Synthesis and Investigation of Anti-tumor Properties of Novel, Bicyclic Furopyrimidine, Pyrrolopyrimidine and Pyrimidopyridazine Nucleoside Analogues

A series of nine hitherto unknown bicylic pyrimidine nucleoside analogues (BCNAs) bearing bicyclic furo[2,3-d]pyrimidin-2(3H)-one, 3H-pyrrolo[2,3-d]pyrimidin-2(7H)-one and 5,6-dihydropyrimido[4,5-c]pyridazin-7(8H)-one bases were prepared in a straightforward approach. The synthesised compounds posses β-D-rybofuranose or β-D-2-deoxyrybofuranose or β-D-arabinofuranose moieties attached to each of the heterocylic ring systems. This is one of a few examples of synthesis of pyrrolo[2,3-d]pyrimidin-2(7H)-one and dihydropyrimido[4,5-c]pyridazin-7(8H)-one nucleosides, and the first example of such nucleosides possessing arabinose moiety. A key synthetic step was a Sonogashira coupling reaction. For coupling with 4-phenyl-1-butyne, we used deprotected 5-iodouridine, 2’-deoxy-5-iodouridine, and 5-iodoarabinouridine and this reaction was followed by cycloisomerization and subsequent conversion of the furane ring into a pyrole ring or a pyridiazine. This approach resulted in the creation of small library of compounds, which were evaluated for their antiproliferative properties against HL-60 and Jurkat E6.1 cell lines. Of all tested compounds, only 3-(β-D-rybofuranosyl)-6-(2-phenylethyl)furo[2,3-d]pyrimidin-2(3H)-one exhibited weak anti-proliferative activity, with IC50 values of 54 and 81 µM for HL-60 and Jurkat E6.1 cells, respectively

Immunogenic epitope scanning in bacteriolytic enzymes Pal and Cpl-1 and engineering Pal to escape antibody responses

Bacteriolytic enzymes are promising antibacterial agents, but they can cause a typical immune response in vivo. In this study, we used a targeted modification method for two antibacterial endolysins, Pal and Cpl-1. We identified the key immunogenic amino acids, and designed and tested new, bacteriolytic variants with altered immunogenicity. One new variant of Pal (257-259 MKS → TFG) demonstrated decreased immunogenicity while a similar mutant (257-259 MKS → TFK) demonstrated increased immunogenicity. A third variant (280-282 DKP → GGA) demonstrated significantly increased antibacterial activity and it was not cross-neutralized by antibodies induced by the wild-type enzyme. We propose this variant as a new engineered endolysin with increased antibacterial activity that is capable of escaping cross-neutralization by antibodies induced by wild-type Pal. We show that efficient antibacterial enzymes that avoid cross-neutralization by IgG can be developed by epitope scanning, in silico design, and substitutions of identified key amino acids with a high rate of success. Importantly, this universal approach can be applied to many proteins beyond endolysins and has the potential for design of numerous biological drugs

Bicyclic cytarabine analogues: synthesis and investigation of antitumor properties of novel, 6-aryl- and 6-alkyl-3H-pyrrolo[2,3-d] pyrimidin-2(7H)-one arabinosides.

A series of sixteen hitherto unknown Cytarabine analogues bearing a bicyclic 3H-pyrrolo[2,3-d]pyrimidin-2(7H)-one base modified with aryl, pyridyl, benzyl and alkyl substituents was prepared in a straightforward approach. This is the one of the few examples of the synthesis of pyrrolo[2,3-d]pyrimidin-2(7H)-one nucleosides and the first example of pyrrolo[2,3-d]pyrimidin-2(7H)-one nucleosides possessing arabinose moiety. For the first time, the conversion of the furopyrimidine arabinoside products to a series of novel pyrrolopyrimidines by ammonolysis reaction was thoroughly investigated using aqueous and methanolic reaction conditions under classical and micro-wave heating. This approach resulted in a small library of compounds, which were evaluated for their antiproliferative properties against HL-60 and Jurkat E6.1 cell lines. All synthesised compounds exhibited a weaker cytotoxic effect in comparision to the mother compound. Of all the tested compounds, the derivative bearing a 4-n-pentylphenyl substituent exhibited the highest antiproliferative activity

DataSheet_1_Immunogenic epitope scanning in bacteriolytic enzymes Pal and Cpl-1 and engineering Pal to escape antibody responses.docx

Bacteriolytic enzymes are promising antibacterial agents, but they can cause a typical immune response in vivo. In this study, we used a targeted modification method for two antibacterial endolysins, Pal and Cpl-1. We identified the key immunogenic amino acids, and designed and tested new, bacteriolytic variants with altered immunogenicity. One new variant of Pal (257-259 MKS → TFG) demonstrated decreased immunogenicity while a similar mutant (257-259 MKS → TFK) demonstrated increased immunogenicity. A third variant (280-282 DKP → GGA) demonstrated significantly increased antibacterial activity and it was not cross-neutralized by antibodies induced by the wild-type enzyme. We propose this variant as a new engineered endolysin with increased antibacterial activity that is capable of escaping cross-neutralization by antibodies induced by wild-type Pal. We show that efficient antibacterial enzymes that avoid cross-neutralization by IgG can be developed by epitope scanning, in silico design, and substitutions of identified key amino acids with a high rate of success. Importantly, this universal approach can be applied to many proteins beyond endolysins and has the potential for design of numerous biological drugs.</p