Co-reporter:Megan Garland, Sebastian Loscher, and Matthew Bogyo
Chemical Reviews 2017 Volume 117(Issue 5) pp:
Publication Date(Web):February 24, 2017
DOI:10.1021/acs.chemrev.6b00676
Antibiotic resistance is a significant emerging health threat. Exacerbating this problem is the overprescription of antibiotics as well as a lack of development of new antibacterial agents. A paradigm shift toward the development of nonantibiotic agents that target the virulence factors of bacterial pathogens is one way to begin to address the issue of resistance. Of particular interest are compounds targeting bacterial AB toxins that have the potential to protect against toxin-induced pathology without harming healthy commensal microbial flora. Development of successful antitoxin agents would likely decrease the use of antibiotics, thereby reducing selective pressure that leads to antibiotic resistance mutations. In addition, antitoxin agents are not only promising for therapeutic applications, but also can be used as tools for the continued study of bacterial pathogenesis. In this review, we discuss the growing number of examples of chemical entities designed to target exotoxin virulence factors from important human bacterial pathogens.
Co-reporter:Caroline L. Ng, David A. Fidock, Matthew Bogyo
Trends in Parasitology 2017 Volume 33, Issue 9(Issue 9) pp:
Publication Date(Web):1 September 2017
DOI:10.1016/j.pt.2017.05.009
Artemisinin (ART)-based combination therapies are the most efficacious treatment of uncomplicated Plasmodium falciparum malaria. Alarmingly, P. falciparum strains have acquired resistance to ART across much of Southeast Asia. ART creates widespread protein and lipid damage inside intraerythrocytic parasites, necessitating macromolecule degradation. The proteasome is the main engine of Plasmodium protein degradation. Indeed, proteasome inhibition and ART have shown synergy in ART-resistant parasites. Moreover, ubiquitin modification is associated with altered parasite susceptibility to multiple antimalarials. Targeting the ubiquitin–proteasome system (UPS), therefore, is an attractive avenue to combat drug resistance. Here, we review recent advances leading to specific targeting of the Plasmodium proteasome. We also highlight the potential for targeting other nonproteasomal protein degradation systems as an additional strategy to disrupt protein homeostasis.
Co-reporter:Wouter A. van der Linden, Christopher J. Schulze, Andrew S. Herbert, Tyler B. Krause, Ariel A. Wirchnianski, John M. Dye, Kartik Chandran, and Matthew Bogyo
ACS Infectious Diseases 2016 Volume 2(Issue 3) pp:173
Publication Date(Web):January 20, 2016
DOI:10.1021/acsinfecdis.5b00130
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.Keywords: cathepsin inhibitor; Ebola virus; filovirus; inhibition of host cell entry
Co-reporter:Kristina Oresic Bender; Leslie Ofori; Wouter A. van der Linden; Elliot D. Mock; Gopal K. Datta; Somenath Chowdhury; Hao Li; Ehud Segal; Mateo Sanchez Lopez; Jonathan A. Ellman; Carl G. Figdor; Matthew Bogyo;Martijn Verdoes
Journal of the American Chemical Society 2015 Volume 137(Issue 14) pp:4771-4777
Publication Date(Web):March 18, 2015
DOI:10.1021/jacs.5b00315
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.
Co-reporter:Wouter A. van der Linden, Ehud Segal, Matthew A. Child, Anna Byzia, Marcin Drąg, Matthew Bogyo
Chemistry & Biology 2015 Volume 22(Issue 8) pp:995-1001
Publication Date(Web):20 August 2015
DOI:10.1016/j.chembiol.2015.07.010
•Application of substrate screening to identify bleomycin hydrolase-specific scaffolds•Identification of cell-permeable irreversible inhibitors for bleomycin hydrolase•Identification of cell-permeable activity-based probes for bleomycin hydrolaseBleomycin hydrolase (BLMH) is a neutral cysteine aminopeptidase that has been ascribed roles in many physiological and pathological processes, yet its primary biological function remains enigmatic. In this work, we describe the results of screening of a library of fluorogenic substrates to identify non-natural amino acids that are optimally recognized by BLMH. This screen identified several substrates with kcat/KM values that are substantially improved over the previously reported fluorogenic substrates for this enzyme. The substrate sequences were used to design activity-based probes that showed potent labeling of recombinant BLMH as well as endogenously expressed BLMH in cell extracts, and in intact cells. Importantly, we identify potent BLMH inhibitors that are able to fully inhibit endogenous BLMH activity in intact cells. These probes and inhibitors will be valuable new reagents to study BLMH function in cellular and animal models of human diseases where BLMH is likely to be involved.Figure optionsDownload full-size imageDownload high-quality image (179 K)Download as PowerPoint slide
Co-reporter:Leslie O. Ofori, Nimali P. Withana, Tyler R. Prestwood, Martijn Verdoes, Jennifer J. Brady, Monte M. Winslow, Jonathan Sorger, and Matthew Bogyo
ACS Chemical Biology 2015 Volume 10(Issue 9) pp:1977
Publication Date(Web):June 3, 2015
DOI:10.1021/acschembio.5b00205
There is a need for new molecular-guided contrast agents to enhance surgical procedures such as tumor resection that require a high degree of precision. Cysteine cathepsins are highly up-regulated in a wide variety of cancers, both in tumor cells and in the tumor-supporting cells of the surrounding stroma. Therefore, tools that can be used to dynamically monitor their activity in vivo could be used as imaging contrast agents for intraoperative fluorescence image guided surgery (FGS). Although multiple classes of cathepsin-targeted substrate probes have been reported, most suffer from overall fast clearance from sites of protease activation, leading to reduced signal intensity and duration in vivo. Here we describe the design and synthesis of a series of near-infrared fluorogenic probes that exploit a latent cationic lysosomotropic effect (LLE) to promote cellular retention upon protease activation. These probes show tumor-specific retention, fast activation kinetics, and rapid systemic distribution. We demonstrate that they are suitable for detection of diverse cancer types including breast, colon and lung tumors. Most importantly, the agents are compatible with the existing, FDA approved, da Vinci surgical system for fluorescence guided tumor resection. Therefore, our data suggest that the probes reported here can be used with existing clinical instrumentation to detect tumors and potentially other types of inflammatory lesions to guide surgical decision making in real time.
Co-reporter:Ehud Segal, Tyler R. Prestwood, Wouter A. van der Linden, Yaron Carmi, Nupur Bhattacharya, Nimali Withana, Martijn Verdoes, Aida Habtezion, Edgar G. Engleman, Matthew Bogyo
Chemistry & Biology 2015 22(1) pp: 148-158
Publication Date(Web):
DOI:10.1016/j.chembiol.2014.11.008
Co-reporter:Kristina Oresic Bender;Megan Garland;Jessica A. Ferreyra;Andrew J. Hryckowian;Matthew A. Child;Aaron W. Puri;David E. Solow-Cordero;Steven K. Higginbottom;Ehud Segal;Niaz Banaei;Aimee Shen;Justin L. Sonnenburg
Science Translational Medicine 2015 Vol 7(306) pp:306ra148
Publication Date(Web):23 Sep 2015
DOI:10.1126/scitranslmed.aac9103
A high-throughput screen against the Clostridium difficile toxin B cysteine protease domain identified a drug in clinical trials that reduced C. difficile pathology in a mouse model.
Co-reporter:Hao Li ; Christopher Tsu ; Christopher Blackburn ; Gang Li ; Paul Hales ; Lawrence Dick
Journal of the American Chemical Society 2014 Volume 136(Issue 39) pp:13562-13565
Publication Date(Web):September 16, 2014
DOI:10.1021/ja507692y
We have identified short N,C-capped peptides that selectively inhibit the proteasome of the malaria-causing pathogen Plasmodium falciparum. These compounds are highly potent in culture with no toxicity in host cells. One cyclic biphenyl ether compound inhibited intraerythrocytic growth of P. falciparum with an IC50 of 35 nM, and we show that even a pulse treatment with this cyclic peptide induced parasite death due to proteasome inhibition. These compounds represent promising new antimalarial agents that target the essential proteasomal machinery of the parasite without toxicity toward the host.
Co-reporter:Hao Li, Wouter A. van der Linden, Martijn Verdoes, Bogdan I. Florea, Fiona E. McAllister, Kavitha Govindaswamy, Joshua E. Elias, Purnima Bhanot, Herman S. Overkleeft, and Matthew Bogyo
ACS Chemical Biology 2014 Volume 9(Issue 8) pp:1869
Publication Date(Web):June 11, 2014
DOI:10.1021/cb5001263
The ubiquitin-proteasome system (UPS) is a potential pathway for therapeutic intervention for pathogens such as Plasmodium, the causative agent of malaria. However, due to the essential nature of this proteolytic pathway, proteasome inhibitors must avoid inhibition of the host enzyme complex to prevent toxic side effects. The Plasmodium proteasome is poorly characterized, making rational design of inhibitors that induce selective parasite killing difficult. In this study, we developed a chemical probe that labels all catalytic sites of the Plasmodium proteasome. Using this probe, we identified several subunit selective small molecule inhibitors of the parasite enzyme complex. Treatment with an inhibitor that is specific for the β5 subunit during blood stage schizogony led to a dramatic decrease in parasite replication while short-term inhibition of the β2 subunit did not affect viability. Interestingly, coinhibition of both the β2 and β5 catalytic subunits resulted in enhanced parasite killing at all stages of the blood stage life cycle and reduced parasite levels in vivo to barely detectable levels. Parasite killing was achieved with overall low host toxicity, something that has not been possible with existing proteasome inhibitors. Our results highlight differences in the subunit dependency of the parasite and human proteasome, thus providing a strategy for development of potent antimalarial drugs with overall low host toxicity.
Co-reporter:Junpeng Xiao ; Petr Broz ; Aaron W. Puri ; Edgar Deu ; Montse Morell ; Denise M. Monack
Journal of the American Chemical Society 2013 Volume 135(Issue 24) pp:9130-9138
Publication Date(Web):May 23, 2013
DOI:10.1021/ja403521u
Caspases are cysteine proteases that play essential roles in apoptosis and inflammation. Unfortunately, their highly conserved active sites and overlapping substrate specificities make it difficult to use inhibitors or activity-based probes to study the function, activation, localization, and regulation of individual members of this family. Here we describe a strategy to engineer a caspase to contain a latent nucleophile that can be targeted by a probe containing a suitably placed electrophile, thereby allowing specific, irreversible inhibition and labeling of only the engineered protease. To accomplish this, we have identified a non-conserved residue on the small subunit of all caspases that is near the substrate-binding pocket and that can be mutated to a non-catalytic cysteine residue. We demonstrate that an active-site probe containing an irreversible binding acrylamide electrophile can specifically target this cysteine residue. Here we validate the approach using the apoptotic mediator, caspase-8, and the inflammasome effector, caspase-1. We show that the engineered enzymes are functionally identical to the wild-type enzymes and that the approach allows specific inhibition and direct imaging of the engineered targets in cells. Therefore, this method can be used to image localization and activation as well as the functional contributions of individual caspase proteases to the process of cell death or inflammation.
Co-reporter:Montse Morell, Thinh Nguyen Duc, Amanda L. Willis, Salahuddin Syed, Jiyoun Lee, Edgar Deu, Yang Deng, Junpeng Xiao, Benjamin E. Turk, Jason R. Jessen, Stephen J. Weiss, and Matthew Bogyo
Journal of the American Chemical Society 2013 Volume 135(Issue 24) pp:9139-9148
Publication Date(Web):May 23, 2013
DOI:10.1021/ja403523p
Matrix metalloproteinases (MMPs) are zinc endopeptidases that play roles in numerous pathophysiological processes and therefore are promising drug targets. However, the large size of this family and a lack of highly selective compounds that can be used for imaging or inhibition of specific MMPs members has limited efforts to better define their biological function. Here we describe a protein engineering strategy coupled with small-molecule probe design to selectively target individual members of the MMP family. Specifically, we introduce a cysteine residue near the active-site of a selected protease that does not alter its overall activity or function but allows direct covalent modification by a small-molecule probe containing a reactive electrophile. This specific engineered interaction between the probe and the target protease provides a means to both image and inhibit the modified protease with absolute specificity. Here we demonstrate the feasibility of the approach for two distinct MMP proteases, MMP-12 and MT1-MMP (or MMP-14).
Co-reporter:Martijn Verdoes ; Kristina Oresic Bender ; Ehud Segal ; Wouter A. van der Linden ; Salahuddin Syed ; Nimali P. Withana ; Laura E. Sanman
Journal of the American Chemical Society 2013 Volume 135(Issue 39) pp:14726-14730
Publication Date(Web):August 23, 2013
DOI:10.1021/ja4056068
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.
Co-reporter:Jiyoun Lee, Matthew Bogyo
Current Opinion in Chemical Biology 2013 Volume 17(Issue 1) pp:118-126
Publication Date(Web):February 2013
DOI:10.1016/j.cbpa.2012.12.022
The past decade has seen rapid growth in the use of diverse compound libraries in classical phenotypic screens to identify modulators of a given process. The subsequent process of identifying the molecular targets of active hits, also called ‘target deconvolution’, is an essential step for understanding compound mechanism of action and for using the identified hits as tools for further dissection of a given biological process. Recent advances in ‘omics’ technologies, coupled with in silico approaches and the reduced cost of whole genome sequencing, have greatly improved the workflow of target deconvolution and have contributed to a renaissance of ‘modern’ phenotypic profiling. In this review, we will outline how both new and old techniques are being used in the difficult process of target identification and validation as well as discuss some of the ongoing challenges remaining for phenotypic screening.Highlights► Discussion of the concept of target deconvolution ► Discussion of why new technology is helping to advance target deconvolution ► Recent examples of various methods used to find targets of small molecule hits ► Discussion of the future for phenotypic screening methods.
Co-reporter:Aaron W. Puri and Matthew Bogyo
Biochemistry 2013 Volume 52(Issue 35) pp:5985-5996
Publication Date(Web):August 12, 2013
DOI:10.1021/bi400854d
Elucidating the molecular and biochemical details of bacterial infections can be challenging because of the many complex interactions that exist between a pathogen and its host. Consequently, many tools have been developed to aid the study of bacterial pathogenesis. Small molecules are a valuable complement to traditional genetic techniques because they can be used to rapidly perturb genetically intractable systems and to monitor post-translationally regulated processes. Activity-based probes are a subset of small molecules that covalently label an enzyme of interest based on its catalytic mechanism. These tools allow monitoring of enzyme activation within the context of a native biological system and can be used to dissect the biochemical details of enzyme function. This review describes the development and application of activity-based probes for examining aspects of bacterial infection on both sides of the host–pathogen interface.
Co-reporter:Edgar Deu;Ingrid T. Chen;Erica M. W. Lauterwasser;Juan Valderramos;Hao Li;Laura E. Edgington;Adam R. Renslo
PNAS 2013 Volume 110 (Issue 45 ) pp:18244-18249
Publication Date(Web):2013-11-05
DOI:10.1073/pnas.1312782110
The precise targeting of cytotoxic agents to specific cell types or cellular compartments is of significant interest in medicine,
with particular relevance for infectious diseases and cancer. Here, we describe a method to exploit aberrant levels of mobile
ferrous iron (FeII) for selective drug delivery in vivo. This approach makes use of a 1,2,4-trioxolane moiety, which serves as an FeII-sensitive “trigger,” making drug release contingent on FeII-promoted trioxolane fragmentation. We demonstrate in vivo validation of this approach with the Plasmodium berghei model of murine malaria. Malaria parasites produce high concentrations of mobile ferrous iron as a consequence of their catabolism
of host hemoglobin in the infected erythrocyte. Using activity-based probes, we successfully demonstrate the FeII-dependent and parasite-selective delivery of a potent dipeptidyl aminopeptidase inhibitor. We find that delivery of the compound
in its FeII-targeted form leads to more sustained target inhibition with greatly reduced off-target inhibition of mammalian cathepsins.
This selective drug delivery translates into improved efficacy and tolerability. These findings demonstrate the utility of
a purely chemical means to achieve selective drug targeting in vivo. This approach may find useful application in parasitic
infections and more broadly in any disease state characterized by aberrant production of reactive ferrous iron.
Co-reporter:Laura E. Edgington ; Martijn Verdoes ; Alberto Ortega ; Nimali P. Withana ; Jiyoun Lee ; Salahuddin Syed ; Michael H. Bachmann ; Galia Blum
Journal of the American Chemical Society 2012 Volume 135(Issue 1) pp:174-182
Publication Date(Web):December 8, 2012
DOI:10.1021/ja307083b
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.
Co-reporter:Martijn Verdoes, Laura E. Edgington, Ferenc A. Scheeren, Melissa Leyva, Galia Blum, Kipp Weiskopf, Michael H. Bachmann, Jonathan A. Ellman, Matthew Bogyo
Chemistry & Biology 2012 Volume 19(Issue 5) pp:619-628
Publication Date(Web):25 May 2012
DOI:10.1016/j.chembiol.2012.03.012
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.Graphical AbstractFigure optionsDownload full-size imageDownload high-quality image (222 K)Download as PowerPoint slideHighlights► BMV083 is a cathepsin S directed nonpeptidic quenched activity-based probe ► BMV083 has enhanced in vivo properties relative to reported peptide-based probes ► BMV083 primarily targets tumor promoting M2 type macrophages in vivo
Co-reporter:Hao Li, Elizabeth L. Ponder, Martijn Verdoes, Kristijana H. Asbjornsdottir, Edgar Deu, Laura E. Edgington, Jeong Tae Lee, Christopher J. Kirk, Susan D. Demo, Kim C. Williamson, Matthew Bogyo
Chemistry & Biology 2012 Volume 19(Issue 12) pp:1535-1545
Publication Date(Web):21 December 2012
DOI:10.1016/j.chembiol.2012.09.019
The Plasmodium proteasome has been suggested to be a potential antimalarial drug target; however, toxicity of inhibitors has prevented validation of this enzyme in vivo. We report a screen of a library of 670 analogs of the recent US Food and Drug Administration-approved inhibitor, carfilzomib, to identify compounds that selectively kill parasites. We identified one compound, PR3, that has significant parasite killing activity in vitro but dramatically reduced toxicity in host cells. We found that this parasite-specific toxicity is not due to selective targeting of the Plasmodium proteasome over the host proteasome, but instead is due to a lack of activity against one of the human proteasome subunits. Subsequently, we used PR3 to significantly reduce parasite load in Plasmodium berghei infected mice without host toxicity, thus validating the proteasome as a viable antimalarial drug target.Graphical AbstractFigure optionsDownload full-size imageDownload high-quality image (156 K)Download as PowerPoint slideHighlights► Purification of Plasmodium proteasomes and labeling with an activity-based probe ► Identification of an epoxyketone-based inhibitor with parasite-specific toxicity ► Demonstration that Plasmodium is sensitive to partial inhibition of its proteasome ► Determination of key parameters for selective parasite killing with proteasome inhibitors
Co-reporter:Laura E. Edgington, Bram J. van Raam, Martijn Verdoes, Christoph Wierschem, Guy S. Salvesen, Matthew Bogyo
Chemistry & Biology 2012 Volume 19(Issue 3) pp:340-352
Publication Date(Web):23 March 2012
DOI:10.1016/j.chembiol.2011.12.021
Although significant efforts have been made to understand the mechanisms of caspase activation during apoptosis, many questions remain regarding how and when executioner caspases get activated. We describe the design and synthesis of an activity-based probe that labels caspase-3/-6/-7, allowing direct monitoring of all executioner caspases simultaneously. This probe has enhanced in vivo properties and reduced cross-reactivity compared to our previously reported probe, AB50. Using this probe, we find that caspase-6 undergoes a conformational change and can bind substrates even in the absence of cleavage of the proenzyme. We also demonstrate that caspase-6 activation does not require active caspase-3/-7, suggesting that it may autoactivate or be cleaved by other proteases. Together, our results suggest that caspase-6 activation proceeds through a unique mechanism that may be important for its diverse biological functions.Graphical AbstractFigure optionsDownload full-size imageDownload high-quality image (104 K)Download as PowerPoint slideHighlights► LE22 is an activity-based probe that targets caspase-3, -6, and -7 ► Uncleaved caspase-6 has low but detectable levels of catalytic activity ► Complete caspase-6 activation is achieved through multiple maturation steps ► Caspase-6 undergoes a conformational change upon activation and substrate binding
Co-reporter:Jiyoun Lee, Matthew Bogyo
Bioorganic & Medicinal Chemistry Letters 2012 Volume 22(Issue 3) pp:1340-1343
Publication Date(Web):1 February 2012
DOI:10.1016/j.bmcl.2011.12.079
Legumain or asparaginly endopeptidase (AEP) is a lysosomal cysteine protease with a high level of specificity for cleavage of protein substrates after an asparagine residue. It is also capable of cleaving after aspartic acids sites when in the acidic environment of the lysosome. Legumain expression and activity is linked to a number of pathological conditions including cancer, atherosclerosis and inflammation, yet its biological role in these pathologies is not well-understood. Highly potent and selective inhibitors of legumain would not only be valuable for studying the functional roles of legumain in these conditions, but may have therapeutic potential as well. We describe here the design, synthesis and in vitro evaluation of selective legumain inhibitors based on the aza-asparaginyl scaffold. We synthesized a library of aza-peptidyl inhibitors with various non-natural amino acids and different electrophilic warheads, and characterized the kinetic properties of inactivation of legumain. We also synthesized fluorescently labeled inhibitors to investigate cell permeability and selectivity of the compounds. The inhibitors have second order rate constants of up to 5 × 104 M−1 s−1 and IC50 values as low as 4 nM against recombinant mouse legumain. In addition, the inhibitors are highly selective toward legumain and have little or no cross-reactivity with cathepsins. Overall, we have identified several valuable new inhibitors of legumain that can be used to study legumain function in multiple disease models.
Co-reporter:Laura E Edgington, Martijn Verdoes, Matthew Bogyo
Current Opinion in Chemical Biology 2011 Volume 15(Issue 6) pp:798-805
Publication Date(Web):December 2011
DOI:10.1016/j.cbpa.2011.10.012
Proteases are enzymes that cleave peptide bonds in protein substrates. This process can be important for regulated turnover of a target protein but it can also produce protein fragments that then perform other functions. Because the last few decades of protease research have confirmed that proteolysis is an essential regulatory process in both normal physiology and in multiple disease-associated conditions, there has been an increasing interest in developing methods to image protease activity. Proteases are also considered to be one of the few ‘druggable’ classes of proteins and therefore a large number of small molecule based inhibitors of proteases have been reported. These compounds serve as a starting point for the design of probes that can be used to target active proteases for imaging applications. Currently, several classes of fluorescent probes have been developed to visualize protease activity in live cells and even whole organisms. The two primary classes of protease probes make use of either peptide/protein substrates or covalent inhibitors that produce a fluorescent signal when bound to an active protease target. This review outlines some of the most recent advances in the design of imaging probes for proteases. In particular, it highlights the strengths and weaknesses of both substrate-based and activity-based probes and their applications for imaging cysteine proteases that are important biomarkers for multiple human diseases.Highlights► Explanation of the need for tools to image protease activity. ► Examples of recently developed substrate-based imaging probes for cysteine proteases. ► Examples of recently developed activity-based imaging probes for cysteine proteases. ► Discussion of the pros and cons of substrate and activity based probes.
Co-reporter:Elizabeth L. Ponder, Victoria E. Albrow, Brittany A. Leader, Miklós Békés, Jowita Mikolajczyk, Urša Pečar Fonović, Aimee Shen, Marcin Drag, Junpeng Xiao, Edgar Deu, Amy J. Campbell, James C. Powers, Guy S. Salvesen, Matthew Bogyo
Chemistry & Biology 2011 Volume 18(Issue 6) pp:711-721
Publication Date(Web):24 June 2011
DOI:10.1016/j.chembiol.2011.04.010
Small ubiquitin-related modifier (SUMO) is implicated in the regulation of numerous biological processes including transcription, protein localization, and cell cycle control. Protein modification by SUMO is found in Plasmodium falciparum; however, its role in the regulation of the parasite life cycle is poorly understood. Here we describe functional studies of a SUMO-specific protease (SENP) of P. falciparum, PfSENP1 (PFL1635w). Expression of the catalytic domain of PfSENP1 and biochemical profiling using a positional scanning substrate library demonstrated that this protease has unique cleavage sequence preference relative to the human SENPs. In addition, we describe a class of small molecule inhibitors of this protease. The most potent lead compound inhibited both recombinant PfSENP1 activity and P. falciparum replication in infected human blood. These studies provide valuable new tools for the study of SUMOylation in P. falciparum.Graphical AbstractFigure optionsDownload full-size imageDownload high-quality image (110 K)Download as PowerPoint slideHighlights► Expression of the primary SENP protease from Plasmodium falciparum ► Biochemical characterization of the primary substrate specificity of PfSENP1 ► Development of a screening assay to identify inhibitors of SENP proteases ► Identification of a new class of small molecule inhibitor of SENP proteases
Co-reporter:Victoria E. Albrow, Elizabeth L. Ponder, Domenico Fasci, Miklós Békés, Edgar Deu, Guy S. Salvesen, Matthew Bogyo
Chemistry & Biology 2011 Volume 18(Issue 6) pp:722-732
Publication Date(Web):24 June 2011
DOI:10.1016/j.chembiol.2011.05.008
Sentrin specific proteases (SENPs) are responsible for activating and deconjugating SUMO (Small Ubiquitin-like MOdifier) from target proteins. It remains difficult to study this posttranslational modification due to the lack of reagents that can be used to block the removal of SUMO from substrates. Here, we describe the identification of small molecule SENP inhibitors and active site probes containing aza-epoxide and acyloxymethyl ketone (AOMK) reactive groups. Both classes of compounds are effective inhibitors of hSENPs 1, 2, 5, and 7 while only the AOMKs efficiently inhibit hSENP6. Unlike previous reported peptide vinyl sulfones, these compounds covalently labeled the active site cysteine of multiple recombinantly expressed SENP proteases and the AOMK probe showed selective labeling of these SENPs when added to complex protein mixtures. The AOMK compounds therefore represent promising new reagents to study the process of SUMO deconjugation.Highlights► Identification of new classes of small molecule inhibitors of human SENP proteases ► Evaluation of the selectivity of inhibitors against multiple SENPS ► Identification of new classes of small molecule activity-based probes for SENPs
Co-reporter:Margot G. Paulick and Matthew Bogyo
ACS Chemical Biology 2011 Volume 6(Issue 6) pp:563
Publication Date(Web):February 15, 2011
DOI:10.1021/cb100392r
Cathepsin X is a lysosomal cysteine protease that functions as a carboxypeptidase with broad substrate specificity. Cathepsin X was discovered only recently, and its physiological roles are still not well understood. A number of studies suggest that cathepsin X may be involved in a variety of biological processes, including cancer, aging and degenerative conditions of the brain, inflammation, and cellular communication. Here we present the synthesis and characterization of several activity-based probes (ABPs) that target active cathepsin X. These ABPs were used to label cathepsin X in complex lysates, whole cells, and in vivo. Furthermore, we have developed a method for selectively labeling and visualizing active cathepsin X in vitro and in vivo. Overall, the probes developed in this study are valuable tools for the study of cathepsin X function.
Co-reporter:Carolyn I. Hall;Eranthie Weerapana;Jeralyn D. Haraldsen;Michael L. Reese;Paul W. Bowyer;Victoria E. Albrow;Gary E. Ward;Edgar Deu Sandoval;Matthew A. Child;MacDonald R. Phillips;Benjamin F. Cravatt;John C. Boothroyd
PNAS 2011 Volume 108 (Issue 26 ) pp:10568-10573
Publication Date(Web):2011-06-28
DOI:10.1073/pnas.1105622108
Toxoplasma gondii is a member of the phylum Apicomplexa that includes several important human pathogens, such as Cryptosporidium and Plasmodium falciparum, the causative agent of human malaria. It is an obligate intracellular parasite that can cause severe disease in congenitally
infected neonates and immunocompromised individuals. Despite the importance of attachment and invasion to the success of the
parasite, little is known about the underlying mechanisms that drive these processes. Here we describe a screen to identify
small molecules that block the process of host cell invasion by the T. gondii parasite. We identified a small molecule that specifically and irreversibly blocks parasite attachment and subsequent invasion
of host cells. Using tandem orthogonal proteolysis–activity-based protein profiling, we determined that this compound covalently
modifies a single cysteine residue in a poorly characterized protein homologous to the human protein DJ-1. Mutation of this
key cysteine residue in the native gene sequence resulted in parasites that were resistant to inhibition of host cell attachment
and invasion by the compound. Further analysis of the invasion phenotype confirmed that modification of Cys127 on TgDJ-1 resulted
in a block of microneme secretion and motility, even in the presence of direct stimulators of calcium release. Together, our
results suggest that TgDJ-1 plays an important role that is likely downstream of the calcium flux required for microneme secretion,
parasite motility, and subsequent invasion of host cells.
Co-reporter:Dr. Sumit S. Mahajan;Dr. Edgar Deu;Dr. Erica M. W. Lauterwasser;Melissa J. Leyva;Dr. Jonathan A. Ellman;Dr. Matthew Bogyo;Dr. Adam R. Renslo
ChemMedChem 2011 Volume 6( Issue 3) pp:
Publication Date(Web):
DOI:10.1002/cmdc.201190006
Co-reporter:Dr. Sumit S. Mahajan;Dr. Edgar Deu;Dr. Erica M. W. Lauterwasser;Melissa J. Leyva;Dr. Jonathan A. Ellman;Dr. Matthew Bogyo;Dr. Adam R. Renslo
ChemMedChem 2011 Volume 6( Issue 3) pp:415-419
Publication Date(Web):
DOI:10.1002/cmdc.201100002
Co-reporter:Aaron W. Puri, Patrick J. Lupardus, Edgar Deu, Victoria E. Albrow, K. Christopher Garcia, Matthew Bogyo, Aimee Shen
Chemistry & Biology 2010 Volume 17(Issue 11) pp:1201-1211
Publication Date(Web):24 November 2010
DOI:10.1016/j.chembiol.2010.09.011
Clostridium difficile is a leading cause of nosocomial infections. The major virulence factors of this pathogen are the multi-domain toxins TcdA and TcdB. These toxins contain a cysteine protease domain (CPD) that autoproteolytically releases a cytotoxic effector domain upon binding intracellular inositol hexakisphosphate. Currently, there are no known inhibitors of this protease. Here, we describe the rational design of covalent small molecule inhibitors of TcdB CPD. We identified compounds that inactivate TcdB holotoxin function in cells and solved the structure of inhibitor-bound protease to 2.0 Å. This structure reveals the molecular basis of CPD substrate recognition and informed the synthesis of activity-based probes for this enzyme. The inhibitors presented will guide the development of therapeutics targeting C. difficile, and the probes will serve as tools for studying the unique activation mechanism of bacterial toxin CPDs.Graphical AbstractFigure optionsDownload full-size imageDownload high-quality image (118 K)Download as PowerPoint slideHighlights► Identification of inhibitors of C. difficile TcdB cysteine protease domain (CPD) ► Determination of the crystal structure of toxin CPD bound to inhibitor ► Demonstration that most potent inhibitors protect cells from TcdB intoxication ► Development of activity-based probes to study allosteric activation of TcdB CPD by InsP6
Co-reporter:Edgar Deu, Melissa J. Leyva, Victoria E. Albrow, Mark J. Rice, Jonathan A. Ellman, Matthew Bogyo
Chemistry & Biology 2010 Volume 17(Issue 8) pp:808-819
Publication Date(Web):27 August 2010
DOI:10.1016/j.chembiol.2010.06.007
The widespread resistance of malaria parasites to all affordable drugs has made the identification of new targets urgent. Dipeptidyl aminopeptidases (DPAPs) represent potentially valuable new targets that are involved in hemoglobin degradation (DPAP1) and parasite egress (DPAP3). Here we use activity-based probes to demonstrate that specific inhibition of DPAP1 by a small molecule results in the formation of an immature trophozoite that leads to parasite death. Using computational methods, we designed stable, nonpeptidic covalent inhibitors that kill Plasmodium falciparum at low nanomolar concentrations. These compounds show signs of slowing parasite growth in a murine model of malaria, which suggests that DPAP1 might be a viable antimalarial target. Interestingly, we found that resynthesis and activation of DPAP1 after inhibition is rapid, suggesting that effective drugs would need to sustain DPAP1 inhibition for a period of 2–3 hr.Graphical AbstractFigure optionsDownload full-size imageDownload high-quality image (184 K)Download as PowerPoint slideHighlights► Specific DPAP1 inhibition impairs malaria parasites growth ► P. falciparum is able to restore DPAP1 activity after a short period of inhibition ► Nonpeptidic DPAP inhibitors are more potent and stable than peptidic inhibitors
Co-reporter:Jiyoun Lee and Matthew Bogyo
ACS Chemical Biology 2010 Volume 5(Issue 2) pp:233
Publication Date(Web):December 17, 2009
DOI:10.1021/cb900232a
Asparaginyl endopeptidase, or legumain, is a lysosomal cysteine protease that was originally identified in plants and later found to be involved in antigen presentation in higher eukaryotes. Legumain is also up-regulated in a number of human cancers, and recent studies suggest that it may play important functional roles in the process of tumorigenesis. However, detailed functional studies in relevant animal models of human disease have been hindered by the lack of suitably selective small molecule inhibitors and imaging reagents. Here we present the design, optimization, and in vivo application of fluorescently labeled activity-based probes (ABPs) for legumain. We demonstrate that optimized aza-peptidyl Asn epoxides are highly selective and potent inhibitors that can be readily converted into near-infrared fluorophore-labeled ABPs for whole body, noninvasive imaging applications. We show that these probes specifically label legumain in various normal tissues as well as in solid tumors when applied in vivo. Interestingly, addition of cell-penetrating peptides to the probes enhanced cellular uptake but resulted in increased cross-reactivity toward other lysosomal proteases as the result of their accumulation in lysosomes. Overall, we find that aza-peptidyl Asn ABPs are valuable new tools for the future study of legumain function in more complex models of human disease.
Co-reporter:Matthew Bogyo
PNAS 2010 Volume 107 (Issue 6 ) pp:2379-2380
Publication Date(Web):2010-02-09
DOI:10.1073/pnas.0914955107
Co-reporter:Aaron W. Puri and Matthew Bogyo
ACS Chemical Biology 2009 Volume 4(Issue 8) pp:603
Publication Date(Web):July 16, 2009
DOI:10.1021/cb9001409
Understanding the ways in which pathogens invade and neutralize their hosts is of great interest from both an academic and a clinical perspective. However, in many cases genetic tools are unavailable or insufficient to fully characterize the detailed mechanisms of pathogenesis. Small molecule approaches are particularly powerful due to their ability to modulate specific biological functions in a highly controlled manner and their potential to broadly target conserved processes across species. Recently, two approaches that make use of small molecules, activity-based protein profiling and high-throughput phenotypic screening, have begun to find applications in the study of pathways involved in pathogenesis. In this Review we highlight ways in which these techniques have been applied to examine bacterial and parasitic pathogenesis and discuss possible ways in which these efforts can be expanded in the near future.Keywords: ABP: Activity-based probe. A small molecule consisting of a tag, specificity region, and an electrophilic warhead that covalently attaches to a target enzyme in a catalysis-dependant manner.; Bacterial toxin: A secreted bacterial protein that specifically disrupts host cell functions.; Chemical genetics: The use of highly specific small molecules to determine the function of a gene product of interest.; Competitive ABPP: Competitive activity-based protein profiling. Optimization of selective inhibitors within a family of enzymes based on competition of the inhibitor with labeling by a general activity-based probe.Keywords: Gram staining: A procedure using crystal violet dye to differentiate bacteria into two general groups (Gram-negative and Gram-positive) based on the composition of their exterior layers.; Protozoan parasites: Unicellular eukaryotic microorganisms that require a host to complete their lifecycle.; Quorum sensing: A process by which bacteria use secreted small molecules such as N-Acyl Homoserine Lactones (AHLs) to communicate population density.; Schizont: A stage of Plasmodium sp. development within a host red blood cell in which the parasite asexually divides into daughter merezoites prior to cell rupture.; T3SS: Type III secretion system. A syringe-like membrane protein assembly used to transfer effectors from a bacterium to a host cell.
Co-reporter:Zhimou Yang, Marko Fonović, Steven H.L. Verhelst, Galia Blum, Matthew Bogyo
Bioorganic & Medicinal Chemistry 2009 Volume 17(Issue 3) pp:1071-1078
Publication Date(Web):1 February 2009
DOI:10.1016/j.bmc.2008.02.089
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, α,β-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.Activity-based probes based on α,β-unsaturated ketone label papain-family cysteine proteases (Cat B and Cat L) in mouse tissue lysates, cell lysate, and intact cells.
Co-reporter:Jeong Tae Lee, David Y. Chen, Zhimou Yang, Alexander D. Ramos, James J.-D. Hsieh, Matthew Bogyo
Bioorganic & Medicinal Chemistry Letters 2009 Volume 19(Issue 17) pp:5086-5090
Publication Date(Web):1 September 2009
DOI:10.1016/j.bmcl.2009.07.045
Taspase1 is a threonine protease responsible for cleaving MLL (Mixed-Lineage Leukemia) to achieve proper HOX gene expression. Subsequent studies identified additional Taspase1 substrates including Transcription Factor IIA (TFIIA) and Drosophila HCF. Taspase1 is essential for cell proliferation and is overexpressed in many cancer cell lines. Currently no small molecule inhibitors of this enzyme have been described. Here, we report the synthesis and evaluation of vinyl sulfone, vinyl ketone, epoxy ketone, and boronic acid inhibitors designed based on the preferred Taspase1 cleavage site (Ac-Ile-Ser-Gln-Leu-Asp). Specifically, we evaluated compounds in which the reactive warhead is positioned in place of the P1 aspartic acid side chain as well as at the C-terminus of the peptide. Interestingly, both classes of inhibitors were effective and vinyl ketones and vinyl sulfones showed the greatest potency for the target protease. These results suggest that Taspase1 has unique substrate recognition properties that could potentially be exploited in the design of potent and selective inhibitors of this enzyme.The synthesis and evaluation of a series of inhibitors of Taspase1 is reported.
Co-reporter:Aimee Shen, Matthew Bogyo
Chemistry & Biology 2008 Volume 15(Issue 9) pp:879-880
Publication Date(Web):22 September 2008
DOI:10.1016/j.chembiol.2008.09.001
Cystatins are cysteine protease inhibitors that are at the front-line of defense against pathogens that secrete proteases as virulence factors. In this issue, Vincents et al. (2008) reveal how the bacterial protease IdeS from Streptococcus pyogenes hijacks normal cystatin C function to convert it into a cofactor that enhances proteolytic destruction of host-defense antibodies.
Co-reporter:Aimee Shen;Patrick J. Lupardus;K. Christopher Garcia
Science 2008 Volume 322(Issue 5899) pp:265-268
Publication Date(Web):10 Oct 2008
DOI:10.1126/science.1162403
Abstract
Vibrio cholerae RTX (repeats in toxin) is an actin-disrupting toxin that is autoprocessed by an internal cysteine protease domain (CPD). The RTX CPD is efficiently activated by the eukaryote-specific small molecule inositol hexakisphosphate (InsP6), and we present the 2.1 angstrom structure of the RTX CPD in complex with InsP6. InsP6 binds to a conserved basic cleft that is distant from the protease active site. Biochemical and kinetic analyses of CPD mutants indicate that InsP6 binding induces an allosteric switch that leads to the autoprocessing and intracellular release of toxin-effector domains.
Co-reporter:Amir M Sadaghiani, Steven HL Verhelst, Matthew Bogyo
Current Opinion in Chemical Biology 2007 Volume 11(Issue 1) pp:20-28
Publication Date(Web):February 2007
DOI:10.1016/j.cbpa.2006.11.030
The field of activity-based proteomics is a relatively new discipline that makes use of small molecules, termed activity-based probes (ABPs), to tag and monitor distinct sets of proteins within a complex proteome. These activity-dependant labels facilitate analysis of systems-wide changes at the level of enzyme activity rather than simple protein abundance. While the use of small molecule inhibitors to label enzyme targets is not a new concept, the past ten years have seen a rapid expansion in the diversity of probe families that have been developed. In addition to increasing the number and types of enzymes that can be targeted by this method, there has also been an increase in the number of methods used to visualize probes once they are bound to target enzymes. In particular, the use of small organic fluorophores has created a wealth of applications for ABPs that range from biochemical profiling of diverse proteomes to direct imaging of active enzymes in live cells and even whole animals. In addition, the advent of new bioorthogonal coupling chemistries now enables a diverse array of tags to be added after targets are labeled with an ABP. This strategy has opened the door to new in vivo applications for activity-based proteomic methods.
Co-reporter:Amir Masoud Sadaghiani, Steven H.L. Verhelst, Vasilena Gocheva, Kimberly Hill, Eva Majerova, Sherman Stinson, Johanna A. Joyce, Matthew Bogyo
Chemistry & Biology 2007 Volume 14(Issue 5) pp:499-511
Publication Date(Web):29 May 2007
DOI:10.1016/j.chembiol.2007.03.010
The papain-family cathepsins are cysteine proteases that are emerging as promising therapeutic targets for a number of human disease conditions ranging from osteoporosis to cancer. Relatively few selective inhibitors for this family exist, and the in vivo selectivity of most existing compounds is unclear. We present here the synthesis of focused libraries of epoxysuccinyl-based inhibitors and their screening in crude tissue extracts. We identified a number of potent inhibitors that display selectivity for endogenous cathepsin targets both in vitro and in vivo. Importantly, the selectivity patterns observed in crude extracts were generally retained in vivo, as assessed by active-site labeling of tissues from treated animals. Overall, this study identifies several important compound classes and highlights the use of activity-based probes to assess pharmacodynamic properties of small-molecule inhibitors in vivo.
Co-reporter:Kelly B. Sexton, Martin D. Witte, Galia Blum, Matthew Bogyo
Bioorganic & Medicinal Chemistry Letters 2007 Volume 17(Issue 3) pp:649-653
Publication Date(Web):1 February 2007
DOI:10.1016/j.bmcl.2006.10.100
Asparaginyl endopeptidase (AEP), also known as legumain, is a cysteine protease that has been ascribed roles in antigen presentation yet its exact role in human biology remains poorly understood. We report here, the use of a positional scanning combinatorial library of peptide AOMKs containing a P1 aspartic acid to probe the P2, P3, and P4 subsite specificity of endogenous legumain. Using inhibitor specificity profiles of cathepsin B and legumain, we designed fluorescent ABPs that are highly selective, cell-permeable reagents for monitoring legumain activity in complex proteomes.The synthesis of a highly selective, cell-permeable fluorescent label of the lysosomal cysteine protease legumain is reported.
Co-reporter:Steven H. L. Verhelst Dr.;Marko Fonović Dr. Dr.
Angewandte Chemie 2007 Volume 119(Issue 8) pp:
Publication Date(Web):5 JAN 2007
DOI:10.1002/ange.200603811
Im Guten getrennt: Mit niedermolekularen Sonden gelingt die Anreicherung bestimmter Zielproteine aus einem komplexen Proteom. Ein Linkersystem für die milde und hoch selektive Spaltung sondenmarkierter Proteine verhindert die Freisetzung unerwünschter, nichtspezifisch gebundener Proteine.
Co-reporter:Steven H. L. Verhelst Dr.;Marko Fonović Dr. Dr.
Angewandte Chemie International Edition 2007 Volume 46(Issue 8) pp:
Publication Date(Web):5 JAN 2007
DOI:10.1002/anie.200603811
An amicable split: Small-molecule probes can be applied for the enrichment of specific protein targets from a complex proteome. A cleavable linker system, which prevents the release of unwanted, nonspecifically bound proteins, can be used for a very mild and highly selective cleavage of probe-labeled proteins.
Co-reporter:K B Sexton, D Kato, A B Berger, M Fonovic, S H L Verhelst and M Bogyo
Cell Death & Differentiation 2007 14(4) pp:727-732
Publication Date(Web):December 15, 2006
DOI:10.1038/sj.cdd.4402074
Activity-Based Probes (ABPs) are small molecules that form stable covalent bonds with active enzymes thereby allowing detection and quantification of their activities in complex proteomes. A number of ABPs that target proteolytic enzymes have been designed based on well-characterized mechanism-based inhibitors. We describe here the evaluation of a novel series of ABPs based on the aza-aspartate inhibitory scaffold. Previous in vitro kinetic studies showed that this scaffold has a high degree of selectivity for the caspases, clan CD cysteine proteases activated during apoptotic cell death. Aza-aspartate ABPs containing either an epoxide or Michael acceptor reactive group were potent labels of executioner caspases in apoptotic cell extracts. However they were also effective labels of the clan CD protease legumain and showed unexpected crossreactivity with the clan CA protease cathepsin B. Interestingly, related aza peptides containing an acyloxymethyl ketone reactive group were relatively weak but highly selective labels of caspases. Thus azapeptide electrophiles are valuable new ABPs for both detection of a broad range of cysteine protease activities and for selective targeting of caspases. This study also highlights the importance of confirming the specificity of covalent protease inhibitors in crude proteomes using reagents such as the ABPs described here.
Co-reporter:Steven H. L. Verhelst Dr.;Martin D. Witte;Shirin Arastu-Kapur Dr.;Marko Fonovic Dr. Dr.
ChemBioChem 2006 Volume 7(Issue 6) pp:
Publication Date(Web):11 APR 2006
DOI:10.1002/cbic.200600001
Recent characterization of multiple classes of functionalized azapeptides as effective covalent inhibitors of cysteine proteases prompted us to investigate O-acyl hydroxamates and their azapeptide analogues for use as activity-based probes (ABPs). We report here a new class of azaglycine-containing O-acylhydroxamates that form stable covalent adducts with target proteases. This allows them to be used as ABPs for papain family cysteine proteases. A second class of related analogues containing a novel O-acyl hydroxyurea warhead was found to function as covalent inhibitors of papain-like proteases. These inhibitors can be easily synthesized on solid support, which allows rapid optimization of compounds with improved selectivity and potency for a given target enzyme. We present here one such optimized inhibitor that showed selective inhibition of falcipain 1, a protease of the malaria-causing parasite, Plasmodium falciparum.
Co-reporter:Steven H. L. Verhelst Dr. Dr.
ChemBioChem 2005 Volume 6(Issue 5) pp:
Publication Date(Web):18 MAR 2005
DOI:10.1002/cbic.200400377
Checking out cathepsins. A solid-phase protocol was devised in order to synthesize activity-based probes (1) that contain peptides on both ends of an epoxysuccinyl moiety. The feasibility of this strategy was illustrated by the synthesis of selective probes for cathepsin B, one of the major lysosomal Papain family cysteine proteases.
Co-reporter:Margot G Paulick, Matthew Bogyo
Current Opinion in Genetics & Development (February 2008) Volume 18(Issue 1) pp:97-106
Publication Date(Web):1 February 2008
DOI:10.1016/j.gde.2007.12.001
Many tumor cells have elevated levels of hydrolytic and proteolytic enzymes, presumably to aid in key processes such as angiogenesis, cancer cell invasion, and metastasis. Functional roles of enzymes in cancer progression are difficult to study using traditional genomic and proteomic methods because the activities of these enzymes are often regulated by post-translational mechanisms. Thus, methods that allow for the direct monitoring of enzyme activity in a physiologically relevant environment are required to better understand the roles of specific players in the complex process of tumorigenesis. This review highlights advances in the field of activity-based proteomics, which uses small molecules known as activity-based probes (ABPs) that covalently bind to the catalytic site of target enzymes. We discuss the application of ABPs to cancer biology, especially to the discovery of tumor biomarkers, the screening of enzyme inhibitors, and the imaging of enzymes implicated in cancer.
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