Study of anti-fibrillogenic activity of iron(II) clathrochelates Vladyslava B. Kovalska a,⇑ , Mykhaylo Yu. Losytskyy a , Oleg A. Varzatskii b , Vsevolod V. Cherepanov c , Yan Z. Voloshin d , Andriy A. Mokhir e , Sergiy M. Yarmoluk a , Sergiy V. Volkov b a Institute of Molecular Biology and Genetics NASU, 150 Zabolotnogo St., 03143 Kyiv, Ukraine b Institute of General and Inorganic Chemistry NASU, 32/34 Palladin Av., 03680 Kyiv, Ukraine c Institute of Physics NASU, 46 Nauky Av., 03680 Kyiv, Ukraine d Nesmeyanov Institute of Organoelement Compounds RAS, 28 Vavilova St., Moscow, Russia e Friedrich-Alexander-Universität Erlangen-Nürnberg, Department of Chemistry and Pharmacy, Organic Chemistry II, Henkestr. 42, Erlangen, Germany article info Article history: Received 21 November 2013 Revised 23 January 2014 Accepted 27 January 2014 Available online 2 February 2014 Keywords: Iron(II) clathrochelates Amyloid fibrils Fibrillization inhibition Fluorescent detection AFM Flow cytometry abstract The macrocyclic compounds mono- and bis-iron(II) clathrochelates were firstly studied as potential anti- fibrillogenic agents using fluorescent inhibitory assay, atomic force microscopy and flow cytometry. It is shown that presence of the clathrochelates leads to the change in kinetics of insulin fibrillization reaction and reduces the amount of formed fibrils (up to 70%). The nature of ribbed substituent could determine the activity of clathrochelates—the higher inhibitory effect is observed for compounds containing carboxybenzenesulfide groups, while the inhibitory properties only slightly depend on the size of complex species. The mono- and bis-clathrochelate derivatives of meta-mercaptobenzoic acid have close values of IC 50 namely 16 ± 2 and 24 ± 5 lM, respectively. The presence of clathrochelates decreases the fibril diameter from 5-12 nm for free insulin fibrils to 3–8 nm for these formed in the clathrochelate pres- ence, it also prevents the lateral aggregation of mature fibrils and formation of superfibrillar clusters. However the addition of clathrochelate results in more heterogeneous (both by size and structure) insulin aggregates population as compared to the free insulin. This way, cage complexes—iron(II) clathr- ochelates are proposed as efficient agents able to suppress the protein aggregation processes. Ó 2014 Elsevier Ltd. All rights reserved. 1. Introduction A series of human diseases, including neurodegenerative disorders (i.e. Alzheimer’s, Parkinson’s and Creutzfeldt–Jakob’s dis- eases), type II diabetes and amyloidoses, are caused by deposition of insoluble protein beta-pleated formations (aggregates or amy- loid fibrils) both in cells and extracellular space of various organs or tissues. 1 The molecular basis of such disorders is associated with the processes of protein misfolding and uncontrolled aggregation. 2 Among the approaches towards the therapeutics of protein aggregation disorders is the use of low-molecular compounds able to bind to the proteins or to the fibril seeds this way suppressing further aggregation. Planar aromatic macrocyclic molecules such as porphyrins and phthalocyanines have been earlier reported among the compounds with anti-fibrillogenic properties. 3–5 After the successful use of axially coordinated phthalocyanines—complexes of non-planar geometry, for these purposes, 6 we decided to study whether other bulky macrocycles—clathrochelates possess anti-fibrillogenic activity. Clathrochelates are cage metal complexes with a three- dimensional macrobicyclic ligand encapsulating a metal ion. The advantages of the iron(II) clathrochelates are their incredible chemical stability, straightforward synthesis in high yield from available and inexpensive initial reagents and reactive clathroche- late precursors and three-dimensional cage (macrobicyclic) geom- etry, allowing to introduce up to fourteen different substituents in chelating and capping fragments of the macrobicyclic frameworks. The interest to clathrochelates is evoked by their biological activity as so-called ‘topological drugs’: 7 the iron(II) clathrochelates proved to be efficient transcription inhibitors of T7 RNA polymerase 8 and to bind with high affinity to the albumins. 9 In present research the series of mono and bis-iron(II) clathr- ochelates (Scheme 1) are firstly investigated as anti-fibrillogenic agents using model amyloidogenic protein insulin. Fluorescent cyanine dye based inhibitory assay is used to study the effect of cage complexes on the kinetics of fibrillization reaction and to determine the half maximal inhibitory concentrations of the metal complexes. The changes in the structure and composition of the products of the fibrillization as a result of clathrochelates presence were studied by atomic force microscopy (AFM) and flow cytome- try. These methods complete each other and together provide a http://dx.doi.org/10.1016/j.bmc.2014.01.048 0968-0896/Ó 2014 Elsevier Ltd. All rights reserved. ⇑ Corresponding author. Tel.: +380 44 526 34 49. E-mail address: v.kovalska@gmail.com (V.B. Kovalska). Bioorganic & Medicinal Chemistry 22 (2014) 1883–1888 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc