Nowadays, search of the macrocyclic middle-sized molecules of three-dimension structure capable of targeting large protein surface elements by multiple supramolecular interactions is of interest. Application of such compounds spreads from protein-protein interactions modulation to protein-based materials development. The goal of this work is to investigate the interactions between series of globular proteins and bioactive boron containing three-dimensional macrocycles (closo-borates, iron (II) clathrochelates).
The interactions of serum albumins and series of globular proteins (lysozyme, β-lactoglobulin, immunoglobulin G) with non-substituted and functionalized closo-borates were investigated. The complexes formation between serum albumins and closo-borate clusters [B10H10]2-, [B12H12]2- and their functionalized derivatives was confirmed by fluorescent spectroscopy and isothermal titration calorimetry (ITC). The intensity of their binding to albumins is affected by the clusters structure and their substituents nature.
The complexes of albumins with halogen closo-decaborates are characterized by higher binding affinity (K about 104 – 106 M-1) as compared to the functionalized or non-functionalized hydrogen clusters (K about 103 M-1); albumin complexes with halogen/arylamine derivatives are characterized by larger number of boron clusters (4-5) bound per one protein molecule in comparison with hydrogen analogues (2). Due to this, halogen closo-borates are proposed for further studies as agents for using in BNCT method with higher “target delivery” potency comparing with hydrogen closo-borates.
The amyloid protein aggregation with dianionic boron cluster – closo-borate [B12H12]2- – leads to early insulin denaturation; low (10 µМ) cluster concentration speeds up the amyloid fibril formation. In addition, the cluster changes the morphology of the amyloid fibrils: fibrillar structures formed in the cluster presence, are unbranched with larger diameter than that upon fibrillization of free insulin; cluster concentration increase (to 100 µМ) intensifies the fibrils ability to lateral aggregation. Due to these features, the closo-borates are of the interest for studies as agents allowing to modify (i.e. to direct) the fibrillization reaction and for further exploring of the effect of anions on protein conformation changes and aggregation.
The series of amyloid-sensitive trimethine cyanine dyes was characterized. Their ability to intensively increase (up to 70 times) the fluorescent signal in amyloid fibrils presence and to monitor protein fibrillization process was shown.
The interactions between serum albumin and iron (II) clathrochelates were investigated and characterized by fluorescent and circular dichroism spectroscopy, ITC. Upon the protein-ligand interaction, optically inactive iron (II) clathrochelates are able to acquire CD-signal in visible range of spectra (350-600 nm).
The nature, number and isomery of clathrochelate terminal groups affect both binding intensity and properties of the corresponding induced CD-signal (shape, peaks values). According to protein fluorescence quenching studies, the protein binding with the clathrochelates containing two or six carboxyl terminal groups, is the most intensive. However, upon serum albumin binding, more intensive ICD-responses acquire clathrochelates with two carboxyphenyl groups; ICD-responses of different shape – hexa carboxyphenyl substituted macrocycles. For the di-carboxyphenyl clathrochelates, which intensively interact with albumins and acquire the most intensive ICD-signals, low cytotoxicity, ІС50 = 40 – 150 μM, on human promyelocytic leukemia cell line was shown.
The complex formation between bovine serum albumin and hexa-carboxyphenyl substituted iron(II) clathrochelates was studied by isothermal titration calorimetry. Binding constants of complex formation BSA with hexa carboxyphenyl isomers are about 103 - 104 М–1, binding ratio is 1-2 clathrochelates molecules per protein molecule.
Hexa-carboxyphenyl iron(II) clathrochelates discriminate between proteins of similar structure, in this case human and bovine serum albumin, giving distinct ICD-spectra. Also iron(II) clathrochelates bound to albumin could reflect the transitions of the protein conformation (caused by pH change) by the changes of the band profile and intensity of their CD spectra. Thus, cage metal complexes iron(II) clathrochelates have shown potency as molecular three-dimensional scaffolds for the design of CD-sensitive reporters able to recognize specific elements of protein surfaces.
According to supposed binding mode of protein-clathrochelate interaction, the electrostatic (polar) interactions between its carboxyl groups and complementary binding groups of a protein play a key role for clathrochelate-to-protein assembling. The protein binding site geometry defines the clathrochelates optically active conformation.
