Ultrasensitive small molecule fluorogenic probe for human heparanase

Li, Jun; Schleyer, Kelton A.; Bryan, Tyrel; Xie, Changjian; Seabra, Gustavo; Xu, Yongmei; Kafle, Arjun; Cui, Chao; Wang, Ying; Yin, Kunlun; Fetrow, Benjamin; Henderson, Paul K. P.; Fatland, Peter Zannes; Liu, Jian; Li, Chenglong; Guo, Hua; Cui, Lina

doi:10.1101/2020.03.26.008730

Cited by 4 publications

(3 citation statements)

References 45 publications

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“…halides) on the aromatic aglycone may promote formation of a more robust -stack with the electron rich ring of Tyr348, with the additional benefit of improving leaving group stability by lowering its pKA. Whilst this manuscript was under preparation, Cui and coworkers reported that difluoro-coumarins are more efficient aglycone leaving groups for HPSE substrates compared to 4MU, essentially confirming the validity of this strategy 35 .…”

Section: Figure 3 a Michaelis Menten Plot For The Hydrolysis Of 1 By Hpse B Competitive Inhibition Of Hpsementioning

confidence: 55%

A Fluorogenic Heparan Sulfate Disaccharide for the Measurement of Heparanase Activity

Wu¹,

Wimmer²,

Ferro³

et al. 2020

Preprint

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The endo-β-glucuronidase heparanase mediates mammalian heparan sulfate catabolism, and is of considerable medical interest due to its prominent role in cancer aggression and metastasis. Biochemical studies of heparanase are currently hampered by a lack of suitable chromogenic or fluorogenic assay substrates, instead relying on lengthy multistep procedures to measure activity. Herein, we demonstrate that N’,6-O’-bis-sulfated 4-methylumbelliferyl heparan sulfate disaccharide is a competent fluorogenic heparanase substrate. Despite somewhat slow turnover, the high sensitivity of 4-methylumbelliferyl fluorescence provides a wide signal window that enables both enzyme turnover and inhibition kinetics measurements. Crystal structures with heparanase also provide the first ever observation of a substrate in an activated 1S3 conformation, highlighting previously unknown interactions involved in turnover. Our results pave the way for the design of further improved HPSE substrates that may enable rapid assessment of enzyme activity, which in turn will drive development of new heparanase inhibitors for therapeutic use.

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Section: Figure 3 a Michaelis Menten Plot For The Hydrolysis Of 1 By Hpse B Competitive Inhibition Of Hpsementioning

confidence: 55%

A Fluorogenic Heparan Sulfate Disaccharide for the Measurement of Heparanase Activity

Wu¹,

Wimmer²,

Ferro³

et al. 2020

Preprint

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“…We thus hypothesized that CTLAP emission was attenuated somehow, resulting in reduced background emission (Figure S4B). The emission of some fluorophores is altered by assay pH, often due to the existence of a non‐ or low‐emissive conjugate acid or base form of the fluorophore, [36] but no such effect was observed for AMC (Figure S6).…”

Section: Figurementioning

confidence: 99%

Dual‐Mechanism Quenched Fluorogenic Probe Provides Selective and Rapid Detection of Cathepsin L Activity**

et al. 2021

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Cathepsin L (CTL) is a cysteine protease demonstrating upregulated activity in many disease states. Overlapping substrate specificity makes selective detection of CTL activity difficult to parse from that of its close homologue CTV and the ubiquitous CTB. Current probes of CTL activity have limited applications due to either poor contrast or extra assay steps required to achieve selectivity. We have developed a fluorogenic probe, CTLAP, that displays good selectivity for CTL over CTB and CTV while exhibiting low background fluorescence attributed to dual quenching mechanisms. CTLAP achieves optimum CTL selectivity in the first 10 min of incubation, thus suggesting that it is amenable for rapid detection of CTL, even in the presence of competing cathepsins.

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“…A 7-hydroxymethylcoumarin moiety was selected as the donor fluorophore, due to the possibility of functionalization onto a porphyrin scaffold and the excellent spectral overlap with the absorbance spectrum of porphyrin-based compounds ( Figures S1 and S2 ). Additionally, 7-hydroxymethylcoumarin derivatives are also known to be fairly photostable, 41 , 42 including compound 1 ( Figure S3 ), a characteristic that will importantly maximize the observed increase in fluorescence following enzymatic probe cleavage.…”

Section: Introductionmentioning

confidence: 99%

A Coumarin–Porphyrin FRET Break-Apart Probe for Heme Oxygenase-1

et al. 2021

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Heme oxygenase-1 (HO-1) is a vital enzyme in humans that primarily regulates free heme concentrations. The overexpression of HO-1 is commonly associated with cardiovascular and neurodegenerative diseases including atherosclerosis and ischemic stroke. Currently, there are no known chemical probes to detect HO-1 activity, limiting its potential as an early diagnostic/prognostic marker in these serious diseases. Reported here are the design, synthesis, and photophysical and biological characterization of a coumarin–porphyrin FRET break-apart probe to detect HO-1 activity, Fe–L 1 . We designed Fe–L 1 to “break-apart” upon HO-1-catalyzed porphyrin degradation, perturbing the efficient FRET mechanism from a coumarin donor to a porphyrin acceptor fluorophore. Analysis of HO-1 activity using Escherichia coli lysates overexpressing hHO-1 found that a 6-fold increase in emission intensity at 383 nm was observed following incubation with NADPH. The identities of the degradation products following catabolism were confirmed by MALDI-MS and LC–MS, showing that porphyrin catabolism was regioselective at the α-position. Finally, through the analysis of Fe–L 2 , we have shown that close structural analogues of heme are required to maintain HO-1 activity. It is anticipated that this work will act as a foundation to design and develop new probes for HO-1 activity in the future, moving toward applications of live fluorescent imaging.

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Ultrasensitive small molecule fluorogenic probe for human heparanase

Cited by 4 publications

References 45 publications

A Fluorogenic Heparan Sulfate Disaccharide for the Measurement of Heparanase Activity

A Fluorogenic Heparan Sulfate Disaccharide for the Measurement of Heparanase Activity

Dual‐Mechanism Quenched Fluorogenic Probe Provides Selective and Rapid Detection of Cathepsin L Activity**

A Coumarin–Porphyrin FRET Break-Apart Probe for Heme Oxygenase-1

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