Towards the anti-fibrillogenic activity of phthalocyanines with out-of-plane ligands: correlation with self-association proneness

Aim. The activity of five hafnium phthalocyanines containing out-of-plane ligands as inhibitors of reaction of insulin fibril formation is studied and correlation between their inhibitory properties and tendency to self-association is discussed. Methods. Fluorescence and absorption spectroscopy. Res...

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Veröffentlicht in:	Вiopolymers and Cell
Datum:	2013
Hauptverfasser:	Kovalska, V.B., Losytskyy, M.Yu., Chernii, S.V., Chernii, V.Ya., Tretyakova, I.M., Yarmoluk, S.M., Volkov, S.V.
Format:	Artikel
Sprache:	English
Veröffentlicht:	Інститут молекулярної біології і генетики НАН України 2013
Schlagworte:	Structure and Function of Biopolymers
Online Zugang:	https://nasplib.isofts.kiev.ua/handle/123456789/153666
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Назва журналу:	Digital Library of Periodicals of National Academy of Sciences of Ukraine
Zitieren:	Towards the anti-fibrillogenic activity of phthalocyanines with out-of-plane ligands: correlation with self-association proneness / V.B. Kovalska, M.Yu. Losytskyy, S.V. Chernii, V.Ya. Chernii, I.M. Tretyakova, S.M. Yarmoluk, S.V. Volkov // Вiopolymers and Cell. — 2013. — Т. 29, №. 6. — С. 473-479. — Бібліогр.: 12 назв. — англ.

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spelling	Kovalska, V.B. Losytskyy, M.Yu. Chernii, S.V. Chernii, V.Ya. Tretyakova, I.M. Yarmoluk, S.M. Volkov, S.V. 2019-06-14T12:15:39Z 2019-06-14T12:15:39Z 2013 Towards the anti-fibrillogenic activity of phthalocyanines with out-of-plane ligands: correlation with self-association proneness / V.B. Kovalska, M.Yu. Losytskyy, S.V. Chernii, V.Ya. Chernii, I.M. Tretyakova, S.M. Yarmoluk, S.V. Volkov // Вiopolymers and Cell. — 2013. — Т. 29, №. 6. — С. 473-479. — Бібліогр.: 12 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.00083C https://nasplib.isofts.kiev.ua/handle/123456789/153666 577.336 + 667.287.4 Aim. The activity of five hafnium phthalocyanines containing out-of-plane ligands as inhibitors of reaction of insulin fibril formation is studied and correlation between their inhibitory properties and tendency to self-association is discussed. Methods. Fluorescence and absorption spectroscopy. Results. For the complexes with weak proneness to self-association PcHfDbm2, PcHfPyr2, and PcHfBtfa2 the values of inhibitory activity were estimated as 60–73 %. For phthalocyanines with the pronounced tendency to self-association PcHfPiromelit and PcHfCl2 the noticeably higher inhibitory activity values (about 95 %) were shown. In the presence of native or fibrilar insulin the destruction of self-associates of metal complex occurs in buffer pH 7.9, Besides upon the conditions of insulin fibrillization reaction (0.1 M HCl) phthalocyanines exist predominantly as monomers. Conclusions. The phthalocyanines with out-of-plane ligands with higher tendency to self-association have shown higher inhibitory activity in the insulin fibril formation comparing with the poorly aggregated metal complexes. At the same time low-order self-associates are not involved directly in the mechanism of inhibition of insulin fibrillization and the phthalocyanines bind with protein in monomeric form. Tendency of phthalocyanines to self-association in aqueous media seems to be an «indicator» of their proneness to stack with protein aromatic amino-acids and thus of anti-fibrilogenic properties. Мета. Дослідити активність п’яти фталоціанінів гафнію, що містять позаплощинні ліганди як інгібітори реакції фібрилоутворення інсуліну, та встановити кореляцію між інгібіторними властивостями фталоціанінів і їхньою схильністю до самоасоціації. Методи. Флуоресцентна спектроскопія і спектроскопія електронного поглинання. Результати. Для комплексів PcHfDbm2, PcHfPyr2 і PcHfBtfa2, які практично не утворюють агрегатів у водному буфері, інгібіторна активність становить 60–73 %. Фталоціаніни PcHfPiromelit і PcHfCl2 з високою схильністю до утворення агрегатів практично повністю пригнічують формування амілоїдних фібрил інсуліну (інгібіторна активність приблизно 95 %). У водному буфері за присутності мономерного або фібрилярного інсуліну відбувається руйнування агрегатів металокомплексів. Крім того, за умов проведення реакції фібрилізації інсуліну (0,1 М HCl) фталоціаніни існують переважно у вигляді мономерів. Висновки. Фталоціаніни з позаплощинними лігандами з тенденцією до самоасоціації демонструють вищу інгібувальну активність у реакції фібрилізації інсуліну порівняно з металокомплексами, які агрегують гірше. Фталоціанінові самоасоціати низького порядку безпосередньо не залучені до механізму інгібування агрегатоутворення інсуліну, тобто фталоціанін зв’язується з білком у мономерній формі. Тенденцію до самоасоціації фталоціані- нів у водному середовищі можна розглядати як «індикатор» їхньої схильності до взаємодії з ароматичними амінокислотами і, отже, як показник їхньої антифібрилогенної активності. Цель. Исследовать активность пяти фталоцианинов гафния, содержащих внеплоскостные лиганды в качестве ингибиторов реакции фибриллообразования инсулина, и установить корреляцию между их ингибиторными свойствами и склонностью к самоассоциации. Методы. Флуоресцентная спектроскопия и спектроскопия электронного поглощения. Результаты. Для комплексов PcHfDbm2, PcHfPyr2 и PcHfBtfa2, практически не образующих агрегатов в водном буфере, ингибиторная активность составляет 60–73 %. Фталоцианины PcHfPiromelit и PcHfCl2 с высокой склонностью к образованию агрегатов практически полностью подавляют формирование амилоидных фибрилл инсулина (ингибиторная активность около 95 %). В водном буфере в присутствии мономерного или фибриллярного инсулина происходит разрушение агрегатов металлокомплексов. Кроме того, в условиях проведения реакции фибриллизации инсулина (0,1 М HCl) фталоцианины существуют преимущественно в виде мономеров. Выводы. Фталоцианины с внеплоскостными лигандами с тенденцией к самоассоциации демонстрируют высокую ингибирующую активность в реакции фибриллизации инсулина по сравнению с металлокомплексами, агрегирующими хуже. Фталоцианиновые самоассоциаты низкого порядка непосредственно не вовлечены в механизм ингибирования агрегатообразования инсулина, т. е. фталоцианин связывается с белком в мономерной форме. Тенденцию к самоассоциации фталоцианинов в водной среде можно рассматривать как «индикатор» их склонности ко взаимодействию с ароматическими аминокислотами и, следовательно, как показатель их антифибриллогенной активности. en Інститут молекулярної біології і генетики НАН України Вiopolymers and Cell Structure and Function of Biopolymers Towards the anti-fibrillogenic activity of phthalocyanines with out-of-plane ligands: correlation with self-association proneness Антифібрилогенна активність фталоціанінів з позаплощинними лігандами: кореляція зі схильністю до самоасоціації Антифибриллогенная активность фталоцианинов с внеплоскостными лигандами: корреляция со склонностью к самоассоциации Article published earlier
institution	Digital Library of Periodicals of National Academy of Sciences of Ukraine
collection	DSpace DC
title	Towards the anti-fibrillogenic activity of phthalocyanines with out-of-plane ligands: correlation with self-association proneness
spellingShingle	Towards the anti-fibrillogenic activity of phthalocyanines with out-of-plane ligands: correlation with self-association proneness Kovalska, V.B. Losytskyy, M.Yu. Chernii, S.V. Chernii, V.Ya. Tretyakova, I.M. Yarmoluk, S.M. Volkov, S.V. Structure and Function of Biopolymers
title_short	Towards the anti-fibrillogenic activity of phthalocyanines with out-of-plane ligands: correlation with self-association proneness
title_full	Towards the anti-fibrillogenic activity of phthalocyanines with out-of-plane ligands: correlation with self-association proneness
title_fullStr	Towards the anti-fibrillogenic activity of phthalocyanines with out-of-plane ligands: correlation with self-association proneness
title_full_unstemmed	Towards the anti-fibrillogenic activity of phthalocyanines with out-of-plane ligands: correlation with self-association proneness
title_sort	towards the anti-fibrillogenic activity of phthalocyanines with out-of-plane ligands: correlation with self-association proneness
author	Kovalska, V.B. Losytskyy, M.Yu. Chernii, S.V. Chernii, V.Ya. Tretyakova, I.M. Yarmoluk, S.M. Volkov, S.V.
author_facet	Kovalska, V.B. Losytskyy, M.Yu. Chernii, S.V. Chernii, V.Ya. Tretyakova, I.M. Yarmoluk, S.M. Volkov, S.V.
topic	Structure and Function of Biopolymers
topic_facet	Structure and Function of Biopolymers
publishDate	2013
language	English
container_title	Вiopolymers and Cell
publisher	Інститут молекулярної біології і генетики НАН України
format	Article
title_alt	Антифібрилогенна активність фталоціанінів з позаплощинними лігандами: кореляція зі схильністю до самоасоціації Антифибриллогенная активность фталоцианинов с внеплоскостными лигандами: корреляция со склонностью к самоассоциации
description	Aim. The activity of five hafnium phthalocyanines containing out-of-plane ligands as inhibitors of reaction of insulin fibril formation is studied and correlation between their inhibitory properties and tendency to self-association is discussed. Methods. Fluorescence and absorption spectroscopy. Results. For the complexes with weak proneness to self-association PcHfDbm2, PcHfPyr2, and PcHfBtfa2 the values of inhibitory activity were estimated as 60–73 %. For phthalocyanines with the pronounced tendency to self-association PcHfPiromelit and PcHfCl2 the noticeably higher inhibitory activity values (about 95 %) were shown. In the presence of native or fibrilar insulin the destruction of self-associates of metal complex occurs in buffer pH 7.9, Besides upon the conditions of insulin fibrillization reaction (0.1 M HCl) phthalocyanines exist predominantly as monomers. Conclusions. The phthalocyanines with out-of-plane ligands with higher tendency to self-association have shown higher inhibitory activity in the insulin fibril formation comparing with the poorly aggregated metal complexes. At the same time low-order self-associates are not involved directly in the mechanism of inhibition of insulin fibrillization and the phthalocyanines bind with protein in monomeric form. Tendency of phthalocyanines to self-association in aqueous media seems to be an «indicator» of their proneness to stack with protein aromatic amino-acids and thus of anti-fibrilogenic properties. Мета. Дослідити активність п’яти фталоціанінів гафнію, що містять позаплощинні ліганди як інгібітори реакції фібрилоутворення інсуліну, та встановити кореляцію між інгібіторними властивостями фталоціанінів і їхньою схильністю до самоасоціації. Методи. Флуоресцентна спектроскопія і спектроскопія електронного поглинання. Результати. Для комплексів PcHfDbm2, PcHfPyr2 і PcHfBtfa2, які практично не утворюють агрегатів у водному буфері, інгібіторна активність становить 60–73 %. Фталоціаніни PcHfPiromelit і PcHfCl2 з високою схильністю до утворення агрегатів практично повністю пригнічують формування амілоїдних фібрил інсуліну (інгібіторна активність приблизно 95 %). У водному буфері за присутності мономерного або фібрилярного інсуліну відбувається руйнування агрегатів металокомплексів. Крім того, за умов проведення реакції фібрилізації інсуліну (0,1 М HCl) фталоціаніни існують переважно у вигляді мономерів. Висновки. Фталоціаніни з позаплощинними лігандами з тенденцією до самоасоціації демонструють вищу інгібувальну активність у реакції фібрилізації інсуліну порівняно з металокомплексами, які агрегують гірше. Фталоціанінові самоасоціати низького порядку безпосередньо не залучені до механізму інгібування агрегатоутворення інсуліну, тобто фталоціанін зв’язується з білком у мономерній формі. Тенденцію до самоасоціації фталоціані- нів у водному середовищі можна розглядати як «індикатор» їхньої схильності до взаємодії з ароматичними амінокислотами і, отже, як показник їхньої антифібрилогенної активності. Цель. Исследовать активность пяти фталоцианинов гафния, содержащих внеплоскостные лиганды в качестве ингибиторов реакции фибриллообразования инсулина, и установить корреляцию между их ингибиторными свойствами и склонностью к самоассоциации. Методы. Флуоресцентная спектроскопия и спектроскопия электронного поглощения. Результаты. Для комплексов PcHfDbm2, PcHfPyr2 и PcHfBtfa2, практически не образующих агрегатов в водном буфере, ингибиторная активность составляет 60–73 %. Фталоцианины PcHfPiromelit и PcHfCl2 с высокой склонностью к образованию агрегатов практически полностью подавляют формирование амилоидных фибрилл инсулина (ингибиторная активность около 95 %). В водном буфере в присутствии мономерного или фибриллярного инсулина происходит разрушение агрегатов металлокомплексов. Кроме того, в условиях проведения реакции фибриллизации инсулина (0,1 М HCl) фталоцианины существуют преимущественно в виде мономеров. Выводы. Фталоцианины с внеплоскостными лигандами с тенденцией к самоассоциации демонстрируют высокую ингибирующую активность в реакции фибриллизации инсулина по сравнению с металлокомплексами, агрегирующими хуже. Фталоцианиновые самоассоциаты низкого порядка непосредственно не вовлечены в механизм ингибирования агрегатообразования инсулина, т. е. фталоцианин связывается с белком в мономерной форме. Тенденцию к самоассоциации фталоцианинов в водной среде можно рассматривать как «индикатор» их склонности ко взаимодействию с ароматическими аминокислотами и, следовательно, как показатель их антифибриллогенной активности.
issn	0233-7657
url	https://nasplib.isofts.kiev.ua/handle/123456789/153666
citation_txt	Towards the anti-fibrillogenic activity of phthalocyanines with out-of-plane ligands: correlation with self-association proneness / V.B. Kovalska, M.Yu. Losytskyy, S.V. Chernii, V.Ya. Chernii, I.M. Tretyakova, S.M. Yarmoluk, S.V. Volkov // Вiopolymers and Cell. — 2013. — Т. 29, №. 6. — С. 473-479. — Бібліогр.: 12 назв. — англ.
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fulltext	UDC 577.336 + 667.287.4 Towards the anti-fibrillogenic activity of phthalocyanines with out-of-plane ligands: correlation with self-association proneness V. B. Kovalska1, M. Yu. Losytskyy1, S. V. Chernii1, V. Ya. Chernii2, I. M. Tretyakova2, S. M. Yarmoluk1, S. V. Volkov2 1Institute of Molecular Biology and Genetics, NAS of Ukraine 150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680 2Vernadsky Institute of General and Inorganic Chemistry, NAS of Ukraine 32/34, Prospekt Akademika Palladina, Kyiv, Ukraine, 03142 v.kovalska@gmail.com Aim. The activity of five hafnium phthalocyanines containing out-of-plane ligands as inhibitors of reaction of insulin fibril formation is studied and correlation between their inhibitory properties and tendency to self- association is discussed. Methods. Fluorescence and absorption spectroscopy. Results. For the complexes with weak proneness to self-association PcHfDbm 2 , PcHfPyr 2 , and PcHfBtfa 2 the values of inhibitory activity were estimated as 60–73 %. For phthalocyanines with the pronounced tendency to self-association PcHfPiromelit and PcHfCl 2 the noticeably higher inhibitory activity values (about 95 %) were shown. In the presence of native or fibrilar insulin the destruction of self-associates of metal complex occurs in buffer pH 7.9, Besides upon the conditions of insulin fibrillization reaction (0.1 M HCl) phthalocyanines exist predominantly as monomers. Conclusions. The phthalocyanines with out-of-plane ligands with higher tendency to self-association have shown higher inhibitory activity in the insulin fibril formation comparing with the poorly aggregated metal comp- lexes. At the same time low-order self-associates are not involved directly in th] mechanism of inhibition of in- sulin fibrillization and the phthalocyanines bind with protein in monomeric form. Tendency of phthalocyanines to self-association in aqueous media seems to be an «indicator» of their proneness to stack with protein aro- matic amino-acids and thus of anti-fibrilogenic properties. Keywords: amyloid fibril, phthalocyanines, inhibitors of fibril formation, insulin, aggregation of phthalo- cyanines, fluorescent dye. Introduction. Pathogenesis of many diseases, inclu- ding neurodegenerative disorders (Alzheimer’s, Parkin- son’s, Huntington’s chorea) is accompanied by forma- tion of the �-pleated aggregates of proteins, so-called amyloid fibrils [1]. In each of these pathological con- ditions specific proteins or protein fragments alter the natural soluble form to insoluble fibrils that accumulate in various organs and tissues [2]. Among the compounds able to suppress the amyloid fibril formation, planar macrocyclic metal complexes – phthalocyanines were reported [3]. Due to their high in- hibitory activity, low toxicity and ability to overcome blood-brain barrier [4], the compounds of this class are considered as prospective anti-fibrillogenic agents. Among the properties that could affect the anti-fib- rillogenic and anti-prion activity of phthalocyanines is the tendency of metal complex to self-association [5, 6]. Lamberto et al. have shown that the differences in binding capacity and anti-amyloid activity of phthalo- cyanines towards alpha-synuclein are attributed to their relative ability to self-stack through �-� interactions mo- dulated by the nature of a metal ion. The low order sta- cked aggregates of phthalocyanines were identified as the active amyloid inhibitory species [5]. 473 ISSN 0233–7657. Biopolymers and Cell. 2013. Vol. 29. N 6. P. 473–479 doi: 10.7124/bc.00083C � Institute of Molecular Biology and Genetics, NAS of Ukraine, 2013 474 Recently the anti-fibrillogenic activity of phthalo- cyanines with out-of-plane ligands has been reported [7]. In contrast to usually planar porphyrine and phtha- locyanine molecules these metal complexes have a ste- ric content due to the presence of out-of-plane ligands bound to the central metal atom. These complexes are able to inhibit the reaction of insulin fibrillization and to redirect its pathway to the formation of oligomeric aggregates [7]. The current work is aimed at studying the inhibitory activity of series of five hafnium phthalocyanines contai- ning out-of-plane ligands of various chemical nature (Fig. 1) in the reaction of insulin fibrillization and at in- quiring into the role of phthalocyanines self-association in anti-fibrillogenic properties. The inhibitory activity of metal complexes is determined using earlier reported fluorescent cyanine based inhibitory assay [8]. For the study on phthalocyanines self-association the absorp- tion spectra were obtained for both free metal comple- xes and in the presence of native and fibrillar insulin. Materials and methods. Hafnium phthalocyanines with out-of-plane ligands were synthesized as described in [9–11]. These compounds were of intensive green– blue color and had moderate solubility in DMF. Phtha- locyanines were stable under the experimental condi- tions. Composition and structure of complexes were con- firmed by 1H NMR and element analysis on metal. Stock solutions of phthalocyanines in concentration of 2 mM were prepared by dissolving the compound in DMF. Amyloid-specific cyanine dye 7519 [12] was kindly pro- vided by Dr. O. I. Tolmachev and Dr. Yu. L. Slominskii (Institute of Organic Chemistry of NAS of Ukraine). 2 mM dye stock solution was prepared in DMF. Insulin fibril formation. Human insulin (Private Joint Stock Company «On the production of insulin «Indar», Ukraine) was dissolved at 340 µM concentration in 100 mM water solution of HCl (pH 2). Fibrils were for- med by incubating the protein solution in a water bath at 65 °C for about 5 h. Aliquots of the reaction mixture were withdrawn from each tube at about 90, 150, 240, and 300 min intervals to allow spectral measurements using dye 7519. For this, 10 µl aliquots of the aggrega- tion mixture were added to 1 ml of a 2 µM dye solution in 50 mM Tris-HCl buffer, pH 7.9, insulin concentra- tion was thus 3.4 µM [7]. The dye 7519 was earlier shown to bind specifically to the fibrillar form of in- sulin accompanied with sharp increase in fluorescence intensity. Since the dye specifically binds predominant- ly to the aggregated form of insulin (both fibrillar and oligomeric) we believe the dye fluorescence intensity to be an adequate characteristic to estimate the quantity of aggregated protein (the free dye demonstrates much lower emission intensity as compared to its bound form). All spectroscopic measurements were made immediate- ly after mixing the protein and dye solutions. Fluores- cence spectra were registered using fluorescent spectro- photometer Cary Eclipse («Varian», Australia). Inhibitory assay. The phthalocyanines PcHfPirome- lit, PcHfDbm2, PcHfPyr2, PcHfBtfa2 and PcHfCl2 were added to the corresponding insulin solution right prior KOVALSKA V. B. ET AL. N N N N N N N N Hf O O Ph Ph O O Ph Ph N N N N N N N N Hf O O CF 3 Ph O O CF 3 Ph N N N N N N N N Hf N N Ph O O Ph N N Ph O O Ph N N N N N N N N Hf O O O O OH O OH O N N N N N N N N Hf ClCl A C B D E Fig. 1. Chemical structure of hafnium phthalocyanine with out-of-plane ligands: dibenzo- ylmethane (A – PcHfDbm 2 ); pyromellitic acid (B – PcHf Piromelit); benzoyl-1,1,1-tri- fluoroacetone (C – PcHfBtfa 2 ); 1-phenyl-3-methyl-4-benzoyl- pyrazolone-5 (D – PcHfPyr 2 ); chlorine atoms (E – PcHfCl 2 ) the beginning of aggregation reaction. The used con- centration of monomeric insulin was 340 µM, the con- centration of inhibitors was 100 µM. The procedure of insulin fibrillization was performed, and the quantity of the formed aggregates was monitored using the 7519 dye fluorescence as described above. Inhibitory activity was defined as (1 – I/I0) �100 %, where I0 and I are the 7519 fluorescence intensity va- lues for inhibitor-free and inhibitor-containing insulin solutions respectively. The ratio I/I0 was considered to be an adequate estimation of quantity of protein aggre- gated in the presence of certain inhibitor concentration Cinh as compared to noninhibited fibrillization pro- cess. The inhibitor-free sample was used as aggrega- tion reference; corresponding aliquot of DMF was ad- ded to this sample to exclude the solvent effect. Absorption spectroscopy. Absorption spectra were recorded using Specord M-40 spectrophotometer («Carl Zeiss», Germany). Phthalocyanines were studied in 50 mM Tris-HCl buffer, pH 7.9 and in 0.1 M HCl (con- centration range 0.5–10 µM); in DMSO (concentration 5 �M); and in the presence of 34 µM native or fibrillar insulin (concentration 10 µM). Results and discussion. Inhibitory activity of phtha- locyanines in insulin fibrillization reaction. The kine- tics of insulin fibrillization in the presence and in the ab- sence of phthalocyanines was monitored by the chan- ges in fluorescence intensity of amyloid-sensitive dye 7519 according to the method described earlier [6]. With the passing of insulin fibrillization reaction we observed the changes in dye fluorescence intensity upon addition of inhibitor-free samples and samples containing compounds PcHfDbm2, PcHfBtfa2, PcHfPyr2 (Fig. 2). The presence of samples containing complexes PcHfPiromelit and PcHfCl2 slightly affected the fluores- cence intensity of dye during the reaction pathway. Thus it could be concluded that the presence of phtha- locyanines with spatial substituents containing multip- le phenyl groups (PcHfDbm2, PcHfBtfa2, PcHfPyr2) partially suppressed the formation of insulin fibrils. For these compounds the values of inhibition efficiency (de- fined as (1 – I/I0) �100 %; see Experimental section) va- ried in the range PcHfBtfa2 (60 %) < PcHfPyr2 (66 %) < PcHfDbm2 (73 %). At the early stage (t = 150 min) of the insulin aggre- gation reaction in the presence of the sample containing phthalocyanine PcHfDbm2 it was demonstrated a high- er dye emission comparing with inhibitor-free sample. Thus, we can suppose that the presence of PcHfDbm2 promotes the formation of beta-pleated structures at the early stage comparing with inhibitor free sample. The ability of phthalocyanines with out-of-plane ligands to redirect the insulin fibrillization reaction to the forma- tion of spherical aggregates was shown earlier. A higher inhibitory activity was noted for phthalo- cyanines PcHfPiromelit and PcHfCl2. Their presence al- most completely suppressed the formation of both beta- pleated insulin aggregates at the early stages of reaction and amyloid fibrils. The final inhibitory effects for the- se complexes were about 94 and 96 % correspondingly. This observation points to the dependence of inhi- bitory activity of phthalocyanines on the chemical na- ture of out-of-plane ligand. Thus the conclusion can be made about high antifibrillogenic properties of the complexes with «small size» ligand (PcHfCl2) or ligand containing carboxy group (PcHfPiromelit) that allows the electrostatic interaction or hydrogen bonds forma- tion with protein aminoacids. Determination of the efficient inhibitory concentra- tion for PcHfPiromelit. Phthalocyanine PcHfPiromelit demonstrated a high anti-fibrillogenic activity in the insulin fibrillization reaction, thus for this complex the 475 TOWARDS THE ANTI-FIBRILLOGENIC ACTIVITY OF PHTHALOCYANINES 0 50 100 150 200 250 300 -100 0 100 300 500 700 F lu o re sc en ce in te n si ty , a . u . Time, min Fig. 2. Effects of presence of PcHfPiromelit (2), PcHfBtfa 2 (3), PcHf Pyr 2 (4), PcHfDbm 2 (5) and PcHfCl 2 (6) on the kinetics of insulin fib- rils formation, plotted with phthalocyanine-free insulin control (K – 1). The reaction mixture containing 340 µM of insulin in 0.1 M water solution of HCl and 100 µM of corresponding inhibitor was incubated at 65 °C for 5 h. Fibril formation efficiency was assessed with fluores- cence intensity of dye 7519 using 2 µM concentration. Experiment was performed three times. Standard deviation of the mean is presented as the error bars efficient inhibitor concentration was identified accor- ding to the method described in [6]. The efficient inhi- bitor concentration is estimated as a concentration of phthalocyanine at which the dye fluorescence intensity is half concentraion for the inhibitor free solution. It is shown that the inhibitory effect enhances with increa- sing concentration of metal complex in the reaction mix- ture. The dependence of I/I0 on C(inh) for PcHfPirome- lit is presented in Fig. 3. Approximation of experimental dependence of I/I0 on C(inh) with the sigmoid curve gives the efficient inhibi- tor concentration IC50 which characterizes the inhibi- tion of the fibrillization reaction by the studied metal com- plex. The efficient inhibitor concentration for PcHfPiro- melit was determined as IC50 = 2,8 � 0,6 µM, that is more that one order lower than the corresponding value (obtai- ned for inhibition of bovine insulin) for zirconium phtha- locyanine containing out-of-plane lysine ligands PcZr Lys2 (IC50 = 37 �0,6 µM), determined by us earlier [6]. Aggregation of phthalocyanines in buffer, pH 7.9, and DMSO. To study the aggregation ability of phthalo- cya nines, we recorded their absorption spectra in polar solvent DMSO and in 0.05 M Tris-HCl buffer (pH 7.9) (Fig. 4). In the spectra of complexes in DMSO two bands we- re observed, short-wavelength vibronic satellite band with maximum near 615 nm and sharp intensive long- wavelength band with maxima on 679–693 nm. The profiles of spectra and band positions are typical for mo- nomeric phthalocyanines. In absorption spectra of complexes in the buffer the bands are much wider than in DMSO. They contain two bands, short-wavelength band or shoulder in the range 642–646 nm and long-wavelength one in the range 685– 700 nm. It is shown that for PcHfBtfa2 (Fig. 4, B), PcHfD bm2, and PcHfPyr2 the long-wavelength band is the most pronounced and that an increase in the complex concen- tration does not lead to redistribution of intensities between the absorption bands. This behavior points to a low propensity of phthalocyanines to self-associa- tion. For these complexes the absorption spectra corres- pond to the monomeric phthalocyanine molecules, the short-wavelength band could be attributed to the vibro- nic transition. The inhibitory activity of these comple- xes varies in the range 60–73 %. In spectra of phthalocyanines PcHfPiromelit (Fig. 4, A) and PcHfCl2 in buffer, the relative intensity of short-wavelength band increases at higher complex con- centration, while that of long-wavelength band decrea- ses. Starting from about 5 �M concentration the short- wavelength band dominates in the spectra. Such beha- vior is an evidence of high tendency of the PcHfPirome- lit and PcHfCl2 metal complexes to self-association. In this case the short-wavelength band belongs to aggrega- tes of phthalocyanines, while the long-wavelength one corresponds to their monomers. At the same time, these complexes demonstrated a very high inhibitory activity – 96 % for PcHfPiromelit and 94 % for PcHfCl2. In this way we observed a higher anti-fibrillogenic activity for phthalocyanines with pronounced tendency to self-association. An addition of native or fibrillar insulin to the comp- lex with high inhibitory activity PcHfPiromelit leads to the significant decrease of short-wavelength aggrega- tion band and increase of long-wavelength monomeric one (Fig. 5, A). Thus it could be concluded that phtha- locyanine interacts with both native and fibrillar prote- ins and this interaction leads to the destruction of metal complex self-associates. It is supposed that phthalocya- nine molecules bind to the surface of protein in mono- meric form. 476 KOVALSKA V. B. ET AL. 1E-3 0,01 0,1 1 10 100 1000 10000 100000 -0.1 0.0 0.1 0.3 0.5 0.7 0.9 I/I0 C(inh), µM Fig. 3. Dose-dependent inhibition of insulin aggregates formation by PcHfPiromelit. The reaction mixtures containing 340 µM of insulin in 100 mM HCl, and 0, 0.5, 2, 10, or 100 µM PcHfPiromelit were incu- bated at 65 °C for 5 h. Fibrillogenesis efficiency at the given inhibitor concentration was assessed with 7519 fluorescence emission (I). The emission intensity in the absence of inhibitor (I 0 ) was regarded as 100 %. Experimental dependence (squares) was approximated by the sigmoid dependence (line). The approximation parameter X 0 equals (Y = 1/(1 + + (x/x 0 ) � dx); x 0 = 2.8 � 0.6; dx = 0.7 � 0.1) to the efficient inhibitor con- centration Aggregation of phthalocyanines upon conditions of fibrillization reaction. Since the reaction of insulin fibril- lization is performed in acidic media – 0.1 M HCl, the aggregation tendency of phthalocyanines in these con- ditions was studied. In absorption spectra of all studied metal complexes in acidic media the short-wavelength band (near 650 nm) was observed as less intensive shoulder, while the long-wavelength band (maximum about 700 nm) was the most pronounced. Besides, an in- crease in the phthalocyanines concentration up to 10 µM did not lead to the redistribution between intensities of short- and long-wavelength bands (Fig. 6). Thus, the ob- served absorption spectra belong mainly to monomeric form of the phthalocyanine. Therefore, we can suppose that irrespectively of the prone of phthalocyanines to self-association in aqueous buffer at neutral or low-ba- sic pH, these complexes did not form aggregates in aci- dic media at low pH. It is shown that in acidic media for phthalocyanine with high inhibitory activity PcHfPiromelit the presen- ce of native or fibrilar insulin slightly affects the rela- tion between intensities of short- and long-wavelength absorption bands (Fig. 5, B) and thus – the aggregation degree of metal complex. Hence, we can conclude that in acidic media the me- tal complexes exist mostly in monomeric form and their interaction with proteins does not cause the additional des truction of phthalocyanine self-associates or forma- tion of associates on protein surface. The axial coordination with imidazole ring of histi- dine was reported among the mechanisms of complex formation of Zn(II) phthalocyanine tetrasulfonate mole- cules and protein -synuclein [4]. For studied phtha- locyanines we can exclude the possibility of their che- mical binding to aminoacids due to the nature of metal atoms and the presence of spatial out-of-plane ligands. 477 TOWARDS THE ANTI-FIBRILLOGENIC ACTIVITY OF PHTHALOCYANINES 600 650 700 750 800 0.0 0.2 0.4 0.6 0.8 1.0 1.2 A b s Wavelength, nm 600 650 700 750 800 0.0 0.4 0.8 1.2 1.6 A b s Wavelength, nm A B Fig. 4. Absorption spectra PcHfPiromelit (A) and PcHfBtfa 2 (B). Spectra are shown for concentrations of A: 1 – 0.5 µM; 2 – 1 µM; 3 – 2 µM; 4 – 5 µM; 5 – 8 µM; 6 – 10 µM; 7 – 5 µM, DMSO; B: 1 – 1 µM; 2 – 2 µM; 3 – 5 µM; 4 – 8 µM; 5 – 10 µM; 6 – 5 µM, DMSO in Tris-HCl (pH 7.9) and 5 µM in DMSO 550 600 650 700 750 800 -0.1 0.0 0.1 0.2 0.3 0.4 0.5 A b s Wavelength, nm 550 600 650 700 750 800 0.0 0.1 0.2 0.3 0.4 0.5 0.6 In te n si ty Wavelength, nm A B Fig. 5. Absorption spectra of PcHfPiromelit (1, concentration 10 µM) in the presence of native (2) and fibrillar insulin (3, concentration 34 µM) in buffer (A) and in 0.1 M HCl (B) We propose that high self-association ability of phtha- locyanines in aqueous media points to a strong stacking interaction between molecules of the metal complex. In the presence of protein the stacking of phthalocyanine molecules with aromatic aminoacids could become mo- re preferable than self-stacking, that results in the des- truction of phthalocyanine aggregates and fixation of the metal complex molecules on the surface of protein. We could further suppose that even in acidic media whe- re the aggregation does not occur, the stronger stacking between metal complexes and aromatic amino-acids still takes place leading to the inhibition of insulin fib- rillization At the same time, the compounds with less pronoun- ced tendency to stacking interactions poorly form self- associates and weakly bind with aromatic amino-acids. Therefore, the tendency of phthalocyanines to self- association in aqueous media is considered as «an in- dicator» of their ability to stack with aromatic amino- acids of protein and thus their anti-fibrilogenic activity. Conclusions. Series of hafnium phthalocyanines containing out-of-plane ligands were firstly studied as anti-fibrillogenic agents using the fluorescent cyanine dye inhibitory assay. For the compounds with weak proneness to self- association in aqueous media (PcHfDbm2, PcHfPyr2, PcHfBtfa2) the value of inhibitory activity varies in the range 60–73 %. For the complexes with the pronoun- ced tendency to self-association in water solution (PcHfPiromelit and PcHfCl2) the inhibitory activity was noticeably higher (about 95 %). For the complex PcHfPiromelit the micromolar efficient inhibitor con- centration (IC50 = 2.8 � 0.6 µM) in the insulin fibril- lization reaction was determined. The addition of native or fibrilar insulin to the ag- gregation-prone phthalocyanines in aqueous buffer leads to the destruction of self-associates of metal complex. We can suppose that phthalocyanines interact with pro- tein through the stacking with aromatic aminoacids in monomeric form. Upon the conditions of insulin fibril- lization reaction (0.1 M HCl) phthalocyanines exist pre- dominantly as monomers, the addition of native or fib- rilar insulin does not affect the degree of self-asso- ciation of metal complexes. Thus, we conclude that low-order self-associates are not involved directly in the mechanism of inhibition of insulin fibrillization, phthalocyanines bind with pro- tein in monomeric form through the stacking to it aro- matic amino-acids. We suggest that the tendency of phthalocyanines to self-association in aqueous media is «an indicator» of the proneness of metal complex mo- lecules to stack with aromatic amino-acids of protein and thus of their anti-fibrilogenic activity. Acknowledgements. This work was supported by STCU-NASU project N 5508. Â. Á. Êîâàëüñüêà, Ì. Þ. Ëîñèöüêèé, Ñ. Â. ×åðí³é, Â. ß. ×åðí³é, ². M. Òðåòüÿêîâà, Ñ. Ì. ßðìîëþê, Ñ. Â. Âîëêîâ Àíòèô³áðèëîãåííà àêòèâí³ñòü ôòàëîö³àí³í³â ç ïîçàïëîùèííèìè ë³ãàíäàìè: êîðåëÿö³ÿ ç³ ñõèëüí³ñòþ äî ñàìîàñîö³àö³¿ Ðåçþìå Ìåòà. Äîñë³äèòè àêòèâí³ñòü ï’ÿòè ôòàëîö³àí³í³â ãàôí³þ, ùî ì³- ñòÿòü ïîçàïëîùèíí³ ë³ãàíäè ÿê ³íã³á³òîðè ðåàêö³¿ ô³áðèëîóòâî- ðåííÿ ³íñóë³íó, òà âñòàíîâèòè êîðåëÿö³þ ì³æ ³íã³á³òîðíèìè âëà- ñòèâîñòÿìè ôòàëîö³àí³í³â ³ ¿õíüîþ ñõèëüí³ñòþ äî ñàìîàñîö³à- 478 KOVALSKA V. B. ET AL. 550 600 650 700 750 800 0.0 0.2 0.4 0.6 0.8 1.0 A b s Wavelength, nm 550 600 650 700 750 800 850 0.0 0.1 0.2 0.3 0.4 0.5 0.6 A b s Wavelength, nm A B Fig. 6. Absorption spectra of PcHfPiromelit (A) and PcHfCl2 (B) in 0.1 M HCl. Spectra are shown for concentrations of: 1 – 0.5 µM; 2 – 1 µM; 3 – 2 µM; 4 – 5 µM; 5 – 8 µM; 6 – 10 µM ö³¿. Ìåòîäè. Ôëóîðåñöåíòíà ñïåêòðîñêîï³ÿ ³ ñïåêòðîñêîï³ÿ åëåê- òðîííîãî ïîãëèíàííÿ. Ðåçóëüòàòè. Äëÿ êîìïëåêñ³â PcHfDbm2, PcHfPyr2 ³ PcHfBtfa2, ÿê³ ïðàêòè÷íî íå óòâîðþþòü àãðåãàò³â ó âîäíîìó áóôåð³, ³íã³á³òîðíà àêòèâí³ñòü ñòàíîâèòü 60–73 %. Ôòàëîö³àí³íè PcHfPiromelit ³ PcHfCl2 ç âèñîêîþ ñõèëüí³ñòþ äî óòâîðåííÿ àãðåãàò³â ïðàêòè÷íî ïîâí³ñòþ ïðèãí³÷óþòü ôîðìó- âàííÿ àì³ëî¿äíèõ ô³áðèë ³íñóë³íó (³íã³á³òîðíà àêòèâí³ñòü ïðè- áëèçíî 95 %). Ó âîäíîìó áóôåð³ çà ïðèñóòíîñò³ ìîíîìåðíîãî àáî ô³áðèëÿðíîãî ³íñóë³íó â³äáóâàºòüñÿ ðóéíóâàííÿ àãðåãàò³â ìåòà- ëîêîìïëåêñ³â. Êð³ì òîãî, çà óìîâ ïðîâåäåííÿ ðåàêö³¿ ô³áðèë³çàö³¿ ³íñóë³íó (0,1 Ì HCl) ôòàëîö³àí³íè ³ñíóþòü ïåðåâàæíî ó âèãëÿä³ ìîíîìåð³â. Âèñíîâêè. Ôòàëîö³àí³íè ç ïîçàïëîùèííèìè ë³ãàíäàìè ç òåíäåíö³ºþ äî ñàìîàñîö³àö³¿ äåìîíñòðóþòü âèùó ³íã³áóâàëüíó àêòèâí³ñòü ó ðåàêö³¿ ô³áðèë³çàö³¿ ³íñóë³íó ïîð³âíÿíî ç ìåòàëîêîìï- ëåêñàìè, ÿê³ àãðåãóþòü ã³ðøå. Ôòàëîö³àí³íîâ³ ñàìîàñîö³àòè íèçü- êîãî ïîðÿäêó áåçïîñåðåäíüî íå çàëó÷åí³ äî ìåõàí³çìó ³íã³áóâàííÿ àãðåãàòîóòâîðåííÿ ³íñóë³íó, òîáòî ôòàëîö³àí³í çâ’ÿçóºòüñÿ ç á³ë- êîì ó ìîíîìåðí³é ôîðì³. Òåíäåíö³þ äî ñàìîàñîö³àö³¿ ôòàëîö³àí³- í³â ó âîäíîìó ñåðåäîâèù³ ìîæíà ðîçãëÿäàòè ÿê «³íäèêàòîð» ¿õ- íüî¿ ñõèëüíîñò³ äî âçàºìîä³¿ ç àðîìàòè÷íèìè àì³íîêèñëîòàìè ³, îòæå, ÿê ïîêàçíèê ¿õíüî¿ àíòèô³áðèëîãåííî¿ àêòèâíîñò³. Êëþ÷îâ³ ñëîâà: àì³ëî¿äí³ ô³áðèëè, ôòàëîö³àí³íè, ³íã³á³òîðè ô³áðèëîóòâîðåííÿ, ³íñóë³í, àãðåãàö³ÿ ôòàëîö³àí³í³â, ôëóîðåñöåí- òíèé áàðâíèê. Â. Á. Êîâàëüñêàÿ, Ì. Þ. Ëîñèöêèé, Ñ. Â. ×åðíèé, Â. ß. ×åðíèé, È. Í. Òðåòüÿêîâà, Ñ. Í. ßðìîëþê, Ñ. Â. Âîëêîâ Àíòèôèáðèëëîãåííàÿ àêòèâíîñòü ôòàëîöèàíèíîâ ñ âíåïëîñêîñòíûìè ëèãàíäàìè: êîððåëÿöèÿ ñî ñêëîííîñòüþ ê ñàìîàññîöèàöèè Ðåçþìå Öåëü. Èññëåäîâàòü àêòèâíîñòü ïÿòè ôòàëîöèàíèíîâ ãàôíèÿ, ñî- äåðæàùèõ âíåïëîñêîñòíûå ëèãàíäû â êà÷åñòâå èíãèáèòîðîâ ðå- àêöèè ôèáðèëëîîáðàçîâàíèÿ èíñóëèíà, è óñòàíîâèòü êîððåëÿöèþ ìåæäó èõ èíãèáèòîðíûìè ñâîéñòâàìè è ñêëîííîñòüþ ê ñàìîàñ- ñîöèàöèè. Ìåòîäû. Ôëóîðåñöåíòíàÿ ñïåêòðîñêîïèÿ è ñïåêòðîñ- êîïèÿ ýëåêòðîííîãî ïîãëîùåíèÿ. Ðåçóëüòàòû. Äëÿ êîìïëåêñîâ PcHfDbm2, PcHfPyr2 è PcHfBtfa2, ïðàêòè÷åñêè íå îáðàçóþùèõ àã- ðåãàòîâ â âîäíîì áóôåðå, èíãèáèòîðíàÿ àêòèâíîñòü ñîñòàâëÿåò 60–73 %. Ôòàëîöèàíèíû PcHfPiromelit è PcHfCl2 ñ âûñîêîé ñêëîí- íîñòüþ ê îáðàçîâàíèþ àãðåãàòîâ ïðàêòè÷åñêè ïîëíîñòüþ ïîäàâ- ëÿþò ôîðìèðîâàíèå àìèëîèäíûõ ôèáðèëë èíñóëèíà (èíãèáèòîð- íàÿ àêòèâíîñòü îêîëî 95 %). Â âîäíîì áóôåðå â ïðèñóòñòâèè ìî- íîìåðíîãî èëè ôèáðèëëÿðíîãî èíñóëèíà ïðîèñõîäèò ðàçðóøåíèå àãðåãàòîâ ìåòàëëîêîìïëåêñîâ. Êðîìå òîãî, â óñëîâèÿõ ïðîâåäå- íèÿ ðåàêöèè ôèáðèëëèçàöèè èíñóëèíà (0,1 Ì HCl) ôòàëîöèàíèíû ñóùåñòâóþò ïðåèìóùåñòâåííî â âèäå ìîíîìåðîâ. Âûâîäû. Ôòà- ëîöèàíèíû ñ âíåïëîñêîñòíûìè ëèãàíäàìè ñ òåíäåíöèåé ê ñàìîàñ- ñîöèàöèè äåìîíñòðèðóþò âûñîêóþ èíãèáèðóþùóþ àêòèâíîñòü â ðåàêöèè ôèáðèëëèçàöèè èíñóëèíà ïî ñðàâíåíèþ ñ ìåòàëëîêîìï- ëåêñàìè, àãðåãèðóþùèìè õóæå. Ôòàëîöèàíèíîâûå ñàìîàññîöèà- òû íèçêîãî ïîðÿäêà íåïîñðåäñòâåííî íå âîâëå÷åíû â ìåõàíèçì èíãèáèðîâàíèÿ àãðåãàòîîáðàçîâàíèÿ èíñóëèíà, ò. å. ôòàëîöèà- íèí ñâÿçûâàåòñÿ ñ áåëêîì â ìîíîìåðíîé ôîðìå. Òåíäåíöèþ ê ñà- ìîàññîöèàöèè ôòàëîöèàíèíîâ â âîäíîé ñðåäå ìîæíî ðàññìàò- ðèâàòü êàê «èíäèêàòîð» èõ ñêëîííîñòè êî âçàèìîäåéñòâèþ ñ àðîìàòè÷åñêèìè àìèíîêèñëîòàìè è, ñëåäîâàòåëüíî, êàê ïîêàçà- òåëü èõ àíòèôèáðèëëîãåííîé àêòèâíîñòè. Êëþ÷åâûå ñëîâà: àìèëîèäíûå ôèáðèëëû, ôòàëîöèàíèíû, èí- ãèáèòîðû ôèáðèëëîîáðàçîâàíèÿ, èíñóëèí, àãðåãàöèÿ ôòàëîöèà- íèíîâ, ôëóîðåñöåíòíûé êðàñèòåëü. REFERENCES 1. Uversky V. N., Fink A. L. Conformational constraints for amy- loid fibrillation: the importance of being unfolded // Biochim. Biophys. Acta.–2004.–1698, N 2.–P. 131–153. 2. Kelly J. W. The alternative conformations of amyloidogenic proteins and their multi-step assembly pathways // Curr. Opin. Struct. Biol.–1998.–8, N 1.–P. 101–106. 3. Lamberto G. R., Binolfi A., Orcellet M. L., Bertonconi C. 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Kovalska V., Losytskyy M., Chernii V., Volkova K., Tretyakova I., Cherepanov V., Yarmoluk S., Volkov S. Studies of anti-fibril- logenic activity of phthalocyanines of zirconium containing out-of-plane ligands // Bioorg. Med. Chem.–2012.–20, N 1.– P. 330–334. 8. Volkova K. D., Kovalska V. B., Inshin D., Slominskii Y. L., Tol- machev O. I., Yarmoluk S. M. Novel fluorescent trimethine cya- nine dye 7519 for amyloid fibril inhibition assay // Biotech. His- tochem.–2011.–86, N 3.– P. 188–191. 9. Chernii V. Ya., Bon V. V., Tretyakova I. N., Severinovskaya O. V., Volkov S. V. Novel zirconium (IV) and hafnium (IV) phthalo- cyanines with dibenzoylmethane as out-of-plane ligand: Syn- thesis, X-ray structure and fluorescent properties // Dyes Pigm.– 2012.–94, N 2.–P. 187–194. 10. Gerasymchuk Y. S., Volkov S. V., Chernii V. Ya., Tomachynski L. A., Radzki St. Synthesis and spectral properties of axially substituted zirconium(IV) and hafnium(IV) water soluble phtha- locyanines in solutions // J. Alloys Compd.–2004.–380, N 1–2.– P. 186–190. 11. Tomachynski L. A., Tretyakova I. N., Chernii V. Ya., Volkov S. V., Kowalska M., Legendziewicz J., Gerasymchuk Y. S., Radzki St. Synthesis and spectral properties of Zr(IV) and Hf(IV) phtha- locyanines with �-diketonates as axial ligands // Inorg. Chim. Acta.–2008.–361, N 9–10.–P. 2569–2581. 12. Volkova K. D., Kovalska V. B., Losytskyy M. Yu., Fal K. O., Derevyanko N. O., Slominskii Y. L., Tolmachov O. I., Yarmoluk S. M. Hydroxy and methoxy substituted thiacarbocyanines for fluorescent detection of amiloid formation // J. Fluoresc.– 2011.–21, N 2.–P. 775–784. Received 18.03.13 479 TOWARDS THE ANTI-FIBRILLOGENIC ACTIVITY OF PHTHALOCYANINES

Towards the anti-fibrillogenic activity of phthalocyanines with out-of-plane ligands: correlation with self-association proneness

Institution

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