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The development of reactive drug carriers that could actively respond to biological signals is a challenging task. Different peptides can self-assemble into biocompatible nanostructures of various functionalities, including drugs carriers. Minimal building blocks, such as diphenylalanine, readily form ordered nanostructures. Here we present the development of self-assembled tetra-peptides that include the diphenylalanine motif, serving as substrates of the cathepsin proteases. This is of great clinical importance as cathepsins, whose activity and expression are highly elevated in cancer and other pathologies, have been shown to serve as efficient enzymes for therapeutic release. Based on the cathepsins affinity around the active site, we generated a library of Phe-Phe-Lys-Phe (FFKF) tetra-peptide substrates (TPSs). We inserted various N-termini capping groups with different chemical properties to investigate the effect on protease affinity and self-assembly. All nine TPSs were cleaved by their targets, cathepsins B and L. However, solvent switching led to nanofibers self-assembly of only seven of them. Due to its rapid self-assembly and complete degradation by cathepsin B, we focused on TPS4, Cbz-FFKF-OH. Degradation of TPS4 nanofibers by cathepsin B led to the release of 91.8±0.3% of the incorporated anti-cancerous drug Doxorubicin from the nanofibers within 8h while only 55±0.2% was released without enzyme treatment. Finally, we demonstrated that tumor lysates fully degraded TPS4 nanofibers. Collectively, these results suggest that tetra-peptide substrates that form nanostructures could serve as a promising platform for targeted drug delivery to pathologies in which protease activity is highly elevated., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: The immune system plays a pivotal role in myocardial homeostasis and response to injury. Interleukins-4 and -13 are anti-inflammatory type-2 cytokines, signaling via the common interleukin-13 receptor α1 chain and the type-2 interleukin-4 receptor. The role of interleukin-13 receptor α1 in the heart is unknown., Methods and Results: We analyzed myocardial samples from human donors (n=136) and patients with end-stage heart failure (n=177). We found that the interleukin-13 receptor α1 is present in the myocardium and, together with the complementary type-2 interleukin-4 receptor chain Il4ra , is significantly downregulated in the hearts of patients with heart failure. Next, we showed that Il13ra1 -deficient mice develop severe myocardial dysfunction and dyssynchrony compared to wild-type mice (left ventricular ejection fraction 29.7±9.9 versus 45.0±8.0; P =0.004, left ventricular end-diastolic diameter 4.2±0.2 versus 3.92±0.3; P =0.03). A bioinformatic analysis of mouse hearts indicated that interleukin-13 receptor α1 regulates critical pathways in the heart other than the immune system, such as extracellular matrix (normalized enrichment score=1.90; false discovery rate q=0.005) and glucose metabolism (normalized enrichment score=-2.36; false discovery rate q=0). Deficiency of Il13ra1 was associated with reduced collagen deposition under normal and pressure-overload conditions., Conclusions: The results of our studies in humans and mice indicate, for the first time, a role of interleukin-13 receptor α1 in myocardial homeostasis and heart failure and suggests a new therapeutic target to treat heart disease., (© 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.)

Atherosclerosis predisposes patients to cardiovascular diseases, such as myocardial infarction and stroke. Instigation of vascular injury is triggered by retention of lipids and inflammatory cells in the vascular endothelium. Whereas these vascular lesions develop in young adults and are mostly considered harmless, over time persistent inflammatory and remodeling processes will ultimately damage the arterial wall and cause a thrombotic event due to exposure of tissue factors into the lumen. Evidence from human tissues and preclinical animal models has clearly established the role of cathepsin cysteine proteases in the development and progression of vascular lesions. Hence, understanding the function of cathepsins in atherosclerosis is important for developing novel therapeutic strategies and advanced point of care diagnostics. In this review we will describe the roles of cysteine cathepsins in different cellular process that become dysfunctional in atherosclerosis, such as lipid metabolism, inflammation and apoptosis, and how they contribute to arterial remodeling and atherogenesis. Finally, we will explore new horizons in protease molecular imaging, which may potentially become a surrogate marker to identify future cardiovascular events., (© The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Cyanoacrylate esters (CA) and their corresponding polymers (PolyCA) are used as general and medical adhesives, biodegradable carriers for controlled drug delivery, and as agents for fingerprint development in forensic science. Most reports of cyanoacrylate are on ethyl or 2-octyl cyanoacrylate ester with little attention to other esters. It is the objective of this study to determine the differences amongst cyanoacrylate esters regarding their synthesis, chemical characterization, hydrolytic degradation, and thermal and mechanical properties. Cyanoacrylate polymers of short and long alkyls or oxy-alkyls, cyclic, and aromatic esters have been synthesized and evaluated. All monomers form polymers when exposed to triethylamine vapours possessing molecular weights in the range of 15,000-150,000Da, where the alkyl esters form high MW polymers. A wide range of hydrolytic degradation rates has been found, as monitored by the release of formaldehyde over time. Alkoxy CAs show faster hydrolytic degradation compared to alkyl CAs. Regarding mechanical properties, CAs are classified into primarily viscous (G'>G″) and primarily elastic (G″>G'). Alkoxy CA polymers have a higher loss modulus (G') than storage modulus (G″). Octyl CA polymer possess a G'≈G″ (phase angle ∼45°) providing appropriate balance between mechanical strength and plasticity. Most alkyl CAs are compact and brittle. Alkoxy CAs show enhanced plasticity, but they lack mechanical strength. In general, the T g for alkoxy CAs is less than alkyl CAs. Alkoxy CAs depolymerise rapidly at temperatures >200°C. Overall, ester sidechains of CA esters strongly affect the polymer property., Statement of Significance: Polycyanoacrylates are an important class of biodegradable polymers mainly used as bioadhesives. The study describes comparative evaluation of different cyanoacrylate polymers with respect to their chemistry, degradation, safety, mechanical, and thermal properties. The study forms the basis for choosing appropriate combination of cyanoacrylate esters for various biomedical uses. Moreover, this study reveals properties of a few new polycyanoacrylates for the first time., (Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Protein lysine methyltransferases (PKMTs) catalyze the methylation of protein substrates, and their dysregulation has been linked to many diseases, including cancer. Accumulated evidence suggests that the reaction path of PKMT-catalyzed methylation consists of the formation of a cofactor(cosubstrate)-PKMT-substrate complex, lysine deprotonation through dynamic water channels, and a nucleophilic substitution (S N 2) transition state for transmethylation. However, the molecular characters of the proposed process remain to be elucidated experimentally. Here we developed a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) method and corresponding mathematic matrix to determine precisely the ratios of isotopically methylated peptides. This approach may be generally applicable for examining the kinetic isotope effects (KIEs) of posttranslational modifying enzymes. Protein lysine methyltransferase SET8 is the sole PKMT to monomethylate histone 4 lysine 20 (H4K20) and its function has been implicated in normal cell cycle progression and cancer metastasis. We therefore implemented the MS-based method to measure KIEs and binding isotope effects (BIEs) of the cofactor S-adenosyl-l-methionine (SAM) for SET8-catalyzed H4K20 monomethylation. A primary intrinsic 13 C KIE of 1.04, an inverse intrinsic α-secondary CD 3 KIE of 0.90, and a small but statistically significant inverse CD 3 BIE of 0.96, in combination with computational modeling, revealed that SET8-catalyzed methylation proceeds through an early, asymmetrical S N 2 transition state with the C-N and C-S distances of 2.35-2.40 Å and 2.00-2.05 Å, respectively. This transition state is further supported by the KIEs, BIEs, and steady-state kinetics with the SAM analog Se-adenosyl-l-selenomethionine (SeAM) as a cofactor surrogate. The distinct transition states between protein methyltransferases present the opportunity to design selective transition-state analog inhibitors., Competing Interests: The authors declare no conflict of interest.

Background: Pelvic organ prolapse is a common medical finding. The use of perineal ultrasound for diagnosis of cystoceles is gaining in importance., Objectives: The purpose of this work was to test whether perineal ultrasound can be used to diagnose a cystocele before surgery and for follow-up examination. Furthermore, patient satisfaction during speculum examination and perineal ultrasound was compared., Materials and Methods: 33 women with cystocele were examined before and after anterior colporrhaphy. Symptoms and satisfaction were documented with questionnaires., Results: Ultrasound measurements of both examiners were correlated before and after colporrhaphy. Also, the degree of cystocele and ultrasound were correlated during Valsalva after surgery. There was no clear relation between typical symptoms of the cystocele and ultrasound measurements. The patient's comfort is higher during ultrasound than during speculum examination (r = 0.45; p = 0.04. t = 4,418; p < 0.01)., Conclusion: The results of the perineal ultrasound are reproducible before and after colporrhaphy. Patients prefer ultrasound to the speculum examination. A sonographic scale of the cystocele would extend the use of perineal ultrasound.

While chemotherapy strongly restricts or reverses tumor growth, the response of host tissue to therapy can counteract its anti-tumor activity by promoting tumor re-growth and/or metastases, thus limiting therapeutic efficacy. Here, we show that vascular endothelial growth factor receptor 3 (VEGFR3)-expressing macrophages infiltrating chemotherapy-treated tumors play a significant role in metastasis. They do so in part by inducing lymphangiogenesis as a result of cathepsin release, leading to VEGF-C upregulation by heparanase. We found that macrophages from chemotherapy-treated mice are sufficient to trigger lymphatic vessel activity and structure in naive tumors in a VEGFR3-dependent manner. Blocking VEGF-C/VEGFR3 axis inhibits the activity of chemotherapy-educated macrophages, leading to reduced lymphangiogenesis in treated tumors. Overall, our results suggest that disrupting the VEGF-C/VEGFR3 axis not only directly inhibits lymphangiogenesis but also blocks the pro-metastatic activity of macrophages in chemotherapy-treated mice., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background and Purpose: Cardiovascular disease is the leading cause of death worldwide, mainly due to an increasing prevalence of atherosclerosis characterized by inflammatory plaques. Plaques with high levels of macrophage infiltration are considered "vulnerable" while those that do not have significant inflammation are considered stable; cathepsin protease activity is highly elevated in macrophages of vulnerable plaques and contributes to plaque instability. Establishing novel tools for non-invasive molecular imaging of macrophages in plaques could aid in preclinical studies and evaluation of therapeutics. Furthermore, compounds that reduce the macrophage content within plaques should ultimately impact care for this disease., Methods: We have applied quenched fluorescent cathepsin activity-based probes (ABPs) to a murine atherosclerosis model and evaluated their use for in vivo imaging using fluorescent molecular tomography (FMT), as well as ex vivo fluorescence imaging and fluorescent microscopy. Additionally, freshly dissected human carotid plaques were treated with our potent cathepsin inhibitor and macrophage apoptosis was evaluated by fluorescent microscopy., Results: We demonstrate that our ABPs accurately detect murine atherosclerotic plaques non-invasively, identifying cathepsin activity within plaque macrophages. In addition, our cathepsin inhibitor selectively induced cell apoptosis of 55%±10% of the macrophage within excised human atherosclerotic plaques., Conclusions: Cathepsin ABPs present a rapid diagnostic tool for macrophage detection in atherosclerotic plaque. Our inhibitor confirms cathepsin-targeting as a promising approach to treat atherosclerotic plaque inflammation.

Background and Purpose: Atherosclerosis is a leading cause of mortality worldwide, contributing to both strokes and heart attacks. Macrophages are key players in atherogenesis, promoting vascular inflammation and arterial remodeling through cysteine cathepsin proteases. We used a cathepsin-targeted activity-based probe in human carotid plaque to assess its diagnostic potential and evaluate macrophage subtypes ex vivo., Methods: Carotid plaque specimens surgically removed during endarterectomy from 62 patients (age range, 38% female, 28% symptomatic) were graded pathologically as either stable (Grade 1) or unstable (Grade 2 or 3). A cathepsin activity-based probe was used to quantify individual cathepsins in plaque tissue and macrophage subtypes., Results: Cathepsin B and S activities were increased in unstable carotid plaques. They were quantified using the probe to biochemically investigate individual cathepsins (Cathepsin B and S: 0.97 and 0.90 for grade 3 versus 0.51 and 0.59 for grade 1; P=0.006 and P=0.03 arbitrary units (AU), respectively). Higher cathepsin activity was observed in carotid plaques from symptomatic patients (Cathepsin B and S: 0.65 and 0.77 for asymptomatic, 0.99 and 1.17 for symptomatic; P=0.008 and P=0.005 AU, respectively). Additionally, it was demonstrated that M2 macrophages from unstable plaques express cathepsin activity 5-fold higher than M2 macrophages from stable plaques (25.52 versus 5.22; P=0.008 AU)., Conclusions: Targeting cathepsin activity in human carotid plaques may present a novel diagnostic tool for characterizing high-risk plaques. Novel cathepsin activity patterns within plaques and macrophage subpopulations suggest their involvement in the transition to active disease., (© 2016 American Heart Association, Inc.)

Approximately one-half of the patients who develop clinical atherosclerosis have normal or only modest elevations in plasma lipids, indicating that additional mechanisms contribute to pathogenesis. In view of increasing evidence that inflammation contributes to atherogenesis, we studied the effect of human neutrophil α-defensins on low density lipoprotein (LDL) trafficking, metabolism, vascular deposition, and atherogenesis using transgenic mice expressing human α-defensins in their polymorphonuclear leukocytes (Def(+/+)). Accelerated Def(+/+) mice developed α-defensin·LDL complexes that accelerate the clearance of LDL from the circulation accompanied by enhanced vascular deposition and retention of LDL, induction of endothelial cathepsins, increased endothelial permeability to LDL, and the development of lipid streaks in the aortic roots when fed a regular diet and at normal plasma levels of LDL. Transplantation of bone marrow from Def(+/+) to WT mice increased LDL clearance, increased vascular permeability, and increased vascular deposition of LDL, whereas transplantation of WT bone marrow to Def(+/+) mice prevented these outcomes. The same outcome was obtained by treating Def(+/+) mice with colchicine to inhibit the release of α-defensins. These studies identify a potential new link between inflammation and the development of atherosclerosis., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The caspases are a family of cysteine proteases that are key regulators of apoptosis and their activity may thus serve as a good marker to monitor cell death. We have developed a quenched fluorescent activity-based probe (qABP) that is selective for caspase-3 activity and emits a fluorescent signal after covalently modifying its target. The probe has a wide range of potential applications, e.g. in real-time imaging, FACS analysis or biochemical quantification of caspase activity in intact cells. Application of the probe allowed us to monitor caspase-3 activation after chemotherapy-treatment and to distinguish between apoptosis sensitive and resistant cells. Moreover, it enabled real-time high-resolution visualization of active caspase-3 during apoptosis. This led to the surprising finding that in cancerous cells active caspase-3 is not only found at the familiar cellular locations but also in mitochondria and the endoplasmic reticulum. Thus, our novel covalent probe allows high spatial and temporal resolution imaging of caspase-3 activation and may thus be used as an effective tool to study molecular mechanisms of programmed cell death in healthy and disease states.

Aims: Invasive fungal infections have significantly increased over the past decades in immunocompromised individuals and high-risk patients. Amphotericin B (AmB) exerts a powerful and broad activity against a vast array of fungi and has a remarkably low rate of microbial resistance. However, most isolates of Aspergillus terreus developed an intrinsic resistance against AmB, and during this study, we characterized the mode of action of this polyene antifungal drug in more detail in resistant (ATR) and rare susceptible (ATS) clinical isolates of A. terreus., Results: We illustrate that AmB treatment changes cellular redox status and promotes the generation of high levels of reactive oxygen species (ROS) in ATS. In contrast, ATR isolates were able to cope better with AmB-induced oxidative stress., Innovation: Most importantly, we demonstrate in this study that coapplication of anti- and pro-oxidants significantly affects AmB efficacy in an antithetic manner--antioxidants and ROS-scavenging agents increase AmB tolerance in susceptible strains, while pro-oxidants render formerly resistant isolates considerably susceptible to the antifungal drug also in vivo in a Galleria animal model., Conclusion: Thereby, our study provides novel therapeutic options to treat formerly resistant fungal strains by a combination of AmB and pro-oxidant compounds.

The tumor suppressors BAP1 and ASXL1 interact to form a polycomb deubiquitinase complex that removes monoubiquitin from histone H2A lysine 119 (H2AK119Ub). However, BAP1 and ASXL1 are mutated in distinct cancer types, consistent with independent roles in regulating epigenetic state and malignant transformation. Here we demonstrate that Bap1 loss in mice results in increased trimethylated histone H3 lysine 27 (H3K27me3), elevated enhancer of zeste 2 polycomb repressive complex 2 subunit (Ezh2) expression, and enhanced repression of polycomb repressive complex 2 (PRC2) targets. These findings contrast with the reduction in H3K27me3 levels seen with Asxl1 loss. Conditional deletion of Bap1 and Ezh2 in vivo abrogates the myeloid progenitor expansion induced by Bap1 loss alone. Loss of BAP1 results in a marked decrease in H4K20 monomethylation (H4K20me1). Consistent with a role for H4K20me1 in the transcriptional regulation of EZH2, expression of SETD8-the H4K20me1 methyltransferase-reduces EZH2 expression and abrogates the proliferation of BAP1-mutant cells. Furthermore, mesothelioma cells that lack BAP1 are sensitive to EZH2 pharmacologic inhibition, suggesting a novel therapeutic approach for BAP1-mutant malignancies.

Background: Platelets were recently identified as a part of innate immunity. They are activated by contact with Aspergillus fumigatus; putative consequences include antifungal defense but also thrombosis, excessive inflammation, and thrombocytopenia. We aimed to identify those fungal surface structures that mediate interaction with platelets., Methods: Human platelets were incubated with Aspergillus conidia and hyphae, isolated wall components, or fungal surface mutants. Interaction was visualized microscopically; activation was quantified by flow cytometry of specific markers., Results: The capacity of A. fumigatus conidia to activate platelets is at least partly due to melanin, because this effect can be mimicked with "melanin ghosts"; a mutant lacking melanin showed reduced platelet stimulating potency. In contrast, conidial hydrophobin masks relevant structures, because an A. fumigatus mutant lacking the hydrophobin protein induced stronger platelet activation than wild-type conidia. A. fumigatus hyphae also contain surface structures that interact with platelets. Wall proteins, galactomannan, chitin, and β-glucan are not the relevant hyphal components; instead, the recently identified fungal polysaccharide galactosaminogalactan potently triggered platelet activation., Conclusions: Conidial melanin and hydrophobin as well as hyphal galactosaminogalactan represent important pathogenicity factors that modulate platelet activity and thus might influence immune responses, inflammation, and thrombosis in infected patients., (© The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Vascular endothelial cell (VEC) senescence is considered an early event in the development of atherosclerotic lesions. Stressful stimuli, in particular oxidative stress, have been linked to premature senescence in the vasculature. Foam cells are a major source of reactive oxygen species and may play a role in the induction of VEC senescence; hence, we investigated their involvement in the induction of VEC senescence in a co-culture transwell system. Primary bovine aortic endothelial cells, exposed to the secretome of THP-1 monocyte-derived foam cells, were analysed for the induction of senescence. Senescence associated β-galactosidase activity and the expression of p16 and p21 were increased, whereas phosphorylated retinoblastoma protein was reduced. This senescent phenotype was mediated by 4-hydroxnonenal (4-HNE), a lipid peroxidation product secreted from foam cells; scavenging of 4-HNE in the co-culture medium blunted this effect. Furthermore, both foam cells and 4-HNE increased the expression of the pro-oxidant thioredoxin-interacting protein (TXNIP). Molecular manipulation of TXNIP expression confirmed its involvement in foam cell-induced senescence. Previous studies showed that peroxisome proliferator-activated receptor (PPAR)δ was activated by 4-hydroalkenals, such as 4-HNE. Pharmacological interventions supported the involvement of the 4-HNE-PPARδ axis in the induction of TXNIP and VEC senescence. The association of TXNIP with VEC senescence was further supported by immunofluorescent staining of human carotid plaques in which the expression of both TXNIP and p21 was augmented in endothelial cells. Collectively, these findings suggest that foam cell-released 4-HNE activates PPARδ in VEC, leading to increased TXNIP expression and consequently to senescence., (© 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

The polyene antifungal amphotericin B (AmB) is widely used to treat life-threatening fungal infections. Even though AmB resistance is exceptionally rare in fungi, most Aspergillus terreus isolates exhibit an intrinsic resistance against the drug in vivo and in vitro. Heat shock proteins perform a fundamental protective role against a multitude of stress responses, thereby maintaining protein homeostasis in the organism. In this study, we elucidated the role of heat shock protein 70 (Hsp70) family members and compared resistant and susceptible A. terreus clinical isolates. The upregulation of cytoplasmic Hsp70 members at the transcriptional as well as translational levels was significantly higher with AmB treatment than without AmB treatment, particularly in resistant A. terreus isolates, thereby indicating a role of Hsp70 proteins in the AmB response. We found that Hsp70 inhibitors considerably increased the susceptibility of resistant A. terreus isolates to AmB but exerted little impact on susceptible isolates. Also, in in vivo experiments, using the Galleria mellonella infection model, cotreatment of resistant A. terreus strains with AmB and the Hsp70 inhibitor pifithrin-μ resulted in significantly improved survival compared with that achieved with AmB alone. Our results point to an important mechanism of regulation of AmB resistance by Hsp70 family members in A. terreus and suggest novel drug targets for the treatment of infections caused by resistant fungal isolates., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Elevated cathepsins levels and activities are found in several types of human cancer, making them valuable biomarkers for detection and targeting therapeutics. We designed small molecule quenched activity-based probes (qABPs) that fluoresce upon activity-dependent covalent modification, yielding cell killing by Photodynamic Therapy (PDT). These novel molecules are highly selective theranostic probes that enable both detection and treatment of cancer with minimal side effects. Our qABPs carry a photosensitizer (PS), which is activated by light, resulting in oxidative stress and subsequent cell ablation, and a quencher that when removed by active cathepsins allow the PS to fluoresce and demonstrate PD properties. Our most powerful and stable PS-qABP, YBN14, consists of a selective cathepsin recognition sequence, a QC-1 quencher and a new bacteriochlorin derivative as a PS. YBN14 allowed rapid and selective non-invasive in vivo imaging of subcutaneous tumors and induced specific tumor macrophage apoptosis by light treatment, resulting in a substantial tumor shrinkage in an aggressive breast cancer mouse model. These results demonstrate for the first time that the PS-qABPs technology offers a functional theranostic tool, which can be applied to numerous tumor types and other inflammation-associated diseases.

Fibrinogen has the potential of being used as a material to harvest and grow normal mesenchymal cells (fibroblasts, endothelial cells) or to trap cancer cells from a suspension with blood as a potential circulatory trap.Insoluble fibrinogen particles (iFP) were prepared from commercial Cohn fraction I paste (source: Kedrion). The sized iFP (~60-180 µm) were not soluble in physiologic buffers, exhibited a density of 1.2 ± 0.02, and did not aggregate or clump when mixed with whole blood or thrombin, but were degraded in lytic solutions.Cell culture studies indicated that the iFP could be used to harvest, expand and transfer normal, mammalian, attachment-dependent cells, notably fibroblasts and stem cells from bone marrow, as well as numerous cancer lines. Cells attached to iFP underwent logarithmic growth kinetics and could be transferred without trypsinization. Transplanted cancer cells-on-iFP generated characteristic tumors and retained their surface marker (by Western immuno-blot). An iFP 'cell-affinity' batch column was shown to trap MCF-7 cancer cells in the presence of red blood cells (RBCs) or serum.The scalable process for fabricating iFP retained the cell attachment properties of native fibrinogen. The results indicate that iFP has the potential to be used as a 3D cell culture matrix, and possibly to trap cancer cells from blood.

Introduction: Lysosomal cathepsins have been reported to contribute to Osteoarthritis (OA) pathophysiology due to their increase in pro-inflammatory conditions. Given the causal role of cathepsins in OA, monitoring their specific activity could provide means for assessing OA severity. To this end, we herein sought to assess a cathepsin activity-based probe (ABP), GB123, in vitro and in vivo., Methods: Protein levels and activity of cathepsins B and S were monitored by immunoblot analysis and GB123 labeling in cultured primary chondrocytes and conditioned media, following stimuli with tumor necrosis factor alpha (TNFα) and/or Interleukin 1 beta (IL-1β). Similarly, cathepsin activity was examined in sections of intact cartilage (IC) and degraded cartilage (DC) regions of OA. Finally, synovial fluid (SF) and serum from donors with no signs of diseases, early OA, late OA and rheumatoid arthritis (RA) patients were analyzed with GB123 to detect distinct activity levels of cathepsin B and S., Results: Cathepsin activity in cell lysates, conditioned media explants and DC sections showed enhanced enzymatic activity of cathepsins B and S. Further histological analysis revealed that cathepsin activity was found higher in superficial zones of DC than in IC. Examining serum and SF revealed that cathepsin B is significantly elevated with OA severity in serum and SF, yet levels of cathepsin S are more correlated with synovitis and RA., Conclusions: Based on our data, cathepsin activity monitored by ABPs correlated well with OA severity and joint inflammation, directing towards a novel etiological target for OA, which possesses significant translational potential in developing means for non-invasive detection of early signs of OA.

SETD8/SET8/Pr-SET7/KMT5A is the sole protein lysine methyltransferase (PKMT) known to monomethylate lysine 20 of histone H4 in vivo. SETD8's methyltransferase activity has been implicated in many essential cellular processes including DNA replication, DNA damage response, transcription modulation, and cell cycle regulation. Developing SETD8 inhibitors with cellular activity is a key step toward elucidating the diverse roles of SETD8 via convenient pharmacological perturbation. From the hits of a prior high throughput screen (HTS), SPS8I1-3 (NSC663284, BVT948, and ryuvidine) were validated as potent SETD8 inhibitors. These compounds contain different structural motifs and inhibit SETD8 via distinct modes. More importantly, these compounds show cellular activity by suppressing the H4K20me1 mark of SETD8 and recapitulate characteristic S/G2/M-phase cell cycle defects as observed for RNAi-mediated SETD8 knockdown. The commonality of SPS8I1-3 against SETD8, together with their distinct structures and mechanisms for SETD8 inhibition, argues for the collective application of these compounds as SETD8 inhibitors.

Protein arginine N-methyltransferase 3 (PRMT3) belongs to the family of type I PRMTs and harbors the activity to use S-adenosyl-l-methionine (SAM) as a methyl-donor cofactor for protein arginine labeling. However, PRMT3's functions remain elusive with the lacked knowledge of its target scope in cellular settings. Inspired by the emerging Bioorthogonal Profiling of Protein Methylation (BPPM) using engineered methyltransferases and SAM analogues for target identification, the current work documents the endeavor to systematically explore the SAM-binding pocket of PRMT3 and identify suitable PRMT3 variants for BPPM. The M233G single point mutation transforms PRMT3 into a promiscuous alkyltransferase using sp(2)-β-sulfonium-containing SAM analogues as cofactor surrogates. Here the conserved methionine was defined as a hot spot that can be engineered alone or in combination with nearby residues to render cofactor promiscuity of multiple type I PRMTs. With this promiscuous variant and the matched 4-propargyloxy-but-2-enyl (Pob)-SAM analogue as the BPPM reagents, more than 80 novel proteins were readily uncovered as potential targets of PRMT3 in the cellular context. Subsequent target validation and functional analysis correlated the PRMT3 methylation to several biological processes such as cytoskeleton dynamics, whose roles might be compensated by other PRMTs. These BPPM-revealed substrates are primarily localized but not restricted in cytoplasm, the preferred site of PRMT3. The broad localization pattern may implicate the diverse roles of PRMT3 in the cellular setting. The revelation of PRMT3 targets and the transformative character of BPPM for other PRMTs present unprecedented pathways toward elucidating physiological and pathological roles of diverse PRMTs.

Objectives: This study sought to investigate the hypothesis that the favorable effects of mesenchymal stromal cells (MSCs) on infarct repair are mediated by macrophages., Background: The favorable effects of MSC therapy in myocardial infarction (MI) are complex and not fully understood., Methods: We induced MI in mice and allocated them to bone marrow MSCs, mononuclear cells, or saline injection into the infarct, with and without early (4 h before MI) and late (3 days after MI) macrophage depletion. We then analyzed macrophage phenotype in the infarcted heart by flow cytometry and macrophage secretome in vitro. Left ventricular remodeling and global and regional function were assessed by echocardiography and speckle-tracking based strain imaging., Results: The MSC therapy significantly increased the percentage of reparative M2 macrophages (F4/80(+)CD206(+)) in the infarcted myocardium, compared with mononuclear- and saline-treated hearts, 3 and 4 days after MI. Macrophage cytokine secretion, relevant to infarct healing and repair, was significantly increased after MSC therapy, or incubation with MSCs or MSC supernatant. Significantly, with and without MSC therapy, transient macrophage depletion increased mortality 30 days after MI. Furthermore, early macrophage depletion produced the greatest negative effect on infarct size and left ventricular remodeling and function, as well as a significant incidence of left ventricular thrombus formation. These deleterious effects were attenuated with macrophage restoration and MSC therapy., Conclusions: Some of the protective effects of MSCs on infarct repair are mediated by macrophages, which are essential for early healing and repair. Thus, targeting macrophages could be a novel strategy to improve infarct healing and repair., (Copyright © 2013 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

We compared two PCR methods for molecular typing the medically important filamentous fungus Aspergillus terreus. In a set of 46 strains investigated we found 19 and 12 different fingerprinting types obtained by random amplified polymorphic DNA PCR (RAPD) and semi-automated repetitive element PCR (rep-PCR), respectively.

Aspergillus terreus-induced invasive infections exhibit high lethality, partly due to the intrinsic resistance for amphotericin B (AmB). We compared the virulence and pathogenesis of an AmB-resistant isolate of A. terreus (ATR) with that of a rare variant showing enhanced sensitivity for AMB (ATS). The modifications that result in enhanced AmB sensitivity of isolates are not associated with reduced virulence in vivo; instead, the ATS-infected mice died even faster than the ATR-infected animals. Since A. terreus enters the blood stream in most patients and frequently induces thrombosis, we studied a putative correlation between virulence of the two A. terreus isolates and their effect on thrombocytes. Those mice infected with the more virulent ATS isolate had lower thrombocyte numbers and more phosphatidylserine exposure on platelets than ATR-infected mice. In vitro experiments confirmed that ATS and ATR differ in their effect on thrombocytes. Conidia, aleurioconidia and hyphae of ATS were more potent than ATR to trigger thrombocyte stimulation, and thrombocytes adhered better to ATS than to ATR fungal structures. Furthermore, ATS secreted more soluble factors that triggered platelet stimulation than ATR. Thus, it might be suggested that the capacity of a fungal isolate to modulate thrombocyte parameters contributes to its virulence in vivo.

One of the noncellular microenvironmental factors that contribute to malignancy of solid tumors is acidic peritumoral pH. We have previously demonstrated that extracellular acidosis leads to localization of the cysteine pro-tease cathepsin B on the tumor cell membrane and its secretion. The objective of the present study was to determine if an acidic extracellular pH such as that observed in vivo (i.e., pHe 6.8) affects the activity of proteases, e.g., cathepsin B, that contribute to degradation of collagen IV by tumor cells when grown in biologically relevant three-dimensional (3D) cultures. For these studies, we used 1) 3D reconstituted basement membrane overlay cultures of human carcinomas, 2) live cell imaging assays to assess proteolysis, and 3) in vivo imaging of active tumor proteases. At pHe 6.8, there were increases in pericellular active cysteine cathepsins and in degradation of dye-quenched collagen IV, which was partially blocked by a cathepsin B inhibitor. Imaging probes for active cysteine cathepsins localized to tumors in vivo. The amount of bound probe decreased in tumors in bicarbonate-treated mice, a treatment previously shown to increase peritumoral pHe and reduce local invasion of the tumors. Our results are consistent with the acid-mediated invasion hypothesis and with a role for cathepsin B in promoting degradation of a basement membrane protein substrate, i.e., type IV collagen, in an acidic peritumoral environment.

Marshall and Brown (2006) proposed a Traits as Situational Sensitivities (TASS) Model, which implies a systematic person × situation interaction. We review this model and show that it suffers from several limitations. We extend and modify the model in order to obtain a symmetric pattern of levels and effects for both person and situation factors. Our suggestions result in a general Nonlinear Interaction of Person and Situation (NIPS) Model. The NIPS model bears striking similarities to the Rasch model. Based on the symmetric nature of the NIPS model, we generalize the concept of weak and strong situations to individuals and propose the concepts of weak and strong persons. Finally, we discuss psychological mechanisms that might explain the NIPS pattern and offer ideas for future research.

Amphotericin B (AMB) is the predominant antifungal drug, but the mechanism of resistance is not well understood. We compared the in vivo virulence of an AMB-resistant Aspergillus terreus (ATR) isolate with that of an AMB-susceptible A. terreus isolate (ATS) using a murine model for disseminated aspergillosis. Furthermore, we analyzed the molecular basis of intrinsic AMB resistance in vitro by comparing the ergosterol content, cell-associated AMB levels, AMB-induced intracellular efflux, and prooxidant effects between ATR and ATS. Infection of immunosuppressed mice with ATS or ATR showed that the ATS strain was more lethal than the ATR strain. However, AMB treatment improved the outcome in ATS-infected mice while having no positive effect on the animals infected with ATR. The in vitro data demonstrated that ergosterol content is not the molecular basis for AMB resistance. ATR absorbed less AMB, discharged more intracellular compounds, and had better protection against oxidative damage than the susceptible strain. Our experiments showed that ergosterol content plays a minor role in intrinsic AMB resistance and is not directly associated with intracellular cell-associated AMB content. AMB might exert its antifungal activity by oxidative injury rather than by an increase in membrane permeation.

Activity-based probes are small molecules that can be used to monitor enzyme activity by covalently binding to specific residues in the active site. In this issue of Chemistry & Biology, Lu and colleagues developed a specific fluorescent activity-based probe that targets the papain-like cysteine bacterial type III effector protease AvrPphB and used it to demonstrate the regulation of the protease secretion and pathogenesis., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Protein methyltransferases (PMTs) have emerged as important epigenetic regulators in myriad biological processes in both normal physiology and disease conditions. However, elucidating PMT-regulated epigenetic processes has been hampered by ambiguous knowledge about in vivo activities of individual PMTs particularly because of their overlapping but nonredundant functions. To address limitations of conventional approaches in mapping chromatin modification of specific PMTs, we have engineered the chromatin-modifying apparatus and formulated a novel technology, termed clickable chromatin enrichment with parallel DNA sequencing (CliEn-seq), to probe genome-wide chromatin modification within living cells. The three-step approach of CliEn-seq involves in vivo synthesis of S-adenosyl-L-methionine (SAM) analogues from cell-permeable methionine analogues by engineered SAM synthetase (methionine adenosyltransferase or MAT), in situ chromatin modification by engineered PMTs, subsequent enrichment and sequencing of the uniquely modified chromatins. Given critical roles of the chromatin-modifying enzymes in epigenetics and structural similarity among many PMTs, we envision that the CliEn-seq technology is generally applicable in deciphering chromatin methylation events of individual PMTs in diverse biological settings.

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.

Enzymatic transmethylation from the cofactor S-adenosyl-L-methionine (SAM) to biological molecules has recently garnered increased attention because of the diversity of possible substrates and implications in normal biology and diseases. To reveal the substrates of protein methyltransferases (PMTs), the present article focuses on an alkyne-containing SAM mimic, Se-adenosyl-L-selenomethionine (ProSeAM), and a cleavable azido-azo-biotin probe to profile the targets of endogenous PMTs in cellular contexts. This article describes the stepwise preparation of cell lysates containing active, endogenous PMTs and subsequent target labeling with ProSeAM. The article continues with the enrichment of the ProSeAM-labeled proteins with the azido-azo biotin probe as a pulldown reagent and the subsequent reductive elution with sodium dithionate for proteomic analysis. The protocols provided here were formulated for ProSeAM as a profiling reagent but can be applied to other terminal-alkyne-containing SAM analog cofactors., (© 2013 by John Wiley & Sons, Inc.)

Protein methyltransferases (PMTs) utilize S-adenosyl-L-methionine (SAM) as a cofactor and transfer its sulfonium methyl moiety to diverse substrates. These methylation events can lead to meaningful biological outcomes, from transcriptional activation/silencing to cell cycle regulation. This article describes recently developed technology based on protein engineering in tandem with SAM analog cofactors and bioorthogonal click chemistry to unambiguously profile the substrates of a specific PMT. The protocols encapsulate the logic and methods of selectively profiling the substrates of a candidate PMT by (1) engineering the selected PMT to accommodate a bulky SAM analog; (2) generating a proteome containing the engineered PMT; (3) visualizing the proteome-wide substrates of the designated PMT via bioorthogonal labeling with a fluorescent tag; and finally (4) pulling down the proteome-wide substrates for mass spectrometric analysis.

The expression of the cysteine protease cathepsin B is increased in early stages of human breast cancer.To assess the potential role of cathepsin B in premalignant progression of breast epithelial cells, we employed a 3D reconstituted basement membrane overlay culture model of MCF10A human breast epithelial cells and isogenic variants that replicate the in vivo phenotypes of hyper plasia(MCF10AneoT) and atypical hyperplasia (MCF10AT1). MCF10A cells developed into polarized acinar structures with central lumens. In contrast, MCF10AneoT and MCF10AT1 cells form larger structures in which the lumens are filled with cells. CA074Me, a cell-permeable inhibitor selective for the cysteine cathepsins B and L,reduced proliferation and increased apoptosis of MCF10A, MCF10AneoT and MCF10AT1 cells in 3D culture. We detected active cysteine cathepsins in the isogenic MCF10 variants in 3D culture with GB111, a cell-permeable activity based probe, and established differential inhibition of cathepsin B in our 3D cultures. We conclude that cathepsin B promotes proliferation and premalignant progression of breast epithelial cells. These findings are consistent with studies by others showing that deletion of cathepsin B in the transgenic MMTV-PyMT mice, a murine model that is predisposed to development of mammary cancer, reduces malignant progression.

In vitro susceptibility testing of clinically important fungi becomes more and more essential due to the rising number of fungal infections in patients with impaired immune system. Existing standardized microbroth dilution methods for in vitro testing of molds (CLSI, EUCAST) are not intended for routine testing. These methods are very time-consuming and dependent on sporulating of hyphomycetes. In this multicentre study, a new (independent of sporulation) inoculum preparation method (containing a mixture of vegetative cells, hyphae, and conidia) was evaluated. Minimal inhibitory concentrations (MIC) of amphotericin B, posaconazole, and voriconazole of 180 molds were determined with two different culture media (YST and RPMI 1640) according to the DIN (Deutsches Institut für Normung) microdilution assay. 24 and 48 h MIC of quality control strains, tested per each test run, prepared with the new inoculum method were in the range of DIN. YST and RPMI 1640 media showed similar MIC distributions for all molds tested. MIC readings at 48 versus 24 h yield 1 log(2) higher MIC values and more than 90 % of the MICs read at 24 and 48 h were within ± 2 log(2) dilution. MIC end point reading (log(2 MIC-RPMI 1640)-log(2 MIC-YST)) of both media demonstrated a tendency to slightly lower MICs with RPMI 1640 medium. This study reports the results of a new, time-saving, and easy-to-perform method for inoculum preparation for routine susceptibility testing that can be applied for all types of spore-/non-spore and hyphae-forming fungi.

Epigenetic regulation is involved in numerous physiological and pathogenic processes. Among the key regulators that orchestrate epigenetic signaling are over 50 human protein lysine methyltransferases (PKMTs). Interrogation of the functions of individual PKMTs can be facilitated by target-specific PKMT inhibitors. Given the emerging need for such small molecules, we envisioned an approach to identify target-specific methyltransferase inhibitors by screening privileged small-molecule scaffolds against diverse methyltransferases. In this work, we demonstrated the feasibility of such an approach by identifying the inhibitors of SETD2. N-propyl sinefungin (Pr-SNF) was shown to interact preferentially with SETD2 by matching the distinct transition-state features of SETD2's catalytically active conformer. With Pr-SNF as a structure probe, we further revealed the dual roles of SETD2's post-SET loop in regulating substrate access through a distinct topological reconfiguration. Privileged sinefungin scaffolds are expected to have broad use as structure and chemical probes of methyltransferases.

Posttranslational methylation by S-adenosyl-L-methionine(SAM)-dependent methyltransferases plays essential roles in modulating protein function in both normal and disease states. As such, there is a growing need to develop chemical reporters to examine the physiological and pathological roles of protein methyltransferases. Several sterically bulky SAM analogues have previously been used to label substrates of specific protein methyltransferases. However, broad application of these compounds has been limited by their general incompatibility with native enzymes. Here we report a SAM surrogate, ProSeAM (propargylic Se-adenosyl-l-selenomethionine), as a reporter of methyltransferases. ProSeAM can be processed by multiple protein methyltransferases for substrate labeling. In contrast, sulfur-based propargylic SAM undergoes rapid decomposition at physiological pH, likely via an allene intermediate. In conjunction with fluorescent/affinity-based azide probes, copper-catalyzed azide-alkyne cycloaddition chemistry, in-gel fluorescence visualization and proteomic analysis, we further demonstrated ProSeAM's utility to profile substrates of endogenous methyltransferases in diverse cellular contexts. These results thus feature ProSeAM as a convenient probe to study the activities of endogenous protein methyltransferases.

Protein methyltransferases (PMTs) orchestrate epigenetic modifications through post-translational methylation of various protein substrates including histones. Since dysregulation of this process is widely implicated in many cancers, it is of pertinent interest to screen inhibitors of PMTs, as they offer novel target-based opportunities to discover small molecules with potential chemotherapeutic use. We have thus developed an enzymatic screening strategy, which can be adapted to scintillation proximity imaging assay (SPIA) format, to identify these inhibitors. We took advantage of S-adenosyl-L-[3H-methyl]-methionine availability and monitored the enzymatically catalyzed [3H]-methyl addition on lysine residues of biotinylated peptide substrates. The radiolabeled peptides were subsequently captured by streptavidin coated SPA imaging PS beads. We applied this strategy to four PMTs: SET7/9, SET8, SETD2, and EuHMTase1, and optimized assay conditions to achieve Z' values ranging from 0.48 to 0.91. The robust performance of this SPIA for the four PMTs was validated in a pilot screen of approximately 7,000 compounds. We identified 80 cumulative hits across the four targets. NF279, a suramin analogue, was found to specifically inhibit SET7/9 and SETD2 with IC50 values of 1.9 and 1.1 μM, respectively. Another identified compound, Merbromin, a topical antiseptic, was classified as a pan-active inhibitor of the four PMTs. These findings demonstrate that our proposed SPIA strategy is generic for multiple PMTs and can be successfully implemented to identify novel and specific inhibitors of PMTs. The specific PMT inhibitors may constitute a new class of anti-proliferative agents for potential therapeutic use.

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., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The aim of this study was to investigate the relationship between fungal exposure prior to hospitalisation and ensuing onset of invasive mould infections (IMI) in patients at risk. Patients admitted to the Department of Haematology, Oncology and Transplant Surgery of the Medical University Innsbruck received a questionnaire regarding fungal exposure prior to hospital stay. Questions inquired heavy fungal exposures up to 5 days before hospitalisation. A total of 234 patients were enrolled in this study. Multiple fungus exposures were associated with the onset of community-acquired IMI in patients with haematological malignancies. In univariate analysis, haematological malignancies (P = 0.013) and allergy to dust, pollen or moulds (P = 0.015) were significantly associated with fungal infections. In multivariate analysis, logistic regression showed that haematological patients (P = 0.015) and patients with allergy (P = 0.015) were significantly more frequently infected with fungi. Hospital-independent fungal sources highlight risk-factors for IMI in severe immunocompromised patients and the rate of community-acquired IMI does increase., (© 2011 Blackwell Verlag GmbH.)

Metastasis to bone is a major cause of morbidity in breast cancer patients, emphasizing the importance of identifying molecular drivers of bone metastasis for new therapeutic targets. The endogenous cysteine cathepsin inhibitor stefin A is a suppressor of breast cancer metastasis to bone that is coexpressed with cathepsin B in bone metastases. In this study, we used the immunocompetent 4T1.2 model of breast cancer which exhibits spontaneous bone metastasis to evaluate the function and therapeutic targeting potential of cathepsin B in this setting of advanced disease. Cathepsin B abundancy in the model mimicked human disease, both at the level of primary tumors and matched spinal metastases. RNA interference-mediated knockdown of cathepsin B in tumor cells reduced collagen I degradation in vitro and bone metastasis in vivo. Similarly, intraperitoneal administration of the highly selective cathepsin B inhibitor CA-074 reduced metastasis in tumor-bearing animals, a reduction that was not reproduced by the broad spectrum cysteine cathepsin inhibitor JPM-OEt. Notably, metastasis suppression by CA-074 was maintained in a late treatment setting, pointing to a role in metastatic outgrowth. Together, our findings established a prometastatic role for cathepsin B in distant metastasis and illustrated the therapeutic benefits of its selective inhibition in vivo.

Several investigators have shown the utility of systemically delivered optical imaging probes to image tumors in small animal models of cancer. Here we demonstrate an innovative method for imaging tumors and tumor margins during surgery. Specifically, we show that optical imaging probes topically applied to tumors and surrounding normal tissue rapidly differentiate between tissues. In contrast to systemic delivery of optical imaging probes which label tumors uniformly over time, topical probe application results in rapid and robust probe activation that is detectable as early as 5 minutes following application. Importantly, labeling is primarily associated with peri-tumor spaces. This methodology provides a means for rapid visualization of tumor and potentially infiltrating tumor cells and has potential applications for directed surgical excision of tumor tissues. Furthermore, this technology could find use in surgical resections for any tumors having differential regulation of cysteine cathepsin activity.

Protein methyltransferases (PMTs) catalyze arginine and lysine methylation of diverse histone and nonhistone targets. These posttranslational modifications play essential roles in regulating multiple cellular events in an epigenetic manner. In the recent process of defining PMT targets, S-adenosyl-L-methionine (SAM) analogues have emerged as powerful small molecule probes to label and profile PMT targets. To examine efficiently the reactivity of PMTs and their variants on SAM analogues, we transformed a fluorogenic PMT assay into a ready high throughput screening (HTS) format. The reformulated fluorogenic assay is featured by its uncoupled but more robust character with the first step of accumulation of the commonly-shared reaction byproduct S-adenosyl-L-homocysteine (SAH), followed by SAH-hydrolase-mediated fluorogenic quantification. The HTS readiness and robustness of the assay were demonstrated by its excellent Z' values of 0.83-0.95 for the so-far-examined 8 human PMTs with SAM as a cofactor (PRMT1, PRMT3, CARM1, SUV39H2, SET7/9, SET8, G9a and GLP1). The fluorogenic assay was further implemented to screen the PMTs against five SAM analogues (allyl-SAM, propargyl-SAM, (E)-pent-2-en-4-ynyl-SAM (EnYn-SAM), (E)-hex-2-en-5-ynyl-SAM (Hey-SAM) and 4-propargyloxy-but-2-enyl-SAM (Pob-SAM)). Among the examined 8 × 5 pairs of PMTs and SAM analogues, native SUV39H2, G9a and GLP1 showed promiscuous activity on allyl-SAM. In contrast, the bulky SAM analogues, such as EnYn-SAM, Hey-SAM and Pob-SAM, are inert toward the panel of human PMTs. These findings therefore provide the useful structure-activity guidance to further evolve PMTs and SAM analogues for substrate labeling. The current assay format is ready to screen methyltransferase variants on structurally-diverse SAM analogues., (This journal is © The Royal Society of Chemistry 2011)

The papain family of cysteine cathepsins are actively involved in multiple stages of tumorigenesis. Because elevated cathepsin activity can be found in many types of human cancers, they are promising biomarkers that can be used to target radiological contrast agents for tumor detection. However, currently there are no radiological imaging agents available for these important molecular targets. We report here the development of positron emission tomography (PET) radionuclide-labeled probes that target the cysteine cathepsins by formation of an enzyme activity-dependent bond with the active site cysteine. These probes contain an acyloxymethyl ketone (AOMK) functional group that irreversibly labels the active site cysteine of papain family proteases attached to a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) tag for labeling with (64)Cu for PET imaging studies. We performed biodistribution and microPET imaging studies in nude mice bearing subcutaneous tumors expressing various levels of cysteine cathepsin activity and found that the extent of probe uptake by tumors correlated with overall protease activity as measured by biochemical methods. Furthermore, probe signals could be reduced by pre-treatment with a general cathepsin inhibitor. We also found that inclusion of a Cy5 tag on the probe increased tumor uptake relative to probes lacking this fluorogenic dye. Overall, these results demonstrate that small molecule activity-based probes carrying radio-tracers can be used to image protease activity in living subjects.

Imaging agents that enable direct visualization and quantification of apoptosis in vivo have great potential value for monitoring chemotherapeutic response as well as for early diagnosis and disease monitoring. We describe here the development of fluorescently labeled activity-based probes (ABPs) that covalently label active caspases in vivo. We used these probes to monitor apoptosis in the thymi of mice treated with dexamethasone as well as in tumor-bearing mice treated with the apoptosis-inducing monoclonal antibody Apomab (Genentech). Caspase ABPs provided direct readouts of the kinetics of apoptosis in live mice, whole organs and tissue extracts. The probes produced a maximum fluorescent signal that could be monitored noninvasively and that coincided with the peak in caspase activity, as measured by gel analysis. Overall, these studies demonstrate that caspase-specific ABPs have the potential to be used for noninvasive imaging of apoptosis in both preclinical and clinical settings.

Recent advances in the field of non-invasive optical imaging have included the development of contrast agents that report on the activity of enzymatic targets associated with disease pathology. In particular, proteases have proven to be ideal targets for development of optical sensors for cancer. Recently developed contrast agents for protease activity include both small peptides and large polymer-based quenched fluorescent substrates as well as fluorescently labeled activity based probes (ABPs). While substrates produce a fluorescent signal as a result of processing by a protease, ABPs are retained at the site of proteolysis due to formation of a permanent covalent bond with the active site catalytic residue. Both methods have potential advantages and disadvantages yet a careful comparison of substrates and ABPs has not been performed. Here we present the results of a direct comparison of commercially available protease substrates with several recently described fluorescent ABPs in a mouse model of cancer. The results demonstrate that fluorescent ABPs show more rapid and selective uptake into tumors as well as overall brighter signals compared to substrate probes. These data suggest that the lack of signal amplification for an ABP is offset by the increased kinetics of tissue uptake and prolonged retention of the probes once bound to a protease target. Furthermore, fluorescent ABPs can be used as imaging reagents with similar or better results as the commercially available protease substrates.

Tumors initiate angiogenesis primarily by secreting vascular endothelial growth factor (VEGF-A(164)). The first new vessels to form are greatly enlarged, pericyte-poor sinusoids, called mother vessels (MV), that originate from preexisting venules. We postulated that the venular enlargement necessary to form MV would require a selective degradation of their basement membranes, rigid structures that resist vascular expansion. To identify the specific proteases responsible for MV formation, we induced angiogenesis in mouse tissues with an adenoviral vector expressing VEGF-A(164) (Ad-VEGF-A(164)) or with VEGF-A-secreting TA3/St mammary tumors. We found that MV formation resulted from greatly increased activity of cathepsins (B>S>L) in venules transitioning into MV, as well as from a reciprocal decrease in the expression of several cysteine protease inhibitors (CPI), stefin A and cystatins B and C, by these same venules. Using a fluorescence probe that selectively binds cellular sites of cathepsin protease activity in vivo, we showed that increased cathepsin activity was localized exclusively to perivenular cells, not to venule endothelial cells. CPI strikingly inhibited angiogenesis in the Matrigel assay, and Ad-VEGF-A(164)-induced angiogenesis was reduced by approximately 50% in cathepsin B-null mice. Thus, VEGF-A, whether expressed by interstitial cells infected with an adenoviral vector or by tumor cells, upsets the normal cathepsin-CPI balance in nearby venules, leading to degradation of their basement membranes, an important first step in angiogenesis.

Localization of proteases to the surface of endothelial cells and remodeling of the extracellular matrix (ECM) are essential to endothelial cell tube formation and angiogenesis. Here, we partially localized active cathepsin B and its cell surface binding partners, S100A/p11 (p11) of the annexin II heterotetramer (AIIt), to caveolae of human umbilical vein endothelial cells (HUVEC). Via a live-cell proteolysis assay, we observed that degradation products of quenched-fluorescent (DQ)-proteins (i.e. gelatin and collagen IV) colocalized intracellularly with caveolin-1 (cav-1) of HUVEC grown in either monolayer cultures or in vitro tube formation assays. Activity-based probes that bind covalently to active cysteine cathepsins and degradation products of DQ-collagen IV partially localized to intracellular vesicles that contained cav-1 and active cysteine cathepsins. Biochemical analyses revealed that the distribution of active cathepsin B in caveolar fractions increased during in vitro tube formation. Pro-uPA, uPAR, MMP-2 and MMP-14, which have been linked with cathepsin B to ECM degradation pathways, were also found to increase in caveolar fractions during in vitro tube formation. Our findings are the first to demonstrate through live-cell imaging ECM degradation in association with active cathepsin B in caveolae of endothelial cells during tube formation.

The field of activity-based proteomics makes use of small molecule active site probes to monitor distinct subsets of enzymatic proteins. While a number of reactive functional groups have been applied to activity-based probes (ABPs) that target diverse families of proteases, there remains a continual need for further evaluation of new probe scaffolds and reactive functional groups for use in ABPs. In this study we evaluate the utility of the, alpha,beta-unsaturated ketone reactive group for use in ABPs targeting the papain-family of cysteine proteases. We find that this reactive group shows highly selective labeling of cysteine cathepsins in both intact cells and total cell extracts. We observed a variable degree of background labeling that depended on the type of tag and linker used in the probe synthesis. The relative ease of synthesis of this class of compounds provides the potential for further derivatization to generate new families of cysteine protease ABPs with unique specificity and labeling properties.