Platinum cytostatic agents are a class of extensively studied DNA-damaging compounds which includes potent platinum antitumour drugs (cisplatin, carboplatin and oxaliplatin). Biological activities (antitumor properties, tissue-specific cell resistance, general and specific toxicities) depend strongly structure of coordination sphere of platinum drug. Yet the relationship between structure and activity for platinum drugs is still poorly understood, and many controversies exist concerning the biological role of reactions of platinum drugs with non-DNA targets.
We develop the hypothesis, that the biological activities of a platinum complex depend on the competition between reactions of the (pro)drug with DNA, sulfur-containing groups and inorganic anions (Figure). For this assumption kinetic studies, aiming to determine rate constants of the concurrent processes are the most important. Most information about the mechanisms of platinum drugs with potential biological studies still comes from the physicochemical studies of model systems. So we focus our studies on the mechanisms of interaction of platinum complexes with methionine and its derivatives in direct competition with DNA and the rates of drug inactivation by sulfur-donor groups, as well as interactions of platinum complexes with proteins. For the reasons of low concentrations, relatively high rates of reactions and large molecular mass of interacting biopolymers there is sometimes no options except optical spectroscopy. Usually the bands in optical spectra strongly overlap, which hinders quantitative analysis and extraction of information of physicochemical importance. In our studies we try to overcome this obstacle by applying chemometric techniques - principal component analysis and multivariate curve resolution. The method was applied to study kinetics of the reactions of cisplatin with several model proteins, including core histone proteins. It has been shown that protein platination is also rate-limited by cisplatin hydrolysis and methionine residues are the primary (and in many systems the only) targets for protein platination. DNA-damaging properties of cisplatin is damped by L-methionine in concentration dependent and time dependent way, indicating for a strong non-equilibrium nature of the competition between DNA and Met. The crucial step for inactivation is possibly chelation of methionine, which has characteristic time of 1-2 hours at 37°C.

Synthesis, structure and reactivity of platinum and copper complexes with chiral ligands

Dr. Alexey SKVORTSOV
(in collaboration with Prof. Studentsov E.P., Prof. Skvortsov N.K., Dr. de Vekki D.A., St-Petersburg State Institute of Technology)

One of the main obstacles in studying reactions of platinum complexes in biologically relevant conditions (submicromolar concentrations of platinum) is the absence of characteristic properties in platinum drugs, which limits the number of available techniques. Optical absorption spectroscopy in UV-bands of platinum drugs offers good sensitivity, but it has obvious limitation, as most biomolecules also absorb in UV region. One of the solutions, proposed earlier by our group was the usage of optically active platinum complexes with strong stereogenic centers, which are close structural analogs of platinum drugs. Strong circular dichroism (CD) bands of such ligands, which are easily tractable serve as a 'chiral label' for reaction studies.
Methods for synthesis of ligands with stereogenic centers and/or platinum complexes were developed. The structure of the products was confirmed by X-ray crystallographic analysis, NMR-spectroscopy, UV and CD spectroscopy (Figure). The first ligand is a well known asymmetric sulfoxide Me-p-TolSO, which readily produce biologically active complexes. Possibility to use sulfoxide label for kinetic and equilibrium studies was firmly shown. The second is a binaphtile S-NOBIN, which has much stronger optical activity, but produced complexes with low solubility in water. We also studied 1,3-thiazolidines (produced from cysteine) and oxidized methionine derivatives.

Additional info:  http://biophysics.spbstu.ru/node/57

Financial Support: RFBR-BRFBR grant 10-04-90048-Bel; President grant MK-5692.2008.4

Publications

Skvortsov A.N., Uvarov V. M., de Vekki D. A., Studentsov E. P., Skvortsov N. K. (2010) Conformational Analysis, Spectral and Catalytic Properties of 1,3-Thiazolidines, Ligands for Acetophenone Hydrosilylation with Diphenylsilane // J. Gen. Chem. Rus. 80 (10): 2007-2021.

Skvortsov A.N., Spevak V. N., Studentsov E.P., Sokolova O.V., Skvortsov N.K. (2010) Synthesis and Spectral Properties of Mixed-Ligand Platinum(II) Complexes with (-)-S-NOBIN and Sulfoxides // J. Gen. Chem. Rus. 80 (10): 1963-1971.

Studentsov E. P., Piskunova O. V., Skvortsov A.N., Skvortsov N.K. (2009) Synthesis and Optical Properties of (S)-Nobin // J. Gen. Chem. Rus. 79 (3): 962-965.

Bachurina I.V., Skvortsov A.N., Skvortsov N.K., Il'yushin M.A. (2009) Synthesis and Spectral Characteristics of the Combined Ligand Copper Complex with Proline and Corazole // J. Gen. Chem. Rus. 79 (3): 505-508.

Skvortsov A.N. 2009 [Efficient method of analysis of optical spectra from kinetic studies]. // Tsitologyia. 51 (3): 229-239 (in Russian).

Fridman A.S., Galyuk E.N., Vorob'ev V.I., Skvortsov A.N., Lando D.Y. (2008) Melting of crosslinked DNA: VI. Comparison of influence of interstrand crosslinks and other chemical modifications formed by antitumor compounds on DNA stability. // J. Biomol. Struct. Dyn. 26 (2): 175-185.

Skvortsov A.N., Reznikov A.N., de Vekki D.A., Stash A.I., Belsky V.K., Spevak V.N., Skvortsov N.K. (2006) Synthesis and crystal structures of platinum (II) complexes with phosphine sulfide: cis-Dichloro [dimethylsulfoxide] (triphenylphosphine sulfide) platinum (II) and (–)-cis-dichloro[(S)-methyl-p-tolylsulfoxide] (triphenylphosphine sulfide) platinum (II). // Inorganica Chimica Acta 359 (4): 1031-1040.

Skvortsov A.N., de Vekki D.A., Stash A.I., Belsky V.K., Spevak V.N., Skvortsov N.K. (2002) Synthesis, crystal structures and optical activity of cis- and trans-(–)-dichloro[(S)-methyl p-tolylsulfoxide]pyridyl platinum(II) complexes. // Tetrahedron: Asymmetry 13 (15): 1663-1671.

Transfection of cells is a very important step in genetic engineering of eukaryotic cells, both in research and therapy applications. Efficient DNA carriers should be biocompatible, bind DNA reversibly, protect it in extracellular environment and target it to cell nucleus. For therapy applications the carriers should also be non-toxic and degradable. Polycationic branched polymers of L-lysine and its copolymers with other amino acids are a promising group of DNA carriers.
We studied physicochemical properties and DNA-binding properties of a series of lysine (co)polymers with different topology (see Figure) and various L-amino acid modifiers - Ala, Glu, Arg, His, synthesized in the Institute of Macromolecular Compounds of Russian Academy of Sciences. Hyperbranched polymers, especially dendrimers, are known to have more common properties with globular proteins than random polypeptides. Hyperbranching is achieved by starting new peptide chains at some percent of epsilon-amino groups of L-lysine. The structural changes of the polymers with pH, ionic strength, solvent, and DNA binding were investigated by UV absorption and circular dichroism (CD) spectroscopy. The hyperbranched polymers retain significant conformational freedom and may adopt alpha-helical-type conformations (up to 45% secondary structure). The percent of alpha-type structure depends on changes of charge of modifier amino acid and corresponds well to general rules for proteins. Dendrimeric polymers have a structure with no alpha-like or beta-like regular moieties, this structure is very indifferent to physicochemical conditions.
DNA binding depends strongly on input copolymer/DNA ratio. Generally three binding modes were observed. At low ratios DNA and polymer optical spectra exhibit minor changes, the complex is soluble the electrophoretic mobility of plasmid DNA does not significantly change, the complex is readily stained by EtBr. At low ratios the complex formation is close to a true equilibrium. At some critical ratio (which is close to full compensation of DNA charge by the polycation) the complex flocculates or aggregates, and sediments easily; its optical properties are dominated by light scattering (no typical absorption or CD of DNA are observed). At polymer concentrations above critical ratio the complex again increases its solubility, the scattering decrease and CD bands reappear. However, the corresponding complexes do not stain with EtBr, possibly because of net positive charge. Some His-containing branched copolymers exhibited two successive critical ratios, at which complex solution became turbid.
The results indicate that DNA-binding properties of branched lysine polymers could be controlled by changing topology of the polymer; nature and percentage of modifier aminoacid in the polymer.

Publications

Vlasov G.P., Filippov A.P., Tarasenko I.I., Tarabukina E.B., Pankova G.A., Il'ina I.E., Shpyrkov A.A., Skvortsova E.V., Skvortsov A.N., and Vorob'ev V.I. (2008) Hyperbranched Poly(L-lysine) Modified with Histidine Residues via Lysine Terminal Amino Groups: Synthesis and Structure // Polymer Science, Series A. Polymer Physics 50 (4): 374-381.

Vlasov G.P., Tarasenko I.I., Valueva S.V., Kipper A.I., Tarabukina E.B., Filippov A.P., Avdeeva (Skvortsova) E.V., and Vorob'ev V.I. (2005) Hyperbranched Poly(L-lysine) Containing Additional Amino Acids or Their Oligomers Between Branching Points: Synthesis and Structure // Polymer Science, Series A. Polymer Physics 47 (5): 422-429.

Guryanov I.A., Vlasov G.P., Lesina E.A., Kiselev A.V., Baranov V.S., Avdeeva (Skvortsova) E.V., and Vorob'ev V. I. (2005) Cationic Oligopeptides Modified with Lipophilic Fragments: Use for DNA Delivery to Cells // Russian Journal of Bioorganic Chemistry 31 (1): 18-26.

Shpakov A.O., Gur'ianov I.A., Avdeeva (Skvortsova) E.V., Vorob'ev V.I., Vlasov G.P. [Molecular mechanisms of action of dendrons, containing 48-60 sequence of HIV-1 TAT-protein, on the functional activity of the adenylyl cyclase signaling systems]. // Tsitologiya 46 (11): 1011-1022, in Russian.

Structural organization of DNA in chromatin has been extensively studied for many years. When the structure of the nucleosome was solved, the investigations of the higher levels of the structural organization became one of the most important steps in understanding the functioning of chromatin. A great variety of DNA-binding proteins interact with DNA forming intricate protein-DNA complexes. DNA-bound protein factors normally cooperate to assemble higher-order nucleoprotein structures in which multiple protein-DNA and protein-protein contacts increase the specificity and stability of the final complexes. Interactions between proteins bound to neighboring regulatory elements allow cooperative DNA binding without encountering any serious topological difficulties. However, interactions between DNA-bound proteins that are not directly adjacent require a deformation of the DNA double helix, which is energetically unfavorable for DNA fragments shorter than ~140 nucleotides. Binding of 'architectural' proteins can nonetheless change the situation. Among such proteins, HMGB-domain proteins are particularly intriguing.
The suprafamily of the High Mobility Group known as the HMG proteins can be divided into three subfamilies HMGB, MGBN and HMGI/Y, which are represented by proteins that are different both in structure and functions. The HMGB subfamily is the most abundant among the non-histone chromatin proteins and well-known by such members as HMGB1, LEF-1, SRY, UBF, TCF-1 and many others. Their common features are the DNA-binding domains, often called HMG-Box domains whose structure is highly conservative. Some of the proteins in this family contain only one HMGB-domain (LEF-1, SRY), but a great number of others possess two or even more. All multi-domain HMGB proteins including HMGB1 demonstrate high affinity in binding to some structural distortions in DNA rather than to a particular nucleotide sequence. The members of the family are able to distinguish and bind preferably to DNA four-way junctions, binding sites of the anticancer drug cisplatin, and different bends and crossovers in general. The HMGB-domain proteins also induce DNA bends up to 150° when binding to undistorted DNA.
Another abundant chromosomal protein is histone H1, which is one of the best studied chromatin proteins so far. It binds to linker DNA at the entrance or exit of the nucleosome. This interaction takes place through the major groove of DNA and results in DNA-bending around the protein molecule and so one can say that it is quite different from the interaction of HMGB1 with DNA. Nevertheless, both proteins bind linker DNA in chromatin and there were several attempts to study their mutual influence on DNA binding. The data suggest that these two proteins demonstrate a co-action helping each other to some extent rather than competing in binding. Yet structural information about their complexes with DNA is still not available.

Structural aspects of antitumor activity of platinum coordination compounds.
It is known that HMGB1, histone H1 and some other chromatin proteins can bind specifically to DNA damaged by cis-DDP (cis-diamminedichloroplatinum(II), cisplatin). Cis-DDP is one of the most successful anti-tumor drugs. The biological activity of cisplatin is based on its ability to form stable adducts with DNA. Cis-DDP causes the formation of two major intra-strand DNA adducts, 1,2-d(GpG) and d(ApG) cross-links, in which the two chloride ions of cis-DDP are substituted by the N7 atoms of guanine or adenine. A biochemical and structural analysis of intra- and inter-strand cis-DDP adducts reveals major distortions of the DNA double helix including bending and unwinding. Cis-DDP binding changes the structure of DNA and prohibits its proper functioning in living cell.

Our studies are aimed at the investigation of structure-to-function correlations for DNA-protein complexes of chromatin, using the variety of physical-chemical approaches, including:

• circular dichroism spectroscopy
• UV and visible molecular absorption spectroscopy
• infrared absorption and circular dichroism spectroscopy
• atomic force microscopy
• electrophoretic techniques

Publications

Tsankov D., Polyanichko A., Wieser H. (2010) Vibrational Circular Dichroism: Ensuring Quality of Pharmaceutical Products, In: "Handbook of Analysis and Pharmaceutical Quality. Pharmaceutical Sciences Encyclopedia: Drug Discovery, Development, and Manufacturing", Edited by Shayne C. Gad, John Wiley & Sons, Inc, 1-41. (Online ISBN: 978-0-4705-7122-4).

Polyanichko A.M., Andrushchenko V.V., Bour P., Wieser H. (2011) Vibrational circular dichroism studies of biological macromolecules and their complexes. Chapter 2. In: "Circular Dichroism: Theory and Spectroscopy", Edited by David S. Rodgers, Nova Science Publishers, Inc., New York, USA, 2011, 61 pp. (ISBN: 978-1-61122-522-8).

Polyanichko A. and Wieser H. (2010) Structural organization of DNA-protein complexes of chromatin studied by vibrational and electronic circular dichroism. Spectroscopy: An International Journal, 24 (3-4), 239-244.

Chikhirzhina E., Polyanichko A., Leonenko Z., Wieser H., Vorob'ev V. (2010) C-terminal domain of nonhistone protein HMGB1 as a modulator of HMGB1-DNA structural interactions. Spectroscopy: An International Journal, 24 (3-4), 361-366.

Rodionova, T.Y., Chikhirzhina, E.V., Vorob'yov, V.I., Polyanichko, A.M. (2010) Changes in the secondary structure of HMGB1 protein bonded to DNA. Journal of Structural Chemistry 50 (5), 976-981.

Polyanichko, A. M., Leonenko, Z. V., Cramb, D., Wieser, H., Vorob'ev, V. I., & Chikhirzhina, E. V. (2008). Visualization of DNA complexes with HMGB1 and its C-truncated form HMGB1(A+B). Biophysics 53 (3), 202-206.

Polyanichko, A. M., Chikhirzhina, E. V., Andrushchenko, V. V., Vorob'ev, V. I., & Wieser, H. (2006). The effect of manganese(II) on the structure of DNA/HMGB1/H1 complexes: Electronic and vibrational circular dichroism studies. Biopolymers 83 (2), 182-192.

Polyanichko, A. M., Chikhirzhina, E. V., Andrushchenko, V. V., Wieser, H., & Vorob'ev, V. I. (2005). Spectroscopic investigations of the structure of DNA complexes with mn 2+ in UV and IR regions. Biophysics 50 (5), 710-715.

Polyanichko, A., & Wieser, H. (2005). Fourier transform infrared/vibrational circular dichroism spectroscopy as an informative tool for the investigation of large supramolecular complexes of biological macromolecules. Biopolymers 78(6), 329-339.

Polyanichko, A. M., Chikhirzhina, E. V., Andrushchenko, V. V., Kostyleva, E. I., Wieser, H., & Vorob'ev, V. I. (2004). The effect of Ca2+ ions on DNA compaction in the complex with non-histone chromosomal protein HMGB1. Molecular Biology 38 (4), 701-712.

Polyanichko, A. M., Chikhirzhina, E. V., Kostyleva, E. I., & Vorob'ev, V. I. (2004). Structure of DNA complexes with nonhistone chromosomal protein HMGB1 in the presence of manganese ions. Molecular Biology 38 (6), 891-898.

Polyanichko, A. M., Andrushchenko, V. V., Chikhirzhina, E. V., Vorob'ev, V. I., & Wieser, H. (2004). The effect of manganese(II) on DNA structure: Electronic and vibrational circular dichroism studies. Nucleic Acids Research 32 (3), 989-996.

Polyanichko, A. M., Chikhirzhina, E. V., Andrushchenko, V. V., Kostyleva, E. I., Wieser, H., & Vorob'ev, V. I. (2004). The effect of Ca²⁺ ions on DNA compaction in the complex with HMGB1 nonhistone chromosomal protein. Molecular Biology 38 (4), 590-599.

Chikhirzhina, E. V., Polyanichko, A. M., Skvortsov, A. N., Kostyleva, E. I., Houssier, C., & Vorob'ev, V. I. (2002). HMG1 domains: The victims of the circumstances. Molecular Biology 36 (3), 412-418.

Polyanichko, A. M., Chikhirzhina, E. V., Skvortsov, A. N., Kostyleva, E. I., Colson, P., Houssier, C., et al. (2002). The HMG1 ta(i)le. Journal of Biomolecular Structure and Dynamics 19 (6), 1053-1062.

Polyanichko, A. M., Davydenko, S. G., Chikhirzhina, E. V., & Vorob'ev, V. I. (2000). The interaction of supercoiled DNA with nonhistone protein HMG1. Tsitologiya 42 (8), 792-793.

We have developed macro and micro methods to isolate DNA- dependent RNA polymerase III of human placenta nuclei and human epidermoid carcinoma cells A431. RNA polymerase III have been isolated by both methods as two subfractions, IIIa and IIIb, with different buoyant density in gradient of glycerol concentration. Protein kinase activity, phosphorylating in vitro six joint subunits of both subfractions , and two subunits with molecular weight about 30 and 93 kDa of subfraction IIIa and subunit with molecular weight about 95 kDa of -IIIb is co-purified with both RNA polymerase III subfractions. This protein kinase activity was not identified in cells A431 after 48 hours long starvation. The subunit of IIIa subfraction with molecular weigt about 95 kDa is phosphorylated on phophotyrosine as identified by immunoblotting with antibodies against phosphotyrosine, the same subunit of IIIb subfraction is phosphorylated on tyrosine residues not in each enzyme preparation. At least four subunits with molecular weight 90 kDa and lower of the both subfractions of RNA polymerase III of human placenta nuclei are phosporylated in vivo as it has been shown by INDIA Phosphoprobe Kit for phosphorylated polypeptides identificating method. The phosphorylation of RNA polymerase IIIa of human placenta nuclei in vitro causes the activation of specific Alu-repeat, tRNAiMet1 and 5S rRNA genes consisting templates. Transcription activation depends on the presence or absense of specic template during phosphorylation of the enzyme in vitro. The subfractions of RNA polymerase of human placenta nuclei don't include protein phosphotase activity. The co-purified protein kinase utilases ATP and GTP as donors of phophate groups. Dephosphorylation of RNA polymerase IIIa of human placenta nuclei by immobilezed alkaline phosphotase in vitro causes inhibition of Alu- template transcription, dephosphorylated RNA polymerase cann't be substrate of phosphorylation by co-pyrified protein kinase. It may be the copyrified protein kinase losts enzymatic activity through dephosphorylation. Co-pyrified protein kinase aren't numbered to MAP-kinase family as it has been shown by immunobloting method with antibody against MAP-kinase. Two subunits with molecular weight about 200 kDa and 75 kDa were identified in RNA polymerase IIIa and IIIb subfractions of human placenta nuclei by immunobloting method with antibody against TOR- kinase. It is possible that co- pyrified protein kinase, phosphorylating RNA Polymerase III in vitro, belongs to inosytol 3-kinase (PI3- kinase) family thus its activity is inhibited by vortmanin - specific inhibitor of PI3 kinase. The levels of 5S rRNA, initiator tRNAiMet1 and Alu-gene expression in human epidermoid carcinoma cells A431 being in different physiological states upon growing in serum-free media causes the prolongation of generation period, activated to proliferation by low concentration of epidermoid growth factor (EGF) - 0.1 ng/ml, and high concentrations of EGF - 10-100 ng/ml, cause apoptosis. were investigated by real-time RT-PCR method. It was showed that studied Alu-repeat wasn't expressed in cells A431. The level of initiator tRNAiMet1 in actively proliferating cells was 1.7 times higher than in slowly proliferating cells and was remained just high in apoptotic cells. The proportion of 5S rRNA was practically the same in slowly and actively proliferating A431 cells, and was increased about 2.5-fold in apoptotic cells.

The underlying task of investigations is solution of the major problem of fundamental science of paramount significance - creation of the code-based protein physics as a part of the new section of theoretical physics which is destined for description of systems consisted of numerous different elements, connected together. The immediate task of investigations is to develop the algorithm of formation of the three-dimensional structure of water-soluble proteins when an amino acid sequence is given using computer simulation and obtained previously results of simulation of formation of alpha-helices and beta-strands which are supported by experimental data.

Additional info:  http://www.cytspb.rssi.ru/persons/shestopalov/shestopalov_en1.pdf

Publications

Shestopalov B.V. (2007) The code-based physics of formation of a-helices and b-hairpins in water-soluble proteins. Doklady Biochemistry and Biophysics. Vol. 416, pp 245-247.

Shestopalov B.V. (2007) Simulation of formation of a-helices and b-hairpins in water-soluble proteins by the code-based physics. Cell and Tissue Biology. Vol. 1, No. 5, pp. 420-426.

Shestopalov B.V. and G. R. Mavropulo-Stolyarenko (2007) The most complete list of unique protein structures solved by x-ray crystallography. Molecular Biology. Vol. 41, No. 4, pp. 680-681.

Shestopalov B.V. (2005) Comparison of x-ray diffraction analysis and nuclear magnetic resonance from data on the identification of a-helices and b-strands in the same protein. Biophysics. Vol. 50, No. 6, pp. 862-864.

Shestopalov B.V. (2003) Amino acid code of protein secondary structure. Tsitologiya. Vol. 45, No. 7, pp. 702-706.

Shestopalov B.V. (2003) Statistical model of amino acid code of protein secondary structure. Tsitologiya. Vol. 45, No. 7, pp. 707-713.

Kouprina N., Kirillov A., Kroll E., Koryabin M., Shestopalov B., Bannikov V, Zakharyev V, Larionov V. (1993) Identification and cloning of the CHL4 gene controlling chromosome segregation in yeast. Genetics. Vol. 135, № 2, pp 327 - 341.

Kouprina N., Kroll E., Bannikov V., Bliskovsky V., Gizatullin R., Kirillov A., Shestopalov B., Zakharyev V., Hieter P., Spencer F., Larionov V. (1992) CTF4 (CHL15) mutants exhibit defective DNA metabolism in the yeast Saccharomyces cerevisiae. Mol. Cell. Biol. Vol. 12, № 12, pp 5736 - 5747.

Shestopalov B.V. (1988) Amino acid sequence template useful for helix-turn-helix prediction. FEBS Lett. Vol. 233, pp. 105-108.

Gultyaev A.P., Shestopalov B.V. (1988) Structural basis for autogenous regulation of Xenopus laevis ribosomal protein L1 synthesis at the splicing level. FEBS Lett. Vol. 232, № 1, pp 9 - 11.

Shestopalov B.V., Chirgadze Yu.N. (1976) Quantitative study of secondary structure of histones H1, H2A, and H4 in solution by infrared spectroscopy. Eur J Biochem. Vol. 67, № 1, pp 123 - 128.

Chirgadze Yu.N., Shestopalov B.V., Venyaminov S.Yu. (1973) Intensities and other spectral parameters of infrared amide bands of polypeptides in the b- and random forms. Biopolymers. Vol. 12. № 6. pp 1337 - 1351.