized in functional investigations of distinct proteasome subtypes in various biological settings.

High‐Resolution Snapshots of Proteasome Inhibitors In Action Revise Inhibition Paradigms and Inspire Next‐Generation Inhibitor Design

New high-resolution crystal structures reported by Schrader and colleagues refine our understanding of how peptide epoxyketone anticancer drugs inactivate their target: the human proteasome. These findings provide important clues for the design of next-generation proteasome inhibitor drugs.

Abstract 4744: Development of an immunoproteasome fluorescent substrate: A functional proteomics tool

Introduction: This research was conducted to develop the first immunoproteasome fluorescent substrate. The immunoproteasome is an alternate form of the constitutive proteasome with distinct catalytic subunits. While immunoproteasomes appear to generate peptides for MHC class I antigen presentation, their functions are still not completely understood. Antibody-based studies have shown that immunoproteasome catalytic subunits are expressed in a variety of cancers, suggesting they may play important roles in this disease. However, antibodies bind both active and inactive subunits, rendering these studies incapable of correlating immunoproteasome activity with disease progression. Although fluorescent peptide substrates are widely used to monitor proteasome activity, those currently available are cleaved by both forms of the proteasome. Therefore, fluorescent substrates selectively cleaved by the immunoproteasome are needed to correlate their activity with cancer progression. Experimental Procedures: The fluorescent substrate was synthesized by replacing the epoxyketone pharmacophore of the immunoproteasome-selective inhibitor YU-102 with a 7-amino-4-methylcoumarin (AMC) fluorogenic moiety. The rates of hydrolysis of the resulting substrate, YU-102-AMC, by both purified constitutive proteasomes and immunoproteasomes were evaluated by monitoring the release of AMC over time. Hydrolysis of this substrate was also investigated in T1 and T2 cell lysates. Data Summary: The structure of the fluorescent substrate YU-102-AMC was confirmed by mass spectrometry and nuclear magnetic resonance spectroscopy. YU-102-AMC was preferentially cleaved by purified immunoproteasomes in comparison with purified constitutive proteasomes. This preferential cleavage by immunoproteasomes was confirmed in lysates of cancer cell lines T1 and T2. Conclusions: We have successfully developed YU-102-AMC, an immunoproteasome fluorescent substrate which facilitates quantification of immunoproteasome activity in cancer cell lysates. This substrate will be utilized to examine a correlation between immunoproteasome activity and cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4744. doi:1538-7445.AM2012-4744

Abstract 3236: Development of a novel cross-linking strategy to identify distinct proteasome subtypes

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Introduction: This research was conducted to develop a practical method to identify distinct proteasome subtypes. Approval of the proteasome inhibitors bortezomib and carfilzomib for clinical use has validated the proteasome as an anticancer target. These inhibitors target two well-recognized proteasome subtypes, the constitutive proteasome and the immunoproteasome, which comprise distinct sets of catalytic subunits. Additional proteasome subtypes containing mixtures of constitutive and immunoproteasome catalytic subunits have more recently been identified. These intermediate proteasome subtypes display unique proteolytic activity profiles and play important roles in the production of certain tumor antigens. However, much regarding their regulation, functions, and relevance as drug targets remains unknown, largely due to limitations in current experimental methods used to differentiate between proteasome subtypes. We have therefore developed bifunctional activity-based probes capable of cross-linking different catalytic subunit pairs within individual proteasome complexes. These probes allow a straightforward readout of proteasome catalytic subunit composition and facilitate functional studies of distinct proteasome subtypes. Experimental Procedures: Bifunctional proteasome probes were synthesized by coupling pairs of proteasome inhibitors via linkers of varying lengths and compositions. Positions for inhibitor derivatization were selected based on computational modeling, and linkers were chosen based on distances between the active sites of each targeted subunit pair. The ability of these compounds to cross-link distinct catalytic subunit pairs in both purified proteasomes and in cancer cell lysates was visualized by western blotting analysis. Competition assays with subunit-selective proteasome inhibitors were employed to confirm the identities of the cross-linked subunit pairs. Data Summary: The structures of the bifunctional proteasome probes were confirmed by mass spectrometry and nuclear magnetic resonance spectroscopy. Western blotting results demonstrated the ability of these compounds to cross-link multiple pairs of proteasome catalytic subunits, allowing the composition of distinct proteasome subtypes present within cancer cells to be evaluated. Conclusions: We have successfully developed a set of bifunctional proteasome probes for analysis of proteasome catalytic subunit composition. These probes will be used to elucidate the unique functions of distinct proteasome subtypes present within cancer cells. Citation Format: Kimberly C. Carmony, Do-Min Lee, Lalit Kumar Sharma, Jieun Park, Kyung-Bo Kim, Wooin Lee. Development of a novel cross-linking strategy to identify distinct proteasome subtypes. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3236. doi:10.1158/1538-7445.AM2014-3236

Abstract 2231: Development of bifunctional cross-linking agents to identify intermediate proteasomes.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Introduction: This research was conducted to develop bifunctional cross-linking agents that can be used to identify intermediate proteasomes. Two well-characterized proteasome subtypes, the constitutive proteasome and immunoproteasome, differ with respect to their distinct sets of catalytic subunits. Additionally, intermediate proteasome subtypes comprised of non-standard mixtures of catalytic subunits have more recently been discovered. These intermediate proteasomes differ from constitutive and immunoproteasomes in their proteolytic activity profiles, suggesting that they have unique cellular functions. In agreement with this, intermediate proteasomes in cancer cell lines were shown to play important roles in the production of several tumor antigens. However, largely due to limitations of methods currently available to distinguish between subtypes, the detailed functions of intermediate proteasomes remain poorly defined. Bifunctional agents capable of cross-linking two different catalytic subunits within a single proteasome complex will facilitate a simple approach to identify distinct proteasome subtypes present within cells. These compounds will serve as important chemical probes for investigating the functions of individual proteasome subtypes in cancer cells. Experimental Procedures: Bifunctional cross-linking agents were synthesized by coupling two broad-spectrum or subunit-selective proteasome inhibitors through hydrocarbon or polyethylene glycol linkers. Positions for derivatization of these inhibitors were selected based on computational modeling, and linker lengths were varied to increase cross-linking efficiency. The ability of the resulting compounds to cross-link two proteasome catalytic subunits in cancer cell lysates was assessed via western blotting. Competition assays with subunit-selective proteasome inhibitors were conducted to verify the identities of the cross-linked subunit pairs. Data Summary: The structures of the bifunctional cross-linking agents were confirmed by mass spectrometry and nuclear magnetic resonance spectroscopy. Western blotting results revealed the ability of several of these bifunctional agents to cross-link two catalytic subunits within individual proteasome complexes, facilitating compositional analysis of distinct proteasome subtypes. Conclusions: We have successfully developed bifunctional agents capable of cross-linking two catalytic subunits within individual proteasome complexes. These compounds will be utilized in functional studies of intermediate proteasomes present within cancer cells. Citation Format: Kimberly C. Carmony, Lalit Kumar Sharma, Kyung-Bo Kim. Development of bifunctional cross-linking agents to identify intermediate proteasomes. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2231. doi:10.1158/1538-7445.AM2013-2231