dc.description.abstract
Sensitive detection of small molecule fragments binding to defined sites of
biomacromolecules is still a considerable challenge. Here we demonstrate that
protein-binding fragments are able to induce enzymatic reactions on the
protein surface via dynamic fragment ligation. Fragments binding to the S1
pocket of serine proteases containing a nitrogen, oxygen or sulphur
nucleophile are found to activate electrophilic pre-substrates through a
reversible, covalent ligation reaction. The dynamic ligation reaction
positions the pre-substrate molecule at the active site of the protein thereby
inducing its enzymatic cleavage. Catalytic activation of pre-substrates is
confirmed by fluorescence spectroscopy and by high-performance liquid
chromatography. The approach is investigated with 3 pre-substrates and 14
protein-binding fragments and the specific activation and the templating
effect exerted by the enzyme is quantified for each protease–fragment–pre-
substrate combination. The described approach enables the site-specific
identification of protein-binding fragments, the functional characterization
of enzymatic sites and the quantitative analysis of protein template-assisted
ligation reactions. View full text Subject terms: Chemical sciences Chemical
biology Medicinal chemistry At a glance Figures First | 1-4 of 6 | Last View
all figures left The concept of pre-substrate activation by protein-binding
fragments. Figure 1 Proof-of-concept. Figure 2 Structure of potential pre-
substrates 1–3 and S1-binding fragments 4–17. Figure 3 Model for the
activation of pre-substrates by nucleophilic protein-binding fragments. Figure
4 Activation of pre-substrate 3. Figure 5 Three-fragment assembly. Figure 6
right Compounds Genes and Proteins References Abstract• References• Author
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Author information Abstract• References• Author information• Supplementary
information Affiliations Institute of Pharmacy, Medicinal Chemistry,
University of Leipzig, Brüderstraße 34, 04103 Leipzig, Germany Edyta Burda &
Jörg Rademann Institute of Pharmacy, Medicinal Chemistry, Freie Universität
Berlin, Königin-Luise-Straße 2+4, 14195 Berlin, Germany Jörg Rademann
Contributions J.R. and E.B. conceived and designed the experiments, E.B.
performed the experiments. Both authors discussed the results and co-wrote the
manuscript. Competing financial interests The authors declare no competing
financial interests. Corresponding author Correspondence to: Jörg Rademann
Supplementary information Abstract• References• Author information•
Supplementary information PDF files Supplementary Information (915 KB)
Supplementary Figures 1-11, Supplementary Methods and Supplementary
References. Additional data 3-Oxo-(N-(4-methyl-2-oxo-2H-
chromen-7-yl)-propanoylamide N-(4-Methyl-2-oxo-2H-chromen-7-yl)-acrylamide
3-((2-Aminoethyl)thio)-N-(4-methyl-2-oxo-2H-chromen-7-yl)propanamide npj
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Sensitive detection of small molecule fragments binding to defined sites of
biomacromolecules is still a considerable challenge. Here we demonstrate that
protein-binding fragments are able to induce enzymatic reactions on the
protein surface via dynamic fragment ligation. Fragments binding to the S1
pocket of serine proteases containing a nitrogen, oxygen or sulphur
nucleophile are found to activate electrophilic pre-substrates through a
reversible, covalent ligation reaction. The dynamic ligation reaction
positions the pre-substrate molecule at the active site of the protein thereby
inducing its enzymatic cleavage. Catalytic activation of pre-substrates is
confirmed by fluorescence spectroscopy and by high-performance liquid
chromatography. The approach is investigated with 3 pre-substrates and 14
protein-binding fragments and the specific activation and the templating
effect exerted by the enzyme is quantified for each protease–fragment–pre-
substrate combination. The described approach enables the site-specific
identification of protein-binding fragments, the functional characterization
of enzymatic sites and the quantitative analysis of protein template-assisted
ligation reactions.
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