Design of ultrasensitive probes for human neutrophil elastase through hybrid combinatorial substrate library profiling
Open accessAbstract
Significance: The exploration of protease substrate specificity is generally restricted to naturally occurring amino acids, limiting the degree of conformational space that can be surveyed. We substantially enhanced this by incorporating 102 unnatural amino acids to explore the S1–S4 pockets of human neutrophil elastase. This approach provides hybrid natural and unnatural amino acid sequences, and thus we termed it the Hybrid Combinatorial Substrate Library. Using this approach, we have designed an extremely active substrate of NE and subsequently converted it into an ultrasensitive activity-based probe for imaging active elastase during the process of neutrophil extracellular trap formation. Our study could have a substantial effect on the design of substrates, inhibitors, and probes for any endopeptidase.
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The effect of methionine oxidation on HNE cleavage of peptide substrates has a strong positional dependence.
“However, the oxidation of P3 methionine residues in peptide substrates appears to enhance the HNE catalysed cleavage rates of those substrates [20] and suggests a strong positional dependence.”
From Discrimination of Methionine Sulfoxide and Sulfone by Human Neutrophil Elastase (2021) · cites this paper as [20]
The Results report that HNE favors methionine dioxide at P3 and recognizes methionine oxide about fivefold less strongly, supporting an effect of oxidation state on substrate recognition. The cited text does not compare oxidized with unoxidized methionine at P3 or directly establish how methionine oxidation affects cleavage at different positions, so the claimed enhancement and strong positional dependence are not fully supported. (LLM, conf=0.78)
Experimental — accuracy not yet measuredThe previous combinatorial library [20] was constructed of pools of many thousands of peptides, each in very low concentration, and each individual substrate concentration was likely far below its respective Km.
“The previous combinatorial library [20] was constructed of pools of many thousands of peptides, each in very low concentration, and each individual substrate concentration was likely to be far below its respective Km.”
From Discrimination of Methionine Sulfoxide and Sulfone by Human Neutrophil Elastase (2021) · cites this paper as [20]
The Results describe a combinatorial library screened as peptide mixtures at 50 μM total concentration, supporting the point that individual peptides were present at low concentration. But each well contained 361 peptides, not many thousands, and the paper does not establish that each peptide’s concentration was far below its respective Km. (LLM, conf=0.86)
Experimental — accuracy not yet measuredOxidation of P3 methionine residues in peptide substrates enhances HNE-catalysed cleavage rates.
“However, the oxidation of P3 methionine residues in peptide substrates appears to enhance the HNE catalysed cleavage rates of those substrates [20] and suggests a strong positional dependence.”
From Discrimination of Methionine Sulfoxide and Sulfone by Human Neutrophil Elastase (2021) · cites this paper as [20]
HNE has a pronounced preference for peptide substrates with valine at P1, as previously reported.
“A pronounced HNE preference for peptide substrates with valine at P1 was previously reported [20].”
From Discrimination of Methionine Sulfoxide and Sulfone by Human Neutrophil Elastase (2021) · cites this paper as [20]
A combinatorial library screen by Kasperkiewicz et al. showed unexpected HNE selectivity for an oxidised methionine residue at the P3 position of peptide substrates.
“This library screen and analyses showed an unexpected HNE selectivity for an oxidised methionine residue at the P3 position of peptide substrates.”
From Discrimination of Methionine Sulfoxide and Sulfone by Human Neutrophil Elastase (2021) · cites this paper as [20]
PK101 and the optimal HNE peptide substrates selected from the combinatorial library contain unnatural amino acid residues.
“However, PK101 and the optimal HNE peptide substrates selected from the combinatorial library contain unnatural amino acid residues [20], and the inhibitor drugs targeting HNE are small non-proteinaceous synthetic molecules [22,23].”
From Discrimination of Methionine Sulfoxide and Sulfone by Human Neutrophil Elastase (2021) · cites this paper as [20]
The data are consistent with a significant biological role for the previously reported HNE enhancement by substrates containing a P3 methionine sulfone residue.
“The data reported here are consistent with a significant biological role for the previously reported [20] HNE enhancement by substrates containing a P3 methionine sulfone residue.”
From Discrimination of Methionine Sulfoxide and Sulfone by Human Neutrophil Elastase (2021) · cites this paper as [20]
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Cites
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Recent progresses in small-molecule enzymatic fluorescent probes for cancer imaging
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Discrimination of Methionine Sulfoxide and Sulfone by Human Neutrophil Elastase
Leahy, D., Grant, C., Jackson, A., Duff, A., Tardiota, N., Van Haeften, J., Chen, X., Peake, J. M., Kruppa, M. D., Smith, E. T., et al. (2021) · Molecules · doi:10.3390/molecules26175344 · Cited by 2 · see citing works
“[20] screened a combinatorial library of unnatural (not genomically encoded) and natural amino acids to identify and develop an activity-based probe capable of distinguishing between HNE and the closely related protease neutrophil proteinase 3 (PR3).”
BackgroundInfluential
- Authors
- Kasperkiewicz, P., Poreba, M., Snipas, S. J., Parker, H., Winterbourn, C. C., Salvesen, G. S., & Drag, M.
- Affiliations
- Wrocław University of Science and Technology (PL)
- Division of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Technology, Wroclaw 50-370, Poland;
- Sanford Burnham Prebys Medical Discovery Institute (US)
- University of Otago (NZ)
- Venue
- Proceedings of the National Academy of Sciences
- Publisher
- National Academy of Sciences
- Published
- 2014-02-03
- Volume
- 111
- Issue
- 7
- Pages
- 2518-2523
- ISSN
- 0027-8424 (ISSN-L)
- 1091-6490
- PMC
- PMC3932852
- OpenAlex
- W2036699296
- Cited by
- 189
- References
- 1
- Citations by year
- 2026: 8 · 2025: 11 · 2024: 12 · 2023: 12 · 2022: 17 · 2021: 15 · 2020: 40 · 2019: 18 · 2018: 16 · 2017: 9 · 2016: 9 · 2015: 20 · 2014: 2
- FWCI
- 4.38
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Proceedings of the National Academy of Sciences (publishedVersion)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Cancer Institute (R01CA163743, P30CA030199)
- National Institute of General Medical Sciences (R01GM099040, R01-GM09040)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Protease and Inhibitor Mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Chemical Synthesis and Analysis
- Physical Sciences › Chemistry › Organic Chemistry › Click Chemistry and Applications
- Keywords
- human neutrophil elastase, protease specificity, unnatural amino acids, activity-based probes, neutrophil extracellular traps, protease selectivity
- MeSH terms
- Amino Acids, Binding Sites, Humans, Kinetics, Substrate Specificity, Molecular Probes, Molecular Structure, Gene Library, Leukocyte Elastase, Combinatorial Chemistry Techniques
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