Martijn Verdoes

Activity probe for in vivo profiling of the specificity of proteasome inhibitor bortezomib

Proteasome inhibitors, such as the dipeptide boronic acid bortezomib, are emerging as important tools in the treatment of the fatal hematologic malignancy multiple myeloma. Despite the recent US Food and Drug Administration approval of bortezomib (PS341, Velcade) for the treatment of refractory multiple myeloma, many of the basic pharmacologic parameters of bortezomib and its mode of action on myeloma cells remain to be determined. We describe the synthesis and use of a cell-permeant active site–directed probe, which allows profiling of proteasomal activities in living cells. When we compared proteasome activity patterns in cultured cells and crude cell extracts with this probe, we observed substantial differences, stressing the importance for bioassays compatible with live cells to ensure accuracy of such measurements. Using this probe, we investigated the in vivo subunit specificities of bortezomib and another inhibitor, MG132.

Selective Inhibitor of Proteasome's Caspase-like Sites Sensitizes Cells to Specific Inhibition of Chymotrypsin-like Sites

Proteasomes degrade most proteins in mammalian cells and are established targets of anticancer drugs. All eukaryotic proteasomes have three types of active sites: chymotrypsin-like, trypsin-like, and caspase-like. Chymotrypsin-like sites are the most important in protein degradation and are the primary target of most proteasome inhibitors. The biological roles of trypsin-like and caspase-like sites and their potential as cotargets of antineoplastic agents are not well defined. Here we describe the development of site-specific inhibitors and active-site probes of chymotrypsin-like and caspase-like sites. Using these compounds, we show that cytotoxicity of proteasome inhibitors does not correlate with inhibition of chymotrypsin-like sites and that coinhibition of either trypsin-like and/or caspase-like sites is needed to achieve maximal cytotoxicity. Thus, caspase-like and trypsin-like sites must be considered as cotargets of anticancer drugs.

New approaches for dissecting protease functions to improve probe development and drug discovery

Proteases are well-established targets for pharmaceutical development because of their known enzymatic mechanism and their regulatory roles in many pathologies. However, many potent clinical lead compounds have been unsuccessful either because of a lack of specificity or because of our limited understanding of the biological roles of the targeted protease. In order to successfully develop protease inhibitors as drugs, it is necessary to understand protease functions and to expand the platform of inhibitor development beyond active site–directed design and in vitro optimization. Several newly developed technologies will enhance assessment of drug selectivity in living cells and animal models, allowing researchers to focus on compounds with high specificity and minimal side effects in vivo. In this review, we highlight advances in the development of chemical probes, proteomic methods and screening tools that we feel will help facilitate this paradigm shift in drug discovery.

Functional imaging of proteases: Recent advances in the design and application of substrate-based and activity-based probes

Proteases are enzymes that cleave peptide bonds in protein substrates. This process can be important for regulated turnover of a target protein but it can also produce protein fragments that then perform other functions. Because the last few decades of protease research have confirmed that proteolysis is an essential regulatory process in both normal physiology and in multiple disease-associated conditions, there has been an increasing interest in developing methods to image protease activity. Proteases are also considered to be one of the few druggable classes of proteins and therefore a large number of small molecule based inhibitors of proteases have been reported. These compounds serve as a starting point for the design of probes that can be used to target active proteases for imaging applications. Currently, several classes of fluorescent probes have been developed to visualize protease activity in live cells and even whole organisms. The two primary classes of protease probes make use of either peptide/protein substrates or covalent inhibitors that produce a fluorescent signal when bound to an active protease target. This review outlines some of the most recent advances in the design of imaging probes for proteases. In particular, it highlights the strengths and weaknesses of both substrate-based and activity-based probes and their applications for imaging cysteine proteases that are important biomarkers for multiple human diseases.

Characterization of the ubiquitin–proteasome system in bortezomib-adapted cells

Resistance towards the proteasome inhibitor bortezomib is poorly understood. We adapted the HL-60, ARH-77 and AMO-1 cell lines (myeloid leukemia, plasmocytoid lymphoma, myeloma) to bortezomib exceeding therapeutic plasma levels, and compared characteristics of the ubiquitin–proteasome system, alternative proteases and the unfolded protein response (UPR) between adapted cells and parental lines. Adapted cells showed increased transcription rates, activities and polypeptide levels of the bortezomib-sensitive β5, but also of the β2 proteasome subunit and consistently retained elevated levels of active β1/β5-type proteasome subunits in the presence of therapeutic levels of bortezomib. Bortezomib-adapted HL-60 cells showed increased expression and proteasome association of the 11S proteasome activator, and did not accumulate poly-ubiquitinated protein, activate the UPR or UPR-mediated apoptosis in response to bortezomib. The rate of protein biosynthesis was reduced, and the transcription of chaperone genes downmodulated. We did not observe major changes in the activities of TPPII, cathepsins or deubiquitinating proteases. We conclude that different types of bortezomib-adapted cell lines, including myeloma, show similar patterns of changes in the proteasomal machinery which result in residual proteasome activity in the presence of bortezomib and a quantitative balance between protein biosynthesis and destruction.

Improved quenched fluorescent probe for imaging of cysteine cathepsin activity

The cysteine cathepsins are a family of proteases that play important roles in both normal cellular physiology and many human diseases. In cancer, the activity of many of the cysteine cathepsins is upregulated and can be exploited for tumor imaging. Here we present the design and synthesis of a new class of quenched fluorescent activity-based probes (qABPs) containing a phenoxymethyl ketone (PMK) electrophile. These reagents show enhanced in vivo properties and broad reactivity resulting in dramatically improved labeling and tumor imaging properties compared to those of previously reported ABPs.

Functional imaging of legumain in cancer using a new quenched activity-based probe

Legumain is a lysosomal cysteine protease whose biological function remains poorly defined. Legumain activity is up-regulated in most human cancers and inflammatory diseases most likely as the result of high expression in populations of activated macrophages. Within the tumor microenvironment, legumain activity is thought to promote tumorigenesis. To obtain a greater understanding of the role of legumain activity during cancer progression and inflammation, we developed an activity-based probe that becomes fluorescent only upon binding active legumain. This probe is highly selective for legumain, even in the context of whole cells and tissues, and is also a more effective label of legumain than previously reported probes. Here we present the synthesis and application of our probe to the analysis of legumain activity in primary macrophages and in two mouse models of cancer. We find that legumain activity is highly correlated with macrophage activation and furthermore that it is an ideal marker for primary tumor inflammation and early stage metastatic lesions.

Subclassification and Biochemical Analysis of Plant Papain-Like Cysteine Proteases Displays Subfamily-Specific Characteristics

Papain-like cysteine proteases (PLCPs) are a large class of proteolytic enzymes associated with development, immunity, and senescence. Although many properties have been described for individual proteases, the distribution of these characteristics has not been studied collectively. Here, we analyzed 723 plant PLCPs and classify them into nine subfamilies that are present throughout the plant kingdom. Analysis of these subfamilies revealed previously unreported distinct subfamily-specific functional and structural characteristics. For example, the NPIR and KDEL localization signals are distinctive for subfamilies, and the carboxyl-terminal granulin domain occurs in two PLCP subfamilies, in which some individual members probably evolved by deletion of the granulin domains. We also discovered a conserved double cysteine in the catalytic site of SAG12-like proteases and two subfamily-specific disulfides in RD19A-like proteases. Protease activity profiling of representatives of the PLCP subfamilies using novel fluorescent probes revealed striking polymorphic labeling profiles and remarkably distinct pH dependency. Competition assays with peptide-epoxide scanning libraries revealed common and unique inhibitory fingerprints. Finally, we expand the detection of PLCPs by identifying common and organ-specific protease activities and identify previously undetected proteases upon labeling with cell-penetrating probes in vivo. This study provides the plant protease research community with tools for further functional annotation of plant PLCPs.

Specific Cell-Permeable Inhibitor of Proteasome Trypsin-like Sites Selectively Sensitizes Myeloma Cells to Bortezomib and Carfilzomib

Proteasomes degrade the majority of proteins in mammalian cells, are involved in the regulation of multiple physiological functions, and are established targets of anticancer drugs. The proteasome has three types of active sites. Chymotrypsin-like sites are the most important for protein breakdown and have long been considered the only suitable targets for antineoplastic drugs; however, our recent work demonstrated that inhibitors of caspase-like sites sensitize malignant cells to inhibitors of the chymotrypsin-like sites. Here, we describe the development of specific cell-permeable inhibitors and an activity-based probe of the trypsin-like sites. These compounds selectively sensitize multiple myeloma cells to inhibitors of the chymotrypsin-like sites, including antimyeloma agents bortezomib and carfilzomib. Thus, trypsin-like sites are cotargets for anticancers drugs. Together with inhibitors of chymotrypsin- and caspase-like sites developed earlier, we provide the scientific community with a complete set of tools to separately modulate proteasome active sites in living cells.

A Nonpeptidic Cathepsin S Activity-Based Probe for Noninvasive Optical Imaging of Tumor-Associated Macrophages

Macrophage infiltration into tumors has been correlated with poor clinical outcome in multiple cancer types. Therefore, tools to image tumor-associated macrophages could be valuable for diagnosis and prognosis of cancer. Herein, we describe the synthesis and characterization of a cathepsin S-directed, quenched activity-based probe (qABP), BMV083. This probe makes use of an optimized nonpeptidic scaffold leading to enhanced in vivo properties relative to previously reported peptide-based probes. In a syngeneic breast cancer model, BMV083 provides high tumor-specific fluorescence that can be visualized using noninvasive optical imaging methods. Furthermore, analysis of probe-labeled cells demonstrates that the probe primarily targets macrophages with an M2 phenotype. Thus, BMV083 is a potential valuable in vivo reporter for tumor-associated macrophages that could greatly facilitate the future studies of macrophage function in the process of tumorigenesis.