Thermodynamic characterization of a macrocyclic Zika virus NS2B/NS3 protease inhibitor and its acyclic analogs

Hammerschmidt, Stefan; Huber, Simon; Braun, Niklas J; Lander, Marc; Steinmetzer, Torsten; Kersten, Christian

doi:10.1002/ardp.202200518

Cited by 6 publications

(3 citation statements)

References 81 publications

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“…ITCcalc was well-accepted and was considered useful for experimental evaluation. Lastly, the presented guidelines were followed in recent research projects including result evaluation with ITCcalc . , …”

Section: Resultsmentioning

confidence: 99%

“…The deviation from the calculated and the experimentally determined binding heat capacity is a proxy of the possible conformational changes . With this, one can estimate a more negative Δ C P in reactions where hydrophobic residues are buried upon ligand binding or conformational changes. − Those assumptions and calculations should be taken with caution, as several disturbing discrepancies in these simplified perspectives were reported. , …”

Section: Introductionmentioning

confidence: 99%

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Advanced Isothermal Titration Calorimetry for Medicinal Chemists with ITCcalc

Hammerschmidt,

Barthels,

Weldert

et al. 2024

J. Chem. Educ.

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Isothermal titration calorimetry (ITC) is the method of choice for thermodynamic binding profiling. As a label-free in-solution technique, ITC experiments do not require modified or immobilized proteins or ligands and hence can be performed under close-to-native environmental conditions. It directly follows the released heat of the molecular interaction and does not need competing reporter molecules. However, advanced experimental ITC setups are frequently necessary to derive thermodynamic parameters for medicinal chemistry applications. Beyond 1:1 protein−ligand binding, complex systems include displacement titrations to determine thermodynamics for ligands with affinities either too high or too low to be derived from direct titrations, the characterization of protonation changes upon binding, or the elucidation of heat capacities. We present hands-on experiments for graduate medicinal chemists and students familiar with standard ITC methods, reliably highlighting these issues and their respective solutions. The setup can be either a biochemical trypsin−ligand system or a more cost-efficient EDTA-based variant. This demonstration of best practice protocols comes together with the freely accessible ITC web calculator ITCcalc (https://itccalc.uni-mainz.de), guiding through the underlying evaluation of the obtained experimental results.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Advanced Isothermal Titration Calorimetry for Medicinal Chemists with ITCcalc

Hammerschmidt,

Barthels,

Weldert

et al. 2024

J. Chem. Educ.

Self Cite

View full text Add to dashboard Cite

show abstract

“…The authors have cited additional references within the Supporting Information. [39,40,58,[69][70][71][72]…”

Section: Supporting Informationmentioning

confidence: 99%

Conformational Selection and Induced Fit: The Behavior of Two Homologous Proteases

Maus,

Hinze,

Hammerschmidt

et al. 2023

ChemistryEurope

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Protein‐ligand interactions are crucial for many cellular processes, with details of the binding mechanism being discussed as essential for biological functions. Interestingly, protein binding often involves conformational changes between two or more states, whereby different binding mechanisms are possible even with a simple two‐state description. Two models are widely used to portray protein‐ligand interactions: Induced fit and conformational selection. However, distinguishing them experimentally is challenging. Single‐molecule Förster resonance energy transfer (smFRET) has emerged as a powerful tool to resolve structural dynamics at the level of single proteins. Here, we investigated immobilized Zika virus (ZIKV) and dengue virus (DENV2) NS2B‐NS3 proteases using smFRET to compare their conformational changes upon binding to competitive small molecule inhibitors. The analysis of the smFRET data allowed us to distinguish between induced fit and conformational selection models and assign the binding mechanism from the kinetic parameters obtained. Although DENV and ZIKV protease are proteins with high structural similarities, our results reveal that they have opposite binding mechanisms for competitive ligands. While the protein‐ligand interaction in the ZIKV protease follows an induced fit mechanism, the DENV protease follows the conformational selection mechanism.

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Synthesis and structural characterization of new macrocyclic inhibitors of the Zika virus NS2B–NS3 protease

Huber,

Braun,

Schmacke

et al. 2024

Archiv der Pharmazie

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Three new series of macrocyclic active site‐directed inhibitors of the Zika virus (ZIKV) NS2B–NS3 protease were synthesized. First, attempts were made to replace the basic P3 lysine residue of our previously described inhibitors with uncharged and more hydrophobic residues. This provided numerous compounds with inhibition constants between 30 and 50 nM. A stronger reduction of the inhibitory potency was observed when the P2 lysine was replaced by neutral residues, all of these inhibitors possess Ki values >1 µM. However, it is possible to replace the P2 lysine with the less basic 3‐aminomethylphenylalanine, which provides a similarly potent inhibitor of the ZIKV protease (Ki = 2.69 nM). Crystal structure investigations showed that the P2 benzylamine structure forms comparable interactions with the protease as lysine. Twelve additional structures of these inhibitors in complex with the protease were determined, which explain many, but not all, SAR data obtained in this study. All individual modifications in the P2 or P3 position resulted in inhibitors with low antiviral efficacy in cell culture. Therefore, a third inhibitor series with combined modifications was synthesized; all of them contain a more hydrophobic d‐cyclohexylalanine in the linker segment. At a concentration of 40 µM, two of these compounds possess similar antiviral potency as ribavirin at 100 µM. Due to their reliable crystallization in complex with the ZIKV protease, these cyclic compounds are very well suited for a rational structure‐based development of improved inhibitors.

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Thermodynamic characterization of a macrocyclic Zika virus NS2B/NS3 protease inhibitor and its acyclic analogs

Cited by 6 publications

References 81 publications

Advanced Isothermal Titration Calorimetry for Medicinal Chemists with ITCcalc

Advanced Isothermal Titration Calorimetry for Medicinal Chemists with ITCcalc

Conformational Selection and Induced Fit: The Behavior of Two Homologous Proteases

Synthesis and structural characterization of new macrocyclic inhibitors of the Zika virus NS2B–NS3 protease

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