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 ß2-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 ß2 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 ß2-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.

Deletion of the rodent malaria ortholog for falcipain-1 highlights differences between hepatic and blood stage merozoites.

Proteases have been implicated in a variety of developmental processes during the malaria parasite lifecycle. In particular, invasion and egress of the parasite from the infected hepatocyte and erythrocyte, critically depend on protease activity. Although falcipain-1 was the first cysteine protease to be characterized in P. falciparum, its role in the lifecycle of the parasite has been the subject of some controversy. While an inhibitor of falcipain-1 blocked erythrocyte invasion by merozoites, two independent studies showed that falcipain-1 disruption did not affect growth of blood stage parasites. To shed light on the role of this protease over the entire Plasmodium lifecycle, we disrupted berghepain-1, its ortholog in the rodent parasite P. berghei. We found that this mutant parasite displays a pronounced delay in blood stage infection after inoculation of sporozoites. Experiments designed to pinpoint the defect of berghepain-1 knockout parasites found that it was not due to alterations in gliding motility, hepatocyte invasion or liver stage development and that injection of berghepain-1 knockout merosomes replicated the phenotype of delayed blood stage growth after sporozoite inoculation. We identified an additional role for berghepain-1 in preparing blood stage merozoites for infection of erythrocytes and observed that berghepain-1 knockout parasites exhibit a reticulocyte restriction, suggesting that berghepain-1 activity broadens the erythrocyte repertoire of the parasite. The lack of berghepain-1 expression resulted in a greater reduction in erythrocyte infectivity in hepatocyte-derived merozoites than it did in erythrocyte-derived merozoites. These observations indicate a role for berghepain-1 in processing ligands important for merozoite infectivity and provide evidence supporting the notion that hepatic and erythrocytic merozoites, though structurally similar, are not identical.

Tripeptidyl Peptidase II Mediates Levels of Nuclear Phosphorylated ERK1 and ERK2.

Tripeptidyl peptidase II (TPP2) is a serine peptidase involved in various biological processes, including antigen processing, cell growth, DNA repair, and neuropeptide mediated signaling. The underlying mechanisms of how a peptidase can influence this multitude of processes still remain unknown. We identified rapid proteomic changes in neuroblastoma cells following selective TPP2 inhibition using the known reversible inhibitor butabindide, as well as a new, more potent, and irreversible peptide phosphonate inhibitor. Our data show that TPP2 inhibition indirectly but rapidly decreases the levels of active, di-phosphorylated extracellular signal-regulated kinase 1 (ERK1) and ERK2 in the nucleus, thereby down-regulating signal transduction downstream of growth factors and mitogenic stimuli. We conclude that TPP2 mediates many important cellular functions by controlling ERK1 and ERK2 phosphorylation. For instance, we show that TPP2 inhibition of neurons in the hippocampus leads to an excessive strengthening of synapses, indicating that TPP2 activity is crucial for normal brain function.

Legumain is activated in macrophages during pancreatitis.

Pancreatitis is an inflammatory disease of the pancreas characterized by dysregulated activity of digestive enzymes, necrosis, immune infiltration, and pain. Repeated incidence of pancreatitis is an important risk factor for pancreatic cancer. Legumain, a lysosomal cysteine protease, has been linked to inflammatory diseases such as atherosclerosis, stroke, and cancer. Until now, legumain activation has not been studied during pancreatitis. We used a fluorescently quenched activity-based probe to assess legumain activation during caerulein-induced pancreatitis in mice. We detected activated legumain by ex vivo imaging, confocal microscopy, and gel electrophoresis. Compared with healthy controls, legumain activity in the pancreas of caerulein-treated mice was increased in a time-dependent manner. Legumain was localized to CD68(+) macrophages and was not active in pancreatic acinar cells. Using a small-molecule inhibitor of legumain, we found that this protease is not essential for the initiation of pancreatitis. However, it may serve as a biomarker of disease, since patients with chronic pancreatitis show strongly increased legumain expression in macrophages. Moreover, the occurrence of legumain-expressing macrophages in regions of acinar-to-ductal metaplasia suggests that this protease may influence reprogramming events that lead to inflammation-induced pancreatic cancer.

Design of a highly selective quenched activity-based probe and its application in dual color imaging studies of cathepsin S activity localization.

The cysteine cathepsins are a group of 11 proteases whose function was originally believed to be the degradation of endocytosed material with a high degree of redundancy. However, it has become clear that these enzymes are also important regulators of both health and disease. Thus, selective tools that can discriminate between members of this highly related class of enzymes will be critical to further delineate the unique biological functions of individual cathepsins. Here we present the design and synthesis of a near-infrared quenched activity-based probe (qABP) that selectively targets cathepsin S which is highly expressed in immune cells. Importantly, this high degree of selectivity is retained both in vitro and in vivo. In combination with a new green-fluorescent pan-reactive cysteine cathepsin qABP we performed dual color labeling studies in bone marrow-derived immune cells and identified vesicles containing exclusively cathepsin S activity. This observation demonstrates the value of our complementary cathepsin probes and provides evidence for the existence of specific localization of cathepsin S activity in dendritic cells.

The novel ß2-selective proteasome inhibitor LU-102 synergizes with bortezomib and carfilzomib to overcome proteasome inhibitor resistance of myeloma cells.

Proteasome inhibitor resistance is a challenge for myeloma therapy. Bortezomib targets the ß5 and ß1 activity, but not the ß2 activity of the proteasome. Bortezomib-resistant myeloma cells down-regulate the activation status of the unfolded protein response, and up-regulate ß2 proteasome activity. To improve proteasome inhibition in bortezomib-resistant myeloma and to achieve more efficient UPR activation, we have developed LU-102, a selective inhibitor of the ß2 proteasome activity. LU-102 inhibited the ß2 activity in intact myeloma cells at low micromolar concentrations without relevant co-inhibition of ß1 and ß5 proteasome subunits. In proteasome inhibitor-resistant myeloma cells, significantly more potent proteasome inhibition was achieved by bortezomib or carfilzomib in combination with LU-102, compared to bortezomib/carfilzomib alone, resulting in highly synergistic cytotoxic activity of the drug combination via endoplasmatic reticulum stress-induced apoptosis. Combining bortezomib/carfilzomib with LU-102 significantly prolonged proteasome inhibition and increased activation of the unfolded protein response and IRE1-a activity. IRE1-α has recently been shown to control myeloma cell differentiation and bortezomib sensitivity (Leung-Hagesteijn, Cancer Cell 24:3, 289-304). Thus, ß2-selective proteasome inhibition by LU-102 in combination with bortezomib or carfilzomib results in synergistic proteasome inhibition, activation of the unfolded protein response, and cytotoxicity, and overcomes bortezomib/carfilzomib resistance in myeloma cells in vitro.

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.

Bioorthogonal chemistry: applications in activity-based protein profiling.

The close interaction between organic chemistry and biology goes back to the late 18th century, when the modern natural sciences began to take shape. After synthetic organic chemistry arose as a discipline, organic chemists almost immediately began to pursue the synthesis of naturally occurring compounds, thereby contributing to the understanding of their functions in biological processes. Research in those days was often remarkably interdisciplinary; in fact, it constituted chemical biology research before the phrase even existed. For example, histological dyes, both of an organic and inorganic nature, were developed and applied by independent researchers (Gram and Golgi) with the aim of visualizing cellular substructures (the bacterial cell wall and the Golgi apparatus). Over the years, as knowledge within the various fields of the natural sciences deepened, research disciplines drifted apart, becoming rather monodisciplinary. In these years, broadly ranging from the end of World War II to about the 1980s, organic chemistry continued to impact life sciences research, but contributions were of a more indirect nature. As an example, the development of the polymerase chain reaction, from which molecular biology and genetics research have greatly profited, was partly predicated on the availability of synthetic oligonucleotides. These molecules first became available in the late 1960s, the result of organic chemists pursuing the synthesis of DNA oligomers primarily because of the synthetic challenges involved. Today, academic natural sciences research is again becoming more interdisciplinary, and sometimes even multidisciplinary. What was termed "chemical biology" by Stuart Schreiber at the end of the last century can be roughly described as the use of intellectually chemical approaches to shed light on processes that are fundamentally rooted in biology. Chemical tools and techniques that are developed for biological studies in the exciting and rapidly evolving field of chemical biology research include contributions from many areas of the multifaceted discipline of chemistry, and particularly from organic chemistry. Researchers apply knowledge inherent to organic chemistry, such as reactivity and selectivity, to the manipulation of specific biomolecules in biological samples (cell extracts, living cells, and sometimes even animal models) to gain insight into the biological phenomena in which these molecules participate. In this Account, we highlight some of the recent developments in chemical biology research driven by organic chemistry, with a focus on bioorthogonal chemistry in relation to activity-based protein profiling. The rigorous demands of bioorthogonality have not yet been realized in a truly bioorthogonal reagent pair, but remarkable progress has afforded a range of tangible contributions to chemical biology research. Activity-based protein profiling, which aims to obtain information on the workings of a protein (or protein family) within the larger context of the full biological system, has in particular benefited from these advances. Both activity-based protein profiling and bioorthogonal chemistry have been around for approximately 15 years, and about 8 years ago the two fields very profitably intersected. We expect that each discipline, both separately and in concert, will continue to make important contributions to chemical biology research.

Discovery of a potent and highly ß1 specific proteasome inhibitor from a focused library of urea-containing peptide vinyl sulfones and peptide epoxyketones.

Syringolins, a class of natural products, potently and selectively inhibit the proteasome and show promising antitumour activity. To gain insight in the mode of action of syringolins, the ureido structural element present in syringolins is incorporated in oligopeptide vinyl sulfones and peptide epoxyketones yielding a focused library of potent new proteasome inhibitors. The distance of the ureido linkage with respect to the electrophilic trap strongly influences subunit selectivity within the proteasome. Compounds 13 and 15 are ß5 selective and their potency exceeds that of syringolin A. In contrast, 5 may well be the most potent ß1 selective compound active in living cells reported to date.

Two-step bioorthogonal activity-based proteasome profiling using copper-free click reagents: a comparative study.

The development and application of bioorthogonal two-step labeling techniques receives much attention. Employing bifunctional proteasome probe 2 the efficiency of two-step labeling of recently published biotinylated cyclooctynes 3-5 is compared to Staudinger-Bertozzi ligation in cell extracts and living cells. While cyclooctynes 3-5 react faster and at a much lower concentration then the Staudinger-Bertozzi benchmark, background labeling is considerable with these reagents.

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 ß5-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.

Proteasome selectivity towards Michael acceptor containing oligopeptide-based inhibitors.

The synthesis and biological evaluation of ten Michael acceptors containing potential proteasome inhibitors are described. Cellular targets of azide containing inhibitors and were assessed in HEK293T and RAW264.7 cells by a two step labeling strategy, followed by biotin-pulldown, affinity purification, on-bead tryptic digestion and LC-MS(2) identification. This strategy appears to be an attractive alternative to gel-based competition assays.

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.

Development of an activity-based probe for acyl-protein thioesterases.

Protein palmitoylation is a dynamic post-translational modification (PTM) important for cellular functions such as protein stability, trafficking, localization, and protein-protein interactions. S-palmitoylation occurs via the addition of palmitate to cysteine residues via a thioester linkage, catalyzed by palmitoyl acyl transferases (PATs), with removal of the palmitate catalyzed by acyl protein thioesterases (APTs) and palmitoyl-protein thioesterases (PPTs). Tools that target the regulators of palmitoylation-PATs, APTs and PPTs-will improve understanding of this essential PTM. Here, we describe the synthesis and application of a cell-permeable activity-based probe (ABP) that targets APTs in intact mammalian cells and the parasite Toxoplasma gondii. Using a focused library of substituted chloroisocoumarins, we identified a probe scaffold with nanomolar affinity for human APTs (HsAPT1 and HsAPT2) and synthesized a fluorescent ABP, JCP174-BODIPY TMR (JCP174-BT). We use JCP174-BT to profile HsAPT activity in situ in mammalian cells, to detect an APT in T. gondii (TgPPT1). We show discordance between HsAPT activity levels and total protein concentration in some cell lines, indicating that total protein levels may not be representative of APT activity in complex systems, highlighting the utility of this probe.

A fluorescent broad-spectrum proteasome inhibitor for labeling proteasomes in vitro and in vivo.

The proteasome is an essential evolutionary conserved protease involved in many regulatory systems. Here, we describe the synthesis and characterization of the activity-based, fluorescent, and cell-permeable inhibitor Bodipy TMR-Ahx(3)L(3)VS (MV151), which specifically targets all active subunits of the proteasome and immunoproteasome in living cells, allowing for rapid and sensitive in-gel detection. The inhibition profile of a panel of commonly used proteasome inhibitors could be readily determined by MV151 labeling. Administration of MV151 to mice allowed for in vivo labeling of proteasomes, which correlated with inhibition of proteasomal degradation in the affected tissues. This probe can be used for many applications ranging from clinical profiling of proteasome activity, to biochemical analysis of subunit specificity of inhibitors, and to cell biological analysis of the proteasome function and dynamics in living cells.

Toxoplasma DJ-1 Regulates Organelle Secretion by a Direct Interaction with Calcium-Dependent Protein Kinase 1.

Human DJ-1 is a highly conserved and yet functionally enigmatic protein associated with a heritable form of Parkinson's disease. It has been suggested to be a redox-dependent regulatory scaffold, binding to proteins to modulate their function. Here we present the X-ray crystal structure of the Toxoplasma orthologue Toxoplasma gondii DJ-1 (TgDJ-1) at 2.1-Å resolution and show that it directly associates with calcium-dependent protein kinase 1 (CDPK1). The TgDJ-1 structure identifies an orthologously conserved arginine dyad that acts as a phospho-gatekeeper motif to control complex formation. We determined that the binding of TgDJ-1 to CDPK1 is sensitive to oxidation and calcium, and that this interaction potentiates CDPK1 kinase activity. Finally, we show that genetic deletion of TgDJ-1 results in upregulation of CDPK1 expression and that disruption of the CDPK1/TgDJ-1 complex in vivo prevents normal exocytosis of parasite virulence-associated organelles called micronemes. Overall, our data suggest that TgDJ-1 functions as a noncanonical kinase-regulatory scaffold that integrates multiple intracellular signals to tune microneme exocytosis in T. gondiiIMPORTANCE Apicomplexan parasites such as Toxoplasma and Plasmodium are obligate intracellular parasites that require the protective environment of a host cell in order to replicate and survive within a host organism. These parasites secrete effector proteins from specialized apical organelles to select and invade a chosen host cell. The secretion of these organelles is a tightly regulated process coordinated by endogenous small molecules and calcium-dependent protein kinases. We previously identified the Toxoplasma orthologue of the highly conserved protein DJ-1 as a regulator of microneme secretion, but the molecular basis for this was not known. We have now identified the molecular mechanism for how TgDJ-1 regulates microneme secretion. TgDJ-1 interacts with the kinase responsible for the secretion of these organelles (calcium-dependent kinase 1) and synergizes with calcium to potentiate kinase activity. This interaction is direct, phosphodependent, and necessary for the normal secretion of these important organelles.

Bifunctional Probes of Cathepsin Protease Activity and pH Reveal Alterations in Endolysosomal pH during Bacterial Infection.

Cysteine cathepsins are lysosomal proteases involved in regulation of both normal cellular processes and disease. Biochemical studies with peptide substrates indicate that cathepsins have optimal activity at acidic pH and highly attenuated activity at neutral pH. In contrast, there is mounting evidence that cathepsins have biological roles in environments that have non-acidic pH. To further define the specific pH environments where cathepsins act, we designed bifunctional activity-based probes (ABPs) that allow simultaneous analysis of cathepsin protease activity and pH. We use these probes to analyze the steady-state environment of cathepsin activity in macrophages and to measure dynamic changes in activity and pH upon stimulation. We show that Salmonella typhimurium induces a change in lysosomal pH that ultimately impairs cathepsin activity in both infected cells and a fraction of bystander cells, highlighting a mechanism by which Salmonella can simultaneously flourish within host cells and alter the behavior of nearby uninfected cells.

Disruption of glycolytic flux is a signal for inflammasome signaling and pyroptotic cell death.

When innate immune cells such as macrophages are challenged with environmental stresses or infection by pathogens, they trigger the rapid assembly of multi-protein complexes called inflammasomes that are responsible for initiating pro-inflammatory responses and a form of cell death termed pyroptosis. We describe here the identification of an intracellular trigger of NLRP3-mediated inflammatory signaling, IL-1ß production and pyroptosis in primed murine bone marrow-derived macrophages that is mediated by the disruption of glycolytic flux. This signal results from a drop of NADH levels and induction of mitochondrial ROS production and can be rescued by addition of products that restore NADH production. This signal is also important for host-cell response to the intracellular pathogen Salmonella typhimurium, which can disrupt metabolism by uptake of host-cell glucose. These results reveal an important inflammatory signaling network used by immune cells to sense metabolic dysfunction or infection by intracellular pathogens.

Cysteine Cathepsin Inhibitors as Anti-Ebola Agents.

The recent Ebola virus outbreak in western Africa highlights the need for novel therapeutics that target Ebola virus and other filoviruses. Filoviruses require processing by host cell-derived cysteine cathepsins for productive infection. Here we report the generation of a focused library of cysteine cathepsin inhibitors and subsequent screening to identify compounds with potent activity against viral entry and replication. Our top compounds show highly potent and broad-spectrum activity against cysteine cathepsins and were able to effectively block entry of Ebola and Marburg viruses. These agents are promising leads for development as antifilovirus therapeutics.

Non-invasive Imaging of Idiopathic Pulmonary Fibrosis Using Cathepsin Protease Probes.

Idiopathic pulmonary fibrosis (IPF) is a lethal, chronic, progressive disease characterized by formation of scar tissue within the lungs. Because it is a disease of unknown etiology, it is difficult to diagnose, to predict disease course and to devise treatment strategies. Recent evidence suggests that activated macrophages play key roles in the pathology of IPF. Therefore, imaging probes that specifically recognize these pools of activated immune cells could provide valuable information about how these cells contribute to the pathobiology of the disease. Here we demonstrate that cysteine cathepsin-targeted imaging probes can be used to monitor the contribution of macrophages to fibrotic disease progression in the bleomycin-induced murine model of pulmonary fibrosis. Furthermore, we show that the probes highlight regions of macrophage involvement in fibrosis in human biopsy tissues from IPF patients. Finally, we present first-in-human results demonstrating non-invasive imaging of active cathepsins in fibrotic lesions of patients with IPF. Together, our findings validate small molecule cysteine cathepsin probes for clinical PET imaging and suggest that they have the potential to be used to generate mechanistically-informative molecular information regarding cellular drivers of IPF disease severity and progression.