Fluorescence Lifetime-Based Competitive Binding Assays for Measuring the Binding Potency of Protease Inhibitors In Vitro.
Fluorescence lifetime (FLT)-based assays have developed to become highly attractive tools in drug discovery. All recently published examples of FLT-based assays essentially describe their use for monitoring enzyme-mediated peptide modifications, such as proteolytic cleavage or phosphorylation/dephosphorylation. Here we report the development of competitive binding assays as novel, inhibitor-centric assays, principally employing the FLT of the acridone dye Puretime 14 (PT14) as the readout parameter. Exemplified with two case studies on human serine proteases, the details of the rationale for both the design and synthesis of probes (i.e., active site-directed low-molecular-weight inhibitors conjugated to PT14) are provided. Data obtained from testing inhibitors with the novel assay format match those obtained with alternative formats such as FLT-based protease activity and time-resolved fluorescence resonance energy transfer-based competitive binding assays.
Novartis Institutes for BioMedical Research (NIBR), Expertise Platform Proteases, Novartis Pharma AG, Basel, Switzerland.
Andreas
A
Boettcher
Novartis Institutes for BioMedical Research (NIBR), Expertise Platform Proteases, Novartis Pharma AG, Basel, Switzerland.
Nathalie
N
Gradoux
Novartis Institutes for BioMedical Research (NIBR), Expertise Platform Proteases, Novartis Pharma AG, Basel, Switzerland.
Edwige
E
Lorthiois
Novartis Institutes for BioMedical Research (NIBR), Expertise Platform Proteases, Novartis Pharma AG, Basel, Switzerland.
Trixi
T
Brandl
Novartis Institutes for BioMedical Research (NIBR), Global Discovery Chemistry, Novartis Pharma AG, Basel, Switzerland.
David
D
Orain
Novartis Institutes for BioMedical Research (NIBR), Expertise Platform Proteases, Novartis Pharma AG, Basel, Switzerland.
Nikolaus
N
Schiering
Novartis Institutes for BioMedical Research (NIBR), Expertise Platform Proteases, Novartis Pharma AG, Basel, Switzerland.
Frederic
F
Cumin
Novartis Institutes for BioMedical Research (NIBR), Expertise Platform Proteases, Novartis Pharma AG, Basel, Switzerland.
Julian
J
Woelcke
Novartis Institutes for BioMedical Research (NIBR), Expertise Platform Proteases, Novartis Pharma AG, Basel, Switzerland ulrich.hassiepen@novartis.com.
Ulrich
U
Hassiepen
D054831:Acridones / Q000737:chemistry; D001667:Binding, Competitive; D002021:Buffers; D020134:Catalytic Domain; D055808:Drug Discovery / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008168:Lung / Q000201:enzymology; D008968:Molecular Conformation; D008970:Molecular Weight; D010455:Peptides / Q000737:chemistry; D011480:Protease Inhibitors / Q000737:chemistry*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry; D057057:Serine Proteases / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods*; D053802:Tryptases / Q000737:chemistry
D054831:Acridones; D002021:Buffers; D005456:Fluorescent Dyes; D010455:Peptides; D011480:Protease Inhibitors; D011994:Recombinant Proteins; C000612681:puretime 14; C041300:acridone; D057057:Serine Proteases; D053802:Tryptases
assay; compound interference; compound profiling; fluorescence lifetime; inhibitor; protease