Wojciech. T. Markiewicz

Background: Viscum album (the European mistletoe) is a semi-parasitic plant, which is of high medical interest. It is widely found in Europe, Asia, and North America. It contains at least three distinct lectins (i.e. ML-I, II, and III), varying in molecular mass and specificity. Among them, ML-I is in focus of medical research for various activities, including anti-cancer activities. To understand the molecular basis for such medical applications, a few studies have already addressed the structural and functional analysis of ML-I in complex with ligands. In continuation of these efforts, we are reporting the crystal structure of ML from Viscum album in complex with the nucleic acid oxidation product 4-N-furfurylcytosine (FC) refined to 2.85 Å resolution. FC is known to be involved in different metabolic pathways related to oxidative stress and DNA modification. Methods: X-ray suitable hexagonal crystals of the ML-I/FC complex were grown within four days at 294 K using the hanging drop vapor diffusion method. Diffraction data were collected up to a resolution of 2.85 Å. The ligand affinity was verified by in-silico docking. Results: The high-resolution structure was refined subsequently to analyze particularly the active site conformation and a binding epitope of 4-N-furfurylcytosine. A distinct 2Fo-Fc electron density at the active site was interpreted as a single FC molecule. The specific binding of FC is achieved also through hydrophobic interactions involving Tyr76A, Tyr115A, Glu165A, and Leu157A of the ML-I A-chain. The binding energy of FC to the active site of ML-I was calculated as well to be -6.03 kcal mol-1. Conclusion: In comparison to other reported ML-I complexes, we observed distinct differences in the vicinity of the nucleic acid base binding site upon interaction with FC. Therefore, data obtained will provide newinsights in understanding the specificity, inhibition, and cytotoxicity of the ML-I A-chain and related RIPs.

The general idea of regenerative medicine is to fix or replace tissues or organs with alive and patient-specific implants. Pluripotent stem cells are capable of indefinite self-renewal and differentiation into all cell types of body with origin from the three germ layers of the developing embryo, therefore they have a potential to play a substantial role in regenerative medicine. Easily accessible source of induced pluripotent stem cells may allow obtaining and culturing tissues in vitro. Many improvements in the methods leading to obtain such cells have been made by various research groups in order to limit immunogenicity and tumorigenesis, increase efficiency and accelerate kinetics. One of the approaches affecting pluripotency is usage of small molecule compounds including RNA-derivatives – nucleosides analogues. It would be great to assess general character of such molecules and reveal their new derivatives or modifications to improve induced pluripotent stem cells (iPSCs) reprogramming efficiency. Understanding the epigenetic changes during cellular reprogramming will extend understanding of stem cell biology and lead to potential therapeutic approaches. In this digest of compounds found in literature as proven or putative inductors of cells’ reprogramming to pluripotency there are compounds that may substitute for transgenic nucleic acids delivery in order to improve time and efficiency of reprogramming. Nucleic acids’ derivatives or modifications of particular atoms or substitutes influence modulating activities of small molecules, especially their inhibiting activity. Due to dosage-dependent effect of small molecules influence on genes, their application concentration needs to be strictly determined.

The essential idea of regenerative medicine is to fix or replace tissues or organs with alive and patient-specific implants. Pluripotent stem cells are able to indefinitely self-renew and differentiate into all cell types of the body which makes them a potent substantial player in regenerative medicine. The easily accessible source of induced pluripotent stem cells may allow obtaining and cultivating tissues in vitro. Reprogramming refers to regression of mature cells to its initial pluripotent state. One of the approaches affecting pluripotency is the usage of low molecular mass compounds that can modulate enzymes and receptors leading to the formation of pluripotent stem cells (iPSCs). It would be great to assess the general character of such compounds and reveal their new derivatives or modifications to increase the cell reprogramming efficiency. Many improvements in the methods of pluripotency induction have been made by various groups in order to limit the immunogenicity and tumorigenesis, increase the efficiency and accelerate the kinetics. Understanding the epigenetic changes during the cellular reprogramming process will extend the comprehension of stem cell biology and lead to potential therapeutic approaches. There are compounds which have been already proven to be or for now only putative inducers of the pluripotent state that may substitute for the classic reprogramming factors (Oct3/4, Sox2, Klf4, c-Myc) in order to improve the time and efficiency of pluripotency induction. The effect of small molecules on gene expression is dosage-dependent and their application concentration needs to be strictly determined. In this review we analysed the role of small molecules in modulations leading to pluripotency induction, thereby contributing to our understanding of stem cell biology and uncovering the major mechanisms involved in that process.

United States Patent Patent number: US9040490 B2 Application number: US 13/576709 Cytosine analogue, a method of preparation of a cytosine analogue, a DNA methyltransferase 1 inhibitor, a method for DNA methylation inhibition, the use of the analogue in the treatment of diseases associated with deviations from normal DNA methylation Inventors: J. Barciszewski, W.T. Markiewicz, E. Adamska, B. Plitta, M. Giel-Pietraszuk, E. Wyszko, M. Markiewicz, A. Fedoruk-Wyszomirska, T. Kulinski, M. Chmielewski Assignee: Institute of Bioorganic Chemistry, PAS, Poznań Date of Patent: 26. 05. 2015 European Patent Application PCT number: PCT/PL2011/000032 Cytosine analogue, a method of preparation of a cytosine analogue, a DNA methyltransferase 1 inhibitor, a method for DNA methylation inhibition, the use of the analogue in the treatment of diseases associated with deviations from normal DNA methylation Inventors: J. Barciszewski, W.T. Markiewicz, E. Adamska, B. Plitta, M. Giel-Pietraszuk, E. Wyszko, M. Markiewicz, A. Fedoruk-Wyszomirska, T. Kulinski, M. Chmielewski Assignee: Institute of Bioorganic Chemistry, PAS, Poznań Application Date: 17. 03. 2011 Polish Patent Application Polish Application number: P 390 769 Cytosine analogue, a method of preparation of a cytosine analogue, a DNA methyltransferase 1 inhibitor, a method for DNA methylation inhibition, the use of the analogue in the treatment of diseases associated with deviations from normal DNA methylation Inventors: J. Barciszewski, W.T. Markiewicz, E. Adamska, B. Plitta, M. Giel-Pietraszuk, E. Wyszko, M. Markiewicz, A. Fedoruk-Wyszomirska, T. Kulinski, M. Chmielewski Assignee: Institute of Bioorganic Chemistry, PAS, Poznań Application Data: 19. 03. 2010 Published also as: EP2556081A2, US20120322755, WO2011115513A2, WO2011115513A3, WO2011115513A9 Abstract In this study, a cytosine analog and its preparation method and a DNA methyltransferase 1 inhibitor and a method for DNA methylation inhibition are provided for the treatment of diseases associated with deviations from normal DNA methylation. The analog of cytosine comprises of 1, N4, 5, and 6-substituted derivatives of cytosine or 5,6-dihydrocytosine; the analog can be described with a chemical formula where R1 is H, R3, R4, 2-deoxyribosyl, R4 is alkyl or aryl, X is N or C – if X in formula I is N, then R5 is no substituent and if X in formula I and/or II is C or if X in formula II is N, then R5 and R6 are independently alkyl, aryl, hydroxyalkyl, aminoalkyl, hydroxyl, carboxyl, amino group, alkoxyl, aryloxyl, aminoalkyl, aminoaryl, thio group, sulfonyl, sulfinyl, or halogen.

The rationale for the synthesis of cationic modified nucleosides is higher expected nuclease resistance and potentially better cellular uptake due to an overall reduced negative charge based on internal charge compensation. Due to the ideal distance between cationic groups, polyamines are perfect counterions for oligodeoxyribonucleotides. We have synthesized non-nucleosidic analogues built from units that carry different diol structures instead of sugar residues and functionalized with polyamines. The non-nucleosidic analogues were attached as internal or 5′-terminal modifications in oligodeoxyribonucleotide strands. The thermodynamic studies of these polyaminooligonucleotide analogues revealed stabilizing or destabilizing effects that depend on the linker or polyamine used.

Nucleic acid microarrays have recently become one of the basic techniques in the study of gene expression. Owing to progress in the field of miniaturization, thousands of oligonucleotides differing in terms of their sequences can be systematically placed on a small area of a solid support (usually glass). These probes are capable of simultaneously interacting with a large number of longer nucleic acids from particular genes. Nucleic acid microarray construction technology consists, primarily, of the appropriate functionalization of a glass surface with the use of organofunctional silanes. Oligonucleotides known as probes are attached to a functionalized surface using, for instance, the lithography technique and, after being linked to the surface, they are subjected to hybridization with complementary and labelled fragments of nucleic acids known as samples of unknown sequences. In this paper, we present a method for constructing DNA microarrays that is based on the use of microscopic slides modified with epoxy functional group-containing silanes. This study was aimed at optimizing the production of DNA microarrays. The study tested the usefulness of four different epoxy functional silanes with one or three alkoxy groups. In addition, slides were silanized with the use of alkylsilane. The glass slides were characterized using a goniometer and an atomic force microscope (AFM). The synthesized amino linker-containing oligonucleotide probes were printed onto the glass slides in order to check the effectiveness of their attachment to the solid surface.

Synthesis of 5''-phosphate 2'-O-ribosylribonucleosides [Nr(p)] of four common ribonucleosides, and 3'-phosphoramidites of 5''-phosphate 2'-O-ribosyladenosine and 2'-O-ribosylguanosine using the H-phosphonate chemistry is described. An additional ring protected by benzoyl groups was incorporated into the main ribosyl ring in the reaction with 1-O-acetyl-2,3,5-tri-O-benzoyl-β-d-ribofuranose in the presence of SnCl₄. The obtained 2'-O-ribosylribonucleosides (Nr) were applied in the subsequent transformations with selective deprotection. Ethanolamine was applied as a very convenient reagent for selective removal of benzoyl groups. Additionally, the tetraisopropyldisiloxane-1,3-diyl (TIPDSi) group was found to be stable under these deprotection conditions. Thus, the selectively deprotected 5''-hydroxyl group of Nr was transformed into an H-phosphonate monoester which was found to be stable under the following conditions: the removal of the TIPDSi group with triethylammonium fluoride and the dimethoxytritylation of the 5''-hydroxyl function. The 5''-H-phosphonate of Nr precursors was easily transformed to the corresponding dicyanoethyl 5''-O-phosphotriesters before phosphitylation, which gave 3'-phosphoramidite units of Nr(p) in high yield. The derived phosphoramidite units were used in an automated oligonucleotide synthesizer to produce dimer Ar(p)T via the phosphoramidite approach. The obtained products were fully deprotected under standard deprotection conditions giving dimers with a 5''-phosphate monoester function. Application of an alkaline phosphatase to prove the presence of an additional phosphate group was described.

Convenient and efficient methods of the synthesis of N⁶- and N⁴-substituted derivatives of adenine and cytosine and their 2′-deoxyribosides were developed. The reactions of either unprotected nucleobases (adenine, cytosine) or unprotected 2′-deoxyribosides with aryl or alkyl aldehydes give corresponding Schiff bases that can be reduced to the target title compounds with high overall yields. In the case of aryl aldehydes the imine derivatives are obtained in the presence of methoxides in methanol and reduced with sodium borohydride. The corresponding reactions with alkyl aldehydes require the use of acetic acid and borane dimethyl sulfide complex instead.