ABSTRACT
Protein surface mimetics achieve high-affinity binding by exploiting a scaffold to project binding groups over a large area of solvent-exposed protein surface to make multiple cooperative noncovalent interactions. Such recognition is a prerequisite for competitive/orthosteric inhibition of protein-protein interactions (PPIs). This paper describes biophysical and structural studies on ruthenium(II) tris(bipyridine) surface mimetics that recognize cytochrome (cyt) c and inhibit the cytâ c/cytâ c peroxidase (CCP) PPI. Binding is electrostatically driven, with enhanced affinity achieved through enthalpic contributions thought to arise from the ability of the surface mimetics to make a greater number of noncovalent interactions than CCP with surface-exposed basic residues on cytâ c. High-field natural abundance 1 H,15 Nâ HSQC NMR experiments are consistent with surface mimetics binding to cytâ c in similar manner to CCP. This provides a framework for understanding recognition of proteins by supramolecular receptors and informing the design of ligands superior to the protein partners upon which they are inspired.
Subject(s)
Coordination Complexes/metabolism , Cytochrome-c Peroxidase/metabolism , Cytochromes c/metabolism , Ruthenium/chemistry , 2,2'-Dipyridyl/chemistry , Coordination Complexes/chemical synthesis , Coordination Complexes/chemistry , Cytochrome-c Peroxidase/antagonists & inhibitors , Cytochrome-c Peroxidase/genetics , Cytochromes c/antagonists & inhibitors , Cytochromes c/genetics , Hydrogen-Ion Concentration , Magnetic Resonance Spectroscopy , Osmolar Concentration , Protein Binding , Protein Interaction Domains and Motifs , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Spectrometry, Fluorescence , Static Electricity , Surface Properties , ThermodynamicsABSTRACT
Highly functionalised ruthenium(II) tris-bipyridine receptor 1 which acts as a selective sensor for equine cytochrome c (cyt c) is shown to destabilise the native protein conformation by around 25 °C. Receptors 2 and 3 do not exert this effect confirming the behaviour is a specific effect of molecular recognition between 1 and cyt c, whilst the absence of a destabilising effect on 60% acetylated cyt c demonstrates the behaviour of 1 to be protein specific. Molecular recognition also modifies the conformational properties of the target protein at room temperature as evidenced by ion-mobility spectrometry (IMS) and accelerated trypsin proteolysis.
Subject(s)
2,2'-Dipyridyl/chemistry , Cytochromes c/chemistry , Organometallic Compounds/pharmacology , Ruthenium/chemistry , Animals , Binding Sites , Dose-Response Relationship, Drug , Heart , Horses , Models, Molecular , Molecular Structure , Organometallic Compounds/chemical synthesis , Organometallic Compounds/chemistry , Protein Stability , Structure-Activity Relationship , Surface PropertiesABSTRACT
The composition of a dynamic mixture of similar 2,2'-bipyridine complexes of iron(II) bearing either an amide (5-benzylamido-2,2'-bipyridine and 5-(2-methoxyethane)amido-2,2'-bipyridine) or an ester (2,2'-bipyridine-5-carboxylic acid benzylester and 2,2'-bipyridine-5-carboxylic acid 2-methoxyethane ester) side chain have been evaluated by electrospray mass spectroscopy in acetonitrile. The time taken for the complexes to come to equilibrium appears to be dependent on the counteranion, with chloride causing a rapid redistribution of two preformed heteroleptic complexes (of the order of 1 hour), whereas the time it takes in the presence of tetrafluoroborate salts is in excess of 24 h. Similarly the final distribution of products is dependent on the anion present, with the presence of chloride, and to a lesser extent bromide, preferring three amide-functionalized ligands, and a slight preference for an appended benzyl over a methoxyethyl group. Furthermore, for the first time, this study shows that the distribution of a dynamic library of metal complexes monitored by ESI-MS can adapt following the introduction of a different anion, in this case tetrabutylammonium chloride to give the most favoured heteroleptic complex despite the increasing ionic strength of the solution.
Subject(s)
2,2'-Dipyridyl/chemistry , Anions/chemistry , Acetonitriles/chemistry , Amides/chemistry , Coordination Complexes/chemistry , Esters , Ferrous Compounds/chemistry , Ligands , Spectrometry, Mass, Electrospray IonizationABSTRACT
This manuscript describes cell-uptake studies with HEK 293T cells on a series of ruthenium complexes shown previously to act as receptors for protein surface recognition and was motivated by a desire to establish if these receptors represent suitable templates for further elaboration as inhibitors of protein-protein interactions. The results illustrate that large (>3000Da) highly functionalized anionic ruthenium complexes are efficiently transfected via endocytosis to lysosomes with negligible toxicity.
Subject(s)
2,2'-Dipyridyl/analogs & derivatives , Molecular Mimicry/drug effects , 2,2'-Dipyridyl/chemical synthesis , 2,2'-Dipyridyl/chemistry , 2,2'-Dipyridyl/pharmacokinetics , Cell Survival/drug effects , Coordination Complexes , Dose-Response Relationship, Drug , Endocytosis/drug effects , HEK293 Cells , Humans , Lysosomes/drug effects , Molecular Structure , Protein Binding/drug effects , Structure-Activity Relationship , Surface PropertiesABSTRACT
Tetrasodium p-sulfonatocalix[4]arene exists as a hydrate with approximately 14 water molecules and has three polymorphic modifications, all of which contain a water molecule in the molecular cavity that is engaged in OH···π interactions. Single-crystal neutron structures are reported for two of these three forms and reveal a "compressed" water molecule with short OH bonds. Partial atomic charges and hardness analysis (PACHA) calculations based on the neutron coordinates give an OH···π interaction energy of 6.9-7.5 kJ mol(-1). The PACHA analysis also reveals the dominance of the charge-assisted hydrogen bonds from the Na(+)-coordinated water molecules. The instability of the crystal towards dehydration can be traced to an uncoordinated lattice water site. The remarkable calixarene-Na(+)-hydrate motif is conserved almost unchanged across all three polymorphs. A single-crystal neutron structure is also reported for pentasodium p-sulfonatocalix[4]arene·12H(2)O, which exhibits an intracavity water molecule that is engaged in both OH···π and OH···O hydrogen bonding. The shorter covalent bond to the hydrogen atom that forms the interaction with the aromatic ring is again apparent.
ABSTRACT
This manuscript illustrates that the geometric arrangement of protein-binding groups around a ruthenium(II) core leads to dramatic differences in cytochrome c (cyt c) binding highlighting that it is possible to define synthetic receptors with shape complementarity to protein surfaces.
Subject(s)
2,2'-Dipyridyl/analogs & derivatives , Cytochromes c/chemistry , 2,2'-Dipyridyl/chemistry , Binding Sites , Coordination Complexes , Cytochromes c/metabolism , Models, Molecular , Molecular Structure , Surface PropertiesABSTRACT
The synthesis, photophysical, and anion-binding properties of a series of di-, tri-, and tetrapodal anion-binding hosts based on aminopyridinium units with pyrenyl reporter groups are described. The ditopic mesitylene-derived calix[4]arene-based host 4 binds strongly to dicarboxylates, particularly malonate, in a 2:1 anion:host ratio but is essentially nonemissive in the presence of all anions except chloride because of intramolecular quenching by the pyridinium units. Addition of chloride results in a conformational change, giving an initial increase in emission assigned to intramolecular excimer formation. Further chloride addition also results in an increase in the intensity of the pyrenyl monomer emission as chloride binding reduces the acceptor ability of the pyridinium groups. This behavior is not exhibited by control compounds 5 and 6, which lack the ditopic geometry and calixarene spacer unit; however, tripodal 6 forms 1:2 anion:host complexes with a range of anions.
ABSTRACT
Two types of calix[4]arene derived hosts for anions with, respectively, 1,3-alternate and cone conformations have been prepared; the 1,3-alternate system binds dicarboxylate anions in a ditopic manner while the cone compounds are deprotonated by carboxylates.
