Ключові слова: протеїнкіназа СК2, віртуальний скринінг, гнучкий докінг, інгібітор, тип зв’язування, активний сайт.

АННОТАЦИЯ

Голуб А. Г. In silico дизайн ингибиторов протеинкиназы СК2. – Рукопись.

Диссертация на соискание ученой степени кандидата биологических наук по специальности 03.00.03 – молекулярная биология. – Институт молекулярной биологии и генетики НАН Украины, Киев, 2007.

елью работы была разработка новых низкомолекулярных ингибиторов протеинкиназы СК2 человека, исследование их взаимодействия с аминокислотными остатками активного сайта СК2 методами компьютерного моделирования. Используя метод гибкого докинга, найдено ряд новых ингибиторов СК2 среди комбинаторных библиотек 4-аминохиназолинов и 4-аминохинолинов, а также два новых класса высокоактивных специфичных ингибиторов СК2 – 3-карбокси-4-(1Н)-хинолоны и тетрагалогено-1,3-диоксо-2,3-дигидроизоиндолы. На основе результатов гибкого докинга и расчетов молекулярной динамики, для полученных ингибиторов предложена модель взаимодействия с активным сайтом СК2 и объяснено влияние их химических заместителей на биологическую активность. Изучена специфичность новых ингибиторов по отношению к СК2. На основе анализа полученных комплексов “СК2-ингибитор”, анализа соотношения “химическая структура – биологическая активность” и расчетов термодинамической интеграции спрогнозированы направления дальнейшей химической оптимизации новых ингибиторов СК2.

Ключевые слова: протеинкиназа СК2, виртуальный скрининг, гибкий докинг, ингибитор, тип связывания, активный сайт.

SUMMARY

Golub A. G. In silico design of protein kinase CK2 inhibitors. – Manuscript.

Thesis for Philosophy Doctor (PhD) degree in Biology, speciality 03.00.03 – molecular biology. – Institute of Molecular Biology and Genetics of National Academy of Sciences of Ukraine, Kyiv, 2007.

CK2 is a ubiquitous, constitutive, essential serine/threonine kinase whose physiological function remains unclear, but it has been implicated as a regulator of fundamental cellular processes. CK2 is involved in the phosphorylation of a large variety of different cellular proteins, among which are the factors of cell growth and transcription, as well as the regulators of cell cycle and apoptosis. The CK2 holoenzyme is a heterotetramer consisting of 2 catalytic subunits (б and б’) and 2 identical regulatory в-subunits. Interestingly, CK2 demonstrates “dual co-substrate specificity”, using both GTP and ATP as a phosphate donor. CK2 can phosphorylate tyrosine in addition to classic serine/threonine residues, thus this kinase possesses also dual-substrate specificity.

Abnormal CK2 expression and function is associated with a number of pathologies, including inflammatory, infectious and carcinogenic processes. It has been reported that the overexpression of CK2 is associated with cell transformation and neoplasia. It has also been revealed that some viruses use CK2 to phosphorylate functionally critical proteins encoded in their genome; consequently, the important role of CK2 in the development of viral infections has been shown. These studies evoke strong interest in this enzyme as a target for anticancer, anti-inflammatory and anti-infectious drugs. Therefore, small organic CK2 inhibitors, besides of their application in scientific research, may have therapeutic significance.

The goal of the work was to develop novel small molecule inhibitors of CK2 and to study their interactions with amino acid residues of the CK2 ATP binding site by molecular modeling methods.

At the first stage, using flexible docking and molecular dynamics simulation techniques, a number of new CK2 inhibitors were identified among combinatorial libraries of 4-aminoquinazoline and 4-aminoquinoline derivatives. 75 compounds from combinatorial library of 4-aminoquinazoline derivatives have been selected using receptor-based virtual screening technique to study their CK2 inhibitory activity. It has been shown that 26 substances inhibit CK2 activity by more than 50 %. IC50 of these compounds ranged from 7 µM to 20 µM. The most potent inhibitor, 4.57 (2-(3-methylphenyl)-4-(4-carboxyphenylamino)quinazolin, has IC50 7.7 µM. To study the binding mode of the most active 4-aminoquinazoline derivatives, their binding modes in CK2 ATP-binding site obtained with docking have been analyzed. It has been revealed, that the most important interactions are formed between quinazoline core and CK2 residues Phe113, Ile174, Val66, Met163. Especially, the stacking interaction of the core and Phe113 should be emphasized. The carboxyphenile substituents in position R3 contribute much to the inhibitory activity, while bulk substituents in position R1 are not favourable.

52 compounds from combinatorial library of 4-amino-3-carbethoxyquinoline derivatives have been selected using receptor-based virtual screening technique for studying their CK2 inhibition activity. It has been shown that 19 substances inhibit CK2 activity by more than 50 %. IC50 of these compounds ranged from 9 to 19 µM. The most potent inhibitor, 1.32 (3,6-dicarbethoxy-4-(4-acetamidophenylamino)quinoline, has IC50 9 µM. The model of binding of 4-amino-3-carbethoxy-quinoline derivatives to CK2 active site have been developed on the base of docking results. According to the model, the active 4-amino-3-carbethoxyquinolines have almost identical binding mode which is similar to the one for 4-aminoquinazolines. The clamping of the quinoline core between CK2 residues Phe113 and Ile174 has been observed. Moreover, the hydrogen bond between carbethoxy group of the inhibitors and residue Lys 68 was identified by docking engine. An additional hydrogen bond with residue Asp175 also could be potentially formed. Analyzing the structure-activity relationships of the obtained 4-amino-3-carbethoxyquinoline derivatives it was revealed that the position R3 is most critical for their inhibitory activity. The activity is highest when R3 = COOEt. The substituents in position R1 are also make significant contribution to the activity due to their ability to form hydrogen bonds with CK2 active site residues.

At the next stage of the investigations, using receptor-based virtual screening, a new class of highly-active and selective CK2 inhibitors - 3-carboxy-4-(1H) quinolones was identified. It has been revealed that the most active compounds of the class, 5,6,8-trichloro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (IP7) (IC50 = 0.3 µM) and 4-oxo- 1,4-dihydrobenzo[h]quinoline-3-carboxylic acid (IP9) (IC50 = 1 M), are ATP competitive (Ki values are 0.06 and 0.28 µM, respectively). Evaluation of the inhibitors on seven serine/threonine and tyrosine kinases shows their considerable selectivity toward CK2.

For the most active 3-carboxy-4-(1H) quinolone derivatives, IP7 and IP9, molecular dynamics of their complexes with CK2 were studied, and their binding mode models have been proposed. Molecular dynamics simulation revealed, that their binding modes in CK2 active site are somewhat different. The binding mode of IP7 characterized by set of hydrophobic contacts, among them the stacking interaction with Phe113 and contacts with Val66, Ile174 are the most crucial. The hydrogen bonds with residues Lys68, Asp175 and Glu81 were observed in case of IP7. The binding mode of IP9 is distinguished by the absence of stacking interaction with Phe113 and by formation of the hydrogen bonds only with Lys68 and Asp175. The obtained binding modes were compared with those of known CK2 inhibitors (IQA, TBB and its derivatives). The role of carboxyl group and other substituents was explained for these novel CK2 inhibitors. On the base of docking complexes analysis, structure-activity relationships and thermodynamic integration calculations, further directions of 3-carboxy-4-(1H) quinolones structure optimization were predicted.

With the virtual screening of large and diverse compound library (70,000 compounds) the other new class of CK2 inhibitors, tetrahalogeno-1,3-dioxo-2,3-dihydroisoindoles (TID), has been identified. In vitro tests revealed that among TID, the most active compounds are 2-(4,5,6,7-tetrabromo-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)propanoic acid (2.46) and 2-(4,5,6,7-tetrabromo-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)acetic acid (2.43) (IC50 are 0,15 µM and 0,3 µM respectively). The inhibitors are ATP competitive and they only minimally inhibit the activity of protein kinases DYRK1a, MSK1, GSK3 and CDK5. On the base of docking technique, the binding mode of the TID in CK2 active site has been proposed. According to the binding mode, the most critical contacts are hydrogen bonds of the TID with residues Lys68, Trp176 and hydrophobic contacts with Val53, Val66, Ile174 and Phe113. The proposed model agrees with X-ray data on complexes CK2-TBB and TBB derivatives.

The new CK2 inhibitors and their derivatives obtained during this investigation could be used for further structural optimization and biological testing. On the base of these inhibitors, new biologically active substances could be developed and implemented in biological research and clinical practice.

Key words: proteinkinase СК2, virtual screening, flexible docking, inhibitor, binding mode, active site.