Discovery of Pyridyl Bis(oxy)dibenzimidamide Derivatives as Selective Matriptase Inhibitors

Goswami, Rajeev; Mukherjee, Subhendu; Wohlfahrt, Gerd; Chakshusmathi, Ghadiyaram; Nagaraj, Jwala; Chandra, Beeram Ravi; Sistla, Ramesh; Satyam, Leena K.; Samiulla, Dodheri S.; Moilanen, Anu-Maarit; Subramanya, Hosahalli; Ramachandra, Murali

doi:10.1021/ml400213v

Cited by 25 publications

(32 citation statements)

References 24 publications

(45 reference statements)

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“…Interestingly, although matriptase is a trypsin‐like serine proteinase, it did not significantly degrade the aggrecan monomer in vitro, unlike trypsin itself. This suggests that the substrate specificity of matriptase is more restricted than that of trypsin, which is supported by previous studies , such that the observed aggrecanolysis is essentially indirect by other proteinases. This was further supported by the finding that ADAMTS4 (but not ADAMTS5 ) as well as MMPs were induced following matriptase addition to OA cartilage.…”

Section: Discussionsupporting

confidence: 84%

Matriptase Induction of Metalloproteinase‐Dependent Aggrecanolysis In Vitro and In Vivo: Promotion of Osteoarthritic Cartilage Damage by Multiple Mechanisms

Wilkinson

Wang

Habgood

et al. 2017

Arthritis & Rheumatology

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ObjectiveTo assess the ability of matriptase, a type II transmembrane serine proteinase, to promote aggrecan loss from the cartilage of patients with osteoarthritis (OA) and to determine whether its inhibition can prevent aggrecan loss and cartilage damage in experimental OA.MethodsAggrecan release from human OA cartilage explants and human stem cell–derived cartilage discs was evaluated, and cartilage‐conditioned media were used for Western blotting. Gene expression was analyzed by real‐time polymerase chain reaction. Murine OA was induced by surgical destabilization of the medial meniscus, and matriptase inhibitors were administered via osmotic minipump or intraarticular injection. Cartilage damage was scored histologically and aggrecan cleavage was visualized immunohistochemically using specific neoepitope antibodies.ResultsThe addition of soluble recombinant matriptase promoted a time‐dependent release of aggrecan (and collagen) from OA cartilage, which was sensitive to metalloproteinase inhibition and protease‐activated receptor 2 antagonism. Although engineered human (normal) cartilage discs failed to release aggrecan following matriptase addition, both matrix metalloproteinase– and aggrecanase‐mediated cleavages of aggrecan were detected in human OA cartilage. Additionally, while matriptase did not directly degrade aggrecan, it promoted the accumulation of low‐density lipoprotein receptor–related protein 1 (LRP‐1) in conditioned media of the OA cartilage explants. Matriptase inhibition via neutralizing antibody or small molecule inhibitor significantly reduced cartilage damage scores in murine OA, which was associated with reduced generation of metalloproteinase‐mediated aggrecan cleavage.ConclusionMatriptase potently induces the release of metalloproteinase‐generated aggrecan fragments as well as soluble LRP‐1 from OA cartilage. Therapeutic targeting of matriptase proteolytic activity reduces metalloproteinase activity, further suggesting that this serine proteinase may have potential as a disease‐modifying therapy in OA.

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Section: Discussionsupporting

confidence: 84%

Matriptase Induction of Metalloproteinase‐Dependent Aggrecanolysis In Vitro and In Vivo: Promotion of Osteoarthritic Cartilage Damage by Multiple Mechanisms

Wilkinson

Wang

Habgood

et al. 2017

Arthritis & Rheumatology

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“…These membrane‐anchored proteases are structurally defined by a cytoplasmic N‐terminal tail, a transmembrane domain, a stem region that contains various functional domains, and a C‐terminal extracellular serine protease domain, characterized by the catalytic triad, serine, histidine and aspartate, essential for proteolytic activity . Matriptase, one of the best characterized TTSPs, is mainly expressed in epithelia, such as epidermis or thymic stoma . The proteolytic activity of matriptase is regulated by the Kunitz type hepatocyte growth factor activator inhibitors HAI‐1 and HAI‐2 .…”

Section: Methodsmentioning

confidence: 99%

“…One benzguanidine moiety is very likely to be oriented towards the deep, negatively charged S1 pocket . However, the remaining benzguanidine is either able to address the S3/S4 or the S2 pocket of matriptase . The assembly of our probe was inspired by several potent dibasic matriptase inhibitors, such as peptidic ketobenzothiazoles derived from the natural Arg‐Gln‐Ala‐Arg autoactivation sequence of matriptase, bis‐benzamidines, or sulfonylated 3‐amidinophenylalanine derivatives .…”

Section: Methodsmentioning

confidence: 99%

A Fluorescent‐Labeled Phosphono Bisbenzguanidine As an Activity‐Based Probe for Matriptase

Häußler

Schulz-Fincke

Beckmann

et al. 2017

Chemistry A European J

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Activity-based probes are compounds that exclusively form covalent bonds with active enzymes. They can be utilized to profile enzyme activities in vivo, to identify target enzymes and to characterize their function. The design of a new activity-based probe for matriptase, a member of the type II transmembrane serine proteases, is based on linker-connected bis-benzguanidines. An amino acid, introduced as linker, bears the coumarin fluorophore. Moreover, an incorporated phosphonate allows for a covalent interaction with the active-site serine. The resulting irreversible mode of action was demonstrated, leading to enzyme inactivation and, simultaneously, to a fluorescence labeling of matriptase. The ten-step synthetic approach to a coumarin-labeled bis-benzguanidine and its evaluation as activity-based probe for matriptase based on in-gel fluorescence and fluorescence HPLC is reported. HPLC fluorescence detection as a new application for activity-based probes for proteases is demonstrated herein for the first time.

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“…WX-UK1 was evaluated for its potential to inhibit hepsin and uPA using appropriate substrates. 51 (g) A confocal microscope image taken from human breast cancer explant. The sample was processed for Matrigel culture and kept 7 days in culture, followed by immunostaining with indicated antibodies.…”

Section: Wx-uk1 Inhibits Oncogenic Actions Of Hepsin In Mammary Epithmentioning

confidence: 99%

Deregulated hepsin protease activity confers oncogenicity by concomitantly augmenting HGF/MET signalling and disrupting epithelial cohesion

et al. 2015

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Hepsin belongs to a family of cell-surface serine proteases, which have sparked interest as therapeutic targets because of the accessibility of extracellular protease domain for inhibitors. Hepsin is frequently amplified and/or overexpressed in epithelial cancers, but it is not clear how enhanced hepsin expression confers a potential for oncogenicity. We show that hepsin is consistently overexpressed in more than 40% of examined breast cancers, including all major biological subtypes. The effects of doxycycline-induced hepsin overexpression were examined in mammary epithelial organoids, and we found that induced hepsin acutely downmodulates its cognate inhibitor, hepatocyte growth factor (HGF) activator inhibitor type 1 (HAI-1). Hepsin-induced depletion of cellular HAI-1 led to a sharp increase in pericellular serine protease activity. The derepressed hepsin proteolytically activated downstream serine proteases, augmented HGF/MET signalling and caused deterioration of desmosomes and hemidesmosomes; structures important for cell cohesion and cell-basement membrane interaction. Moreover, chronic induction of hepsin considerably shortened the latency of Myc-dependent tumourigenesis in the mouse mammary gland. The serine protease and uPA system inhibitor WX-UK1, identified as a micromolar range hepsin inhibitor, prevented hepsin from augmenting HGF/MET signalling and disrupting desmosomes and hemidesmosomes. The findings suggest that the oncogenic activity of hepsin arises not only from elevated expression level but also from depletion of HAI-1, events which together trigger gain-of-function activity impacting HGF/MET signalling and epithelial cohesion. Thus, hepsin overexpression is a major oncogenic conferrer to a serine protease activity involved in breast cancer dissemination.

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Discovery of Pyridyl Bis(oxy)dibenzimidamide Derivatives as Selective Matriptase Inhibitors

Cited by 25 publications

References 24 publications

Matriptase Induction of Metalloproteinase‐Dependent Aggrecanolysis In Vitro and In Vivo: Promotion of Osteoarthritic Cartilage Damage by Multiple Mechanisms

Matriptase Induction of Metalloproteinase‐Dependent Aggrecanolysis In Vitro and In Vivo: Promotion of Osteoarthritic Cartilage Damage by Multiple Mechanisms

A Fluorescent‐Labeled Phosphono Bisbenzguanidine As an Activity‐Based Probe for Matriptase

Deregulated hepsin protease activity confers oncogenicity by concomitantly augmenting HGF/MET signalling and disrupting epithelial cohesion

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