Selective Identification of Cooperatively Binding Fragments in a High‐Throughput Ligation Assay Enables Development of a Picomolar Caspase‐3 Inhibitor

Schmidt, Marco F.; El-Dahshan, Adeeb; Keller, Sandro; Rademann, Jörg

doi:10.1002/anie.200901647

Cited by 47 publications

(33 citation statements)

References 36 publications

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“…All of these interactions would be absent in a peptide substrate lacking a fluorophore at the P1’ position. It is known from studies on caspase-3 that prime side interactions can lead to a significant increase in inhibitory potency; for instance, the addition of a benzoxazole moiety on the prime side of the Ac-DEVD α-ketoaldehyde peptide inhibitor increases the potency ∼300-fold against caspase-3 [30]. As for the inability of 3 to inhibit Lamin A cleavage, the presence of substrate residues more distal to the scissile bond (P5–P8) may alter the general conformation of the inhibitor binding site to disrupt the key L2–L4 interactions observed in Figure 5B.…”

Section: Discussionmentioning

confidence: 99%

Mechanistic and Structural Understanding of Uncompetitive Inhibitors of Caspase-6

Heise¹,

Murray²,

Augustyn

et al. 2012

PLoS ONE

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Inhibition of caspase-6 is a potential therapeutic strategy for some neurodegenerative diseases, but it has been difficult to develop selective inhibitors against caspases. We report the discovery and characterization of a potent inhibitor of caspase-6 that acts by an uncompetitive binding mode that is an unprecedented mechanism of inhibition against this target class. Biochemical assays demonstrate that, while exquisitely selective for caspase-6 over caspase-3 and -7, the compound’s inhibitory activity is also dependent on the amino acid sequence and P1’ character of the peptide substrate. The crystal structure of the ternary complex of caspase-6, substrate-mimetic and an 11 nM inhibitor reveals the molecular basis of inhibition. The general strategy to develop uncompetitive inhibitors together with the unique mechanism described herein provides a rationale for engineering caspase selectivity.

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Section: Discussionmentioning

confidence: 99%

Mechanistic and Structural Understanding of Uncompetitive Inhibitors of Caspase-6

Heise¹,

Murray²,

Augustyn

et al. 2012

PLoS ONE

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“…In this method, a chemically reactive protein ligand serves as a directing probe for the screening of fragment libraries at a spatially defined protein site 18 . Reversible formation of a covalent bond between the directing probe and a nucleophilic fragment enables the sensitized detection of low-affinity fragments with millimolar activity while testing at lower concentrations of 10-100 mM.…”

mentioning

confidence: 99%

Catalytic activation of pre-substrates via dynamic fragment assembly on protein templates

Burda

Rademann

2014

Nat Commun

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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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“…27 No.9 spectrometry [25][26][27][28]. Recently, fragment detection consistent with established high-throughput methodologies that use standardized microtiter plates and detection by fluorescence, absorption, or fluorescence polarization in DLS have been developed [43]. Finally, the methods described here differ significantly in their scope.…”

Section: Reviewmentioning

confidence: 99%

“…A second subcategory of fragment detection comprises template-assisted strategies such as targetguided synthesis (TGS), in which the chemical reaction used for detection is catalyzed by the protein [31,[35][36][37][38][39][40]. Substrate activity screening (SAS) [41][42][43], which is discussed below, can be used for the transfer of fragment information from substrates to non-substrate protein ligands. Finally, dynamic ligation screening (DLS) [44,45] uses classical bioassays, such as fluorogenic substrate competition or fluorescence polarization [43], for fragment detection and can thus be used for HTS fragment-based drug discovery.…”

Section: Introductionmentioning

confidence: 99%

“…Substrate activity screening (SAS) [41][42][43], which is discussed below, can be used for the transfer of fragment information from substrates to non-substrate protein ligands. Finally, dynamic ligation screening (DLS) [44,45] uses classical bioassays, such as fluorogenic substrate competition or fluorescence polarization [43], for fragment detection and can thus be used for HTS fragment-based drug discovery.…”

Section: Introductionmentioning

confidence: 99%

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Dynamic template-assisted strategies in fragment-based drug discovery

Schmidt

Rademann

2009

Trends in Biotechnology

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Fragment-based methods for drug discovery are increasingly popular because they provide drug leads with greater ligand efficiency than conventional high-throughput screening. However, established methods for fragment detection do not address the central question in fragment-based ligand discovery: how can a primary ligand be optimally extended by a secondary fragment? Dynamic screening methods solve this issue by using a protein target as a template for ligand assembly, thus yielding high-affinity binders from low-affinity fragments. This review summarizes recent work on dynamic screening methodology, which resulted in the development of several high-affinity binders for various targets. Strengths and limitations of the published approaches are discussed and possible contributions of dynamic screening methodology to the drug discovery process are highlighted.

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Selective Identification of Cooperatively Binding Fragments in a High‐Throughput Ligation Assay Enables Development of a Picomolar Caspase‐3 Inhibitor

Cited by 47 publications

References 36 publications

Mechanistic and Structural Understanding of Uncompetitive Inhibitors of Caspase-6

Mechanistic and Structural Understanding of Uncompetitive Inhibitors of Caspase-6

Catalytic activation of pre-substrates via dynamic fragment assembly on protein templates

Dynamic template-assisted strategies in fragment-based drug discovery

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