Wouter A. van der Linden

Proteasome Inhibitors: An Expanding Army Attacking a Unique Target

Proteasomes are large, multisubunit proteolytic complexes presenting multiple targets for therapeutic intervention. The 26S proteasome consists of a 20S proteolytic core and one or two 19S regulatory particles. The 20S core contains three types of active sites. Many structurally diverse inhibitors of these active sites, both natural product and synthetic, have been discovered in the last two decades. One, bortezomib, is used clinically for treatment of multiple myeloma, mantle cell lymphoma, and acute allograft rejection. Five more recently developed proteasome inhibitors are in trials for treatment of myeloma and other cancers. Proteasome inhibitors also have activity in animal models of autoimmune and inflammatory diseases, reperfusion injury, promote bone and hair growth, and can potentially be used as anti-infectives. In addition, inhibitors of ATPases and deubiquitinases of 19S regulatory particles have been discovered in the last decade.

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.

Structure- and function-based design of Plasmodium -selective proteasome inhibitors

The proteasome is a multi-component protease complex responsible for regulating key processes such as the cell cycle and antigen presentation. Compounds that target the proteasome are potentially valuable tools for the treatment of pathogens that depend on proteasome function for survival and replication. In particular, proteasome inhibitors have been shown to be toxic for the malaria parasite Plasmodium falciparum at all stages of its life cycle. Most compounds that have been tested against the parasite also inhibit the mammalian proteasome, resulting in toxicity that precludes their use as therapeutic agents. Therefore, better definition of the substrate specificity and structural properties of the Plasmodium proteasome could enable the development of compounds with sufficient selectivity to allow their use as anti-malarial agents. To accomplish this goal, here we use a substrate profiling method to uncover differences in the specificities of the human and P. falciparum proteasome. We design inhibitors based on amino-acid preferences specific to the parasite proteasome, and find that they preferentially inhibit the β2-subunit. We determine the structure of the P. falciparum 20S proteasome bound to the inhibitor using cryo-electron microscopy and single-particle analysis, to a resolution of 3.6 Å. These data reveal the unusually open P. falciparum β2 active site and provide valuable information about active-site architecture that can be used to further refine inhibitor design. Furthermore, consistent with the recent finding that the proteasome is important for stress pathways associated with resistance of artemisinin family anti-malarials, we observe growth inhibition synergism with low doses of this β2-selective inhibitor in artemisinin-sensitive and -resistant parasites. Finally, we demonstrate that a parasite-selective inhibitor could be used to attenuate parasite growth in vivo without appreciable toxicity to the host. Thus, the Plasmodium proteasome is a chemically tractable target that could be exploited by next-generation anti-malarial agents.

Activity-Based Profiling Reveals Reactivity of the Murine Thymoproteasome-Specific Subunit β5t

Epithelial cells of the thymus cortex express a unique proteasome particle involved in positive T cell selection. This thymoproteasome contains the recently discovered beta5t subunit that has an uncharted activity, if any. We synthesized fluorescent epoxomicin probes that were used in a chemical proteomics approach, entailing activity-based profiling, affinity purification, and LC-MS identification, to demonstrate that the beta5t subunit is catalytically active in the murine thymus. A panel of established proteasome inhibitors showed that the broad-spectrum inhibitor epoxomicin blocks the beta5t activity and that the subunit-specific antagonists bortezomib and NC005 do not inhibit beta5t. We show that beta5t has a substrate preference distinct from beta5/beta5i that might explain how the thymoproteasome generates the MHC class I peptide repertoire needed for positive T cell selection.

Nature of Pharmacophore Influences Active Site Specificity of Proteasome Inhibitors

Proteasomes degrade most proteins in mammalian cells and are established targets of anti-cancer drugs. The majority of proteasome inhibitors are composed of short peptides with an electrophilic functionality (pharmacophore) at the C terminus. All eukaryotic proteasomes have three types of active sites as follows: chymotrypsin-like, trypsin-like, and caspase-like. It is widely believed that active site specificity of inhibitors is determined primarily by the peptide sequence and not the pharmacophore. Here, we report that active site specificity of inhibitors can also be tuned by the chemical nature of the pharmacophore. Specifically, replacement of the epoxyketone by vinyl sulfone moieties further improves the selectivity of β5-specific inhibitors NC-005, YU-101, and PR-171 (carfilzomib). This increase in specificity is likely the basis of the decreased cytotoxicity of vinyl sulfone-based inhibitors to HeLa cells as compared with that of epoxyketone-based inhibitors.

Incorporation of Non-natural Amino Acids Improves Cell Permeability and Potency of Specific Inhibitors of Proteasome Trypsin-like Sites.

Proteasomes degrade the majority of proteins in mammalian cells by a concerted action of three distinct pairs of active sites. The chymotrypsin-like sites are targets of antimyeloma agents bortezomib and carfilzomib. Inhibitors of the trypsin-like site sensitize multiple myeloma cells to these agents. Here we describe systematic effort to develop inhibitors with improved potency and cell permeability, yielding azido-Phe-Leu-Leu-4-aminomethyl-Phe-methyl vinyl sulfone (4a, LU-102), and a fluorescent activity-based probe for this site. X-ray structures of 4a and related inhibitors complexed with yeast proteasomes revealed the structural basis for specificity. Nontoxic to myeloma cells when used as a single agent, 4a sensitized them to bortezomib and carfilzomib. This sensitizing effect was much stronger than the synergistic effects of histone acetylase inhibitors or additive effects of doxorubicin and dexamethasone, raising the possibility that combinations of inhibitors of the trypsin-like site with bortezomib or carfilzomib would have stronger antineoplastic activity than combinations currently used clinically.

A panel of subunit-selective activity-based proteasome probes

Mammals express seven different catalytically active proteasome subunits. In order to determine the roles of the different proteolytically active subunits in antigen presentation and other cellular processes, highly specific inhibitors and activity-based probes that selectively target specific active sites are needed. In this work we present the development of fluorescent activity-based probes that selectively target the beta1 and beta5 sites of the constitutive proteasome.

Detection of Intestinal Cancer by Local, Topical Application of a Quenched Fluorescence Probe for Cysteine Cathepsins

Early detection of colonic polyps can prevent up to 90% of colorectal cancer deaths. Conventional colonoscopy readily detects the majority of premalignant lesions, which exhibit raised morphology. However, lesions that are flat and depressed are often undetected using this method. Therefore, there is a need for molecular-based contrast agents to improve detection rates over conventional colonoscopy. We evaluated a quenched fluorescent activity-based probe (qABP; BMV109) that targets multiple cysteine cathepsins that are overexpressed in intestinal dysplasia in a genetic model of spontaneous intestinal polyp formation and in a chemically induced model of colorectal carcinoma. We found that the qABP selectively targets cysteine cathepsins, resulting in high sensitivity and specificity for intestinal tumors in mice and humans. Additionally, the qABP can be administered by either intravenous injection or by local delivery to the colon, making it a highly valuable tool for improved detection of colorectal lesions using fluorescence-guided colonoscopy.

Azido-BODIPY Acid Reveals Quantitative Staudinger–Bertozzi Ligation in Two-Step Activity-Based Proteasome Profiling

Activity-based protein profiling (ABPP) research is directed towards the development of tools and techniques that report on enzyme activity in complex biological samples.[1–4] With the aid of activity-based probes (ABPs)—small molecules designed to react specifically, covalently, and irreversibly with the active site residues of an enzyme or enzyme family—enzymatic activity levels are detected, rather than the protein expression levels that are measured by means of conventional proteomics techniques. A typical ABP consists of three parts: 1) a “warhead”, the reactive group that binds covalently and irreversibly to the enzyme active site, 2) a recognition element targeting the ABP to a certain enzyme (family), and 3) an affinity tag or a fluorophore for visualization and/or enrichment purposes. In most ABPs that report on enzyme activity, the reporter group is directly attached to the probe, with obvious advantages with respect to experimental design. Incorporation of, for instance, a biotin or large fluorophore in an ABP, however, might have a detrimental effect either on bioavailability (cell permeability) or on enzyme reactivity of the probe, or on both. With the aim of alleviating these problems, the two-step labeling approach is an important alternative in ABPP. We and Cravatt and co-workers simultaneously reported that this approach is also versatile in the profiling of enzyme families: namely the proteasome and serine hydrolases, respectively.[5, 6] In two-step ABPP approaches a small biocompatible reactive group, normally an azide or an acetylene, is introduced into an ABP. After covalent modification of a target protein (family), a reporter group is introduced in a chemoselective manner, by means either of Staudinger–Bertozzi ligation[6–8] or of Huisgen [2+3] cycloaddition (the “click reaction”, of which both copper(I)-catalyzed[5, 9–13] and copper-free[14,15] versions exist). Key to the success of such two-step ABPP experiments are the selectivity (in terms of cross-reactivity towards endogenous functional groups in a biological sample) and efficiency (in terms of chemical yields with which the azide- or acetylene-modified proteins are converted) of the chemoselective ligation step by which the reporter group is attached to the modified proteins. There are several reports on the selectivity of both Staudinger–Bertozzi and click ligations.[11,14] Here we describe a compatible set of one-step and two-step proteasome ABPs 4 and 6 (Scheme 1) and demonstrate that with these the efficiency of the Staudinger–Bertozzi ligation in the two-step ABPP of the proteasome catalytic activities is estimated to proceed in a quantitative fashion. Scheme 1 Reagents and conditions: a) N-hydroxysuccinimide, EDC, DCM, 2 h, 68 %. b) DBU, DMF, 5 min. c) HOBt, 1 min. d) 2, DiPEA, 30 min, 86 %. e) 5, 10 mol % CuSO4, 20 mol % sodium ascorbate, tBuOH/H2O 1:1, RT, 15 h, quant. The design of probes 4 and 6 is based on the new bifunctional azido-BODIPY acid derivative 1, which can be incorporated into ABPs and subsequently functionalized either before or after enzyme labeling by both Staudinger–Bertozzi and click ligation. We have recently demonstrated that the BODIPY-TMR-modified proteasome inhibitor 8 (MV151) labels all proteasome catalytic sites both in cell lysates and in living cells.[16] The capability to introduce a biotin moiety into 4 at will at any time in the profiling experiment provides flexibility in designing the optimal ABP (one-step or two-step), depending on the nature of the ABPP experiment. The title compound, azido-BODIPY acid 1, was synthesized by adaptation of the literature procedures for the synthesis of BODIPY-TMR[16,17] (Supporting Information) and was subsequently converted into the corresponding succinimidyl ester 2 (Scheme 1). Removal of the Fmoc protective group in the hexapeptide vinyl sulfone 3,[16] followed by condensation with azido-BODIPY-OSu 2, gave ABP 4. Copper(I)-catalyzed Huisgen [2+3] cycloaddition[9, 10] with biotin-propargylamide (5) gave rise to the fluorescent and affinity-tagged ABP 6. Having synthesized probes 4 and 6, we assessed their ability to label the proteolytically active proteasome subunits both in cell lysates (Figure 1) and in living cells (Figure 2). EL-4 cell lysates containing both the constitutive proteasome and the immunoproteasome[18] were treated with increasing concentrations of 4 or 6 for 1 h at 37°C. The lysates treated with 4 were then exposed to biotin-phosphane 7 for 1 h at 37°C. All samples were precipitated, and their protein contents were resolved by SDS-PAGE. Direct in-gel read-out of the wet gel slabs showed uniform labeling of the proteasome catalytic subunits (β1, β2, β5, β1i, β2i, β5i) by both ABPs in a concentration-dependent manner. The observed patterns are similar to those demonstrated previously (see the labeling pattern of 8, Figure 1A lane 10 for a representative example).[16] Preincubation with epoxomicin[19,20] (Figure 1A, lane 9, Figure 1C, lane 8) abolished all labeling, which further confirms the activity-based mechanism of ABPs 4 and 6. ABP 4 appears to be slightly more reactive than its biotinylated counterpart 6 (compare Figure 1A, lanes 3–5 and Figure 1C, lanes 3–5). Quantitative Staudinger–Bertozzi ligation on proteasome subunits modified by ABP 4 is evidenced by the gel shift of those samples exposed to 100 μM biotin-phosphane 7 (Figure 1A; compare lanes 3–7 and 8). The efficiency of the ligation is also apparent when the streptavidin blots we prepared from the same gels are compared (Figures 1B and D). Again, the two patterns are highly similar, and the intensities of the signals are similar for those experiments in which we applied 10 μM concentrations of either 4 or 6 (Figures 1B and D, lanes 7). Figure 1 Fluorescence readout (A and C) and streptavidin blot (B and D) of labeled proteasomes in cell lysate. A) and B) EL-4 cell lysates (25 μg total protein) were treated with 4 for 1 h at 37 °C, followed by Staudinger ligation (100 μM ... Figure 2 A) Fluorescence readout, and B) streptavidin blot of labeled proteasomes in living cells. Living EL4 cells were exposed to the indicated probes for 2 h at 37 °C, before being harvested and lysed. Lanes 3–6: 25 μg total protein ... The proteasome labeling potential of ABPs 4 and 6 in living cells was established by incubating EL-4 cells with either of the two probes at various concentrations for 2 h at 37°C. The exposed cells were harvested, washed, and lysed, and the lysates were processed as before (Figure 2). The outcomes of these experiments are highly reminiscent of those involving the ABPP labeling of lysates depicted in Figure 1. However, the main, and important, difference is found in the divergent labeling efficiency now observed for the two probes. In contrast with the proteasome profiling experiments on lysates, in which both probes appeared about equally efficient, we estimate that the two-step ABP 4 is at least five times more efficient in targeting the proteasome catalytic activities in living cells. As both probes are equally efficient in labeling proteasomes in lysates, this difference must be based on the relative cell permeabilities of the two probes. In conclusion, we have demonstrated the versatility of the bifunctional fluorophore azido-BODIPY acid 1 as a new tool in ABPP experiments. We have established that the Staudinger–Bertozzi ligation proceeds in quantitative yield under the conditions applied here. This result essentially means that two-step ABPP may proceed with an efficiency equal to that of contemporary one-step ABPP approaches with respect to protein tagging. The efficiency thus depends on the reactivity of the ABP towards the target protein (family), and not on the chemoselective ligation employed in the second step. The advantage of two-step ABPP is evident from the results presented here demonstrating that ABP 4 is better than biotinylated analogue 6 at labeling proteasomes in living cells. We expect that BODIPY derivative 1 will be useful to the chemical biology community outside the proteasome field for several reasons. Firstly, the system presented here should be of assistance in optimizing Staudinger–Bertozzi ligation conditions, in reaction time and in the amount of phosphane used with respect to the azido modified biomolecule, for instance. Further, azido-BODIPY acid 1 can be readily transposed to different ABPP experimental settings. These include not only those directed towards the profiling of different enzyme families (entailing the incorporation of 1 into other ABPs), but also those directed towards the development or employment of other bio-orthogonal ligation strategies. An obvious extension of the work reported here is evaluation of the efficiency of the Huisgen cycloaddition reaction, but modification of the azide in 1 to encompass reaction partners for new bio-orthogonal ligations are envisaged as well. We are currently pursuing research in these directions.

Mixing of peptides and electrophilic traps gives rise to potent, broad-spectrum proteasome inhibitors

The synthesis and evaluation of hybrid proteasome inhibitors that contain structural elements of the known inhibitors bortezomib, epoxomicin and peptide vinyl sulfones is described. From the panel of 15 inhibitors some structure activity relationships can be deduced with regard to inhibitory activity in relation to peptide recognition element, inhibitor size and nature of the electrophilic trap. Further, the panel contains one of the most potent peptide-based pan-proteasome inhibitors reported to date.