ABSTRACT
In order to develop affinity-based biosensor platforms, appropriate ligands with a functional handle for immobilization onto a biosensor surface are required. To this end, a library of papain inhibitors was designed and synthesized, containing different azide linkers for subsequent immobilization by 'click' chemistry, in this particular case by copper-free, strain-promoted azide-alkyne cycloaddition (SPAAC). Furthermore, a molecular docking study was performed to obtain a better insight as to at which position such azide handles could be tolerated without affecting binding affinity. Although the azide moiety is small, in some cases its introduction strongly influenced the binding affinity. For one class of inhibitors a swapped binding mode was proposed to explain the results. In addition, a specific site for linker introduction was identified, which did not significantly affect the binding affinity.
Subject(s)
Alkynes/pharmacology , Azides/pharmacology , Cysteine Proteinase Inhibitors/pharmacology , Papain/antagonists & inhibitors , Small Molecule Libraries/pharmacology , Alkynes/chemistry , Azides/chemistry , Binding Sites , Cysteine Proteinase Inhibitors/chemical synthesis , Cysteine Proteinase Inhibitors/chemistry , Dose-Response Relationship, Drug , Ligands , Models, Molecular , Molecular Structure , Papain/chemistry , Papain/metabolism , Small Molecule Libraries/chemical synthesis , Small Molecule Libraries/chemistry , Structure-Activity RelationshipABSTRACT
Surface plasmon resonance (SPR) is a powerful label-free diagnostic tool to study biomolecular interactions. However, one of the drawbacks of SPR is the lack of controlled immobilization of ligands on the sensor surface. We have developed a modular platform for the fast, reagent-free and site-specific immobilization of azide-containing ligands by strain-promoted cycloaddition onto a cyclooctyne-modified SPR sensor surface. The usefulness of the concept was shown in a study with a papain model system, and up to 150 experiments were performed without loss of surface quality. Furthermore, azide-containing green fluorescent protein (GFP) was also effectively immobilized. Taken together, cyclooctyne-modified SPR chips enable smooth and site-selective immobilization of ligands and prove to be more robust than traditionally functionalized systems.
Subject(s)
Papain/chemistry , Peptides/chemistry , Surface Plasmon Resonance/instrumentation , Azides/chemistry , Cycloaddition Reaction , Green Fluorescent Proteins/chemistry , Green Fluorescent Proteins/metabolism , Immobilized Proteins/chemistry , Immobilized Proteins/metabolism , Nitrogen Oxides/chemistry , Papain/metabolism , Peptides/metabolism , Surface PropertiesABSTRACT
Kinases present an attractive target for drug development, since they are involved in vital cellular processes and are implicated in a variety of diseases, such as cancer and diabetes. However, obtaining selectivity for a specific kinase over others is difficult since many current kinase inhibitors exclusively target the highly conserved kinase ATP binding domain. Previously, a microarray-based strategy to discover so-called bisubstrate-based inhibitors that target the more specific peptide binding groove in addition to the ATP binding site was described. One attractive feature of this strategy is the opportunity to tune the selectivity of these inhibitors by systematically varying components. In an extension to this previous work, this study explores the potential of this guided selectivity modulation, leading to a series of inhibitors with different selectivity profiles against highly homologous protein kinase C (PKC) isozymes. Of the inhibitors studied, most exhibited improved potency and selectivity compared with their constituent parts. Furthermore, the selectivity was found to be tunable either through modification of the pseudosubstrate peptide (peptide binding groove) or the ATP-competitive part (ATP binding site). In a number of cases, the selectivity of the construct could be predicted from the initial peptide substrate profiling experiment. Since this strategy is applicable to all kinase sets, it could be used to rapidly develop uniquely selective inhibitors.
Subject(s)
Peptides/chemistry , Peptides/pharmacology , Protein Kinase C/antagonists & inhibitors , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Animals , Binding Sites , Click Chemistry , Humans , Isoenzymes/antagonists & inhibitors , Isoenzymes/chemistry , Isoenzymes/metabolism , Peptides/chemical synthesis , Protein Kinase C/chemistry , Protein Kinase C/metabolism , Protein Kinase Inhibitors/chemical synthesis , Solid-Phase Synthesis Techniques , Substrate SpecificityABSTRACT
Efficient strategies for the introduction of arginine residues featuring acetylene or azide moieties in their side chains are described. The substituents are introduced in a way that maintains the basicity of the guanidine moiety. The methodology can be used e.g. for non-invasive labeling of arginine-containing peptides. Its applicability is demonstrated by the introduction of 'click' handles into a Protein Kinase C (PKC) pseudosubstrate peptide, and the subsequent preparation and evaluation of a novel bisubstrate-based inhibitor based on such a peptide.
