Zbigniew J. Leśnikowski

Adenosine and 2'-deoxyadenosine modified with boron cluster pharmacophores as new classes of human blood platelet function modulators.

Novel types of adenosine and 2′‐deoxyadenosine derivatives containing boron clusters at positions C2′, N6, or C8 were synthesized. The effect of these modified compounds on platelet function was studied. Modification of adenosine at the C2′ position with a para‐carborane cluster (C2B10H11) results in efficient inhibition of platelet function, including aggregation, protein secretion, and P‐selectin expression induced by thrombin or ADP. These preliminary findings and the new chemistry proposed form the basis for the development of a new class of adenosine analogues that modulate human blood platelet activities.

Towards new boron carriers for boron neutron capture therapy: metallacarboranes bearing cobalt, iron and chromium and their cholesterol conjugates.

A method for the synthesis of cholesterol-metallacarborane conjugates bearing cobalt, iron and chromium was developed. Effective incorporation of the cholesterol conjugate bearing cobalt into liposome membrane was revealed. Using the metallacarborane-encrusted liposomes as boron delivery system in vivo biodistribution experiments in tumor-bearing mice, high accumulation and selective delivery of boron into tumor tissues was observed. The results demonstrate that the cholesterol-metallacarborane conjugates can be considered as a potential candidate for boron delivery vehicle in BNCT.

Stereospecific synthesis of (Rp)- and (Sp)-thymidylyl(3',5')thymidylyl methanephosphonates

(Rp)- and (Sp)-diastereomers of thymidylyl (3′,5′) thymidylyl methanephosphonate (3) were prepared in the reaction of (Sp)- or (Rp)-isomers of 5′-O-monomethoxytritylthymidine 3′-O-[O-(4-nitrophenyl)methanephosphonate] (1) with activated 3′-O-acetylthymidine (2)1.

Distribution of UL144, US28 and UL55 genotypes in Polish newborns with congenital cytomegalovirus infections

Human cytomegalovirus (HCMV) is the most common congenital infection. HCMV strains display genetic variability in different regions. Distribution of HCMV genotypes in the population of congenitally infected newborns from Central Poland and viral load in newborns’ blood is described and discussed. HCMV isolates were analysed by sequencing at three sites on the genome: the UL144 tumour necrosis factor-alpha (TNFα)-like receptor gene, the US28 beta-chemokine receptor gene and the UL55 envelope glycoprotein B (gB) gene. The newborns’ blood was examined for HCMV DNA with a nested (UL144, UL55) or heminested (US28) polymerase chain reaction, and the genotypes were determined by sequence analysis. HCMV DNA was detectable in 25 out of 55 examined newborns born by HCMV-infected mothers (45.5%). The blood viral load in mother–infant pairs was determined. Most of the newborns had identical virus genotype, gB2 (96%), UL144 B1 (88%) and US28 A2 (84%). These genotypes were detected in all newborns with asymptomatic congenital infection. The occurrence of UL144 B1 or US28 A2 genotypes in the babies examined was significant in comparison to other genotypes (p = 0.0002 and p = 0.040 respectively). There was no association between specific gB subtypes in all patients groups (p = 0.463). There was no correlation between HCMV genotypes and the outcome.

Detection of cytomegalovirus in human placental cells by polymerase chain reaction

Human cytomegalovirus (HCMV) is the most common cause of viral intrauterine infection. Progress in rapid, specific, and dependable detection of HCMV has recently been achieved by the use of DNA hybridization techniques and other molecular methods. We examined 21 placentas after delivery for the presence of HCMV DNA by polymerase chain reaction (PCR). To test the reliability of the PCR for the detection of HCMV DNA in clinical specimens, two simple PCR assays and a real‐time quantitative PCR were used. PCR analysis of villous and decidual cells showed that HCMV DNA was present in 16 placentas (76.2%). Transmission of HCMV infection to chorionic villi was confirmed in 11 organs (52.4%), and congenital infections in newborns were detected in 9 cases (42.8%). These results suggest that HCMV genome detection in placentas at later gestational ages is common. Our results demonstrated that detection of HCMV DNA in placental tissues by DNA amplification provides a specific and sensitive method for diagnosis of intrauterine HCMV infection.

Recent developments with boron as a platform for novel drug design.

ABSTRACT Introduction: After decades of development, the medicinal chemistry of compounds that contain a single boron atom has matured to the present status of having equal rights with other branches of drug discovery, although it remains a relative newcomer. In contrast, the medicinal chemistry of boron clusters is less advanced, but it is expanding and may soon become a productive area of drug discovery. Areas covered: The author reviews the current developments of medicinal chemistry of boron and its applications in drug design. First generation boron drugs that bear a single boron atom and second generation boron drugs that utilize boron clusters as pharmacophores or modulators of bioactive molecules are discussed. The advantages and gaps in our current understanding of boron medicinal chemistry, with a special focus on boron clusters, are highlighted. Expert opinion: Boron is not a panacea for every drug discovery problem, but there is a good chance that it will become a useful addition to the medicinal chemistry tool box. The present status of boron resembles the medicinal chemistry status of fluorine three decades ago; indeed, currently, approximately 20% of pharmaceuticals on the market contain fluorine. The fact that novel boron compounds, especially those based on abiotic polyhedral boron hydrides, are currently unfamiliar could be advantageous because organisms may be less prone to developing resistance against boron cluster-based drugs.

Electrochemical detection of DNA hybridization using metallacarborane unit.

The evaluation of novel electrochemically active label for electrochemical detection of DNA hybridization is presented. Metallacarborane units modified with iron, cobalt or chromium were investigated. The value of redox potential and relatively strong current signal facilitate usage of Fe-carborane as marker covalently attached to the ssDNA. In electrochemical genosensor the sequence complementary to UL55 gene was labeled and used as a target for biosensor device. Interactions were investigated using electrochemical and piezoelectric methods. Obtained results confirm usefulness of the designed label in electrochemical detection of DNA hybridization.

Distribution of cytomegalovirus gN variants and associated clinical sequelae in infants

BACKGROUND Human cytomegalovirus (HCMV) is the most widespread cause of congenital infection. The effects of various viral strains and viral loads on the infection outcome have been under debate. OBJECTIVES To determine the distribution of gN variants in HCMV strains isolated from children with congenital or postnatal infection and to establish the relationship between the viral genotype, the viral load, and the sequelae. STUDY DESIGN The study population included congenitally HCMV-infected newborns and children with postnatal or unproven congenital HCMV infection. The genotyping was performed by RFLP analysis of PCR-amplified fragments, and the viral load was measured by quantitative real-time PCR. RESULTS Our results demonstrated that the HCMV genotypes gN3b, gN4b, and gN4c were prevalent in the patients examined. There were no differences in the distributions of gN genotypes in the congenitally and postnatally infected children. Multiple HCMV strains were detected in both groups of children. A significant association between the HCMV gN4 genotype and the incidence of neurological disorders was observed (p=0.045). Our results suggest that the detection of the gN2 or the gN4 genotype may be indicative of serious manifestations in children. In contrast, the gN3b and the gN1 genotypes represent less pathogenic HCMV strains. The HCMV load in urine was significantly higher in children with congenital infection compared with children with postnatal infection. No correlation was found between the viral load and the genotype. CONCLUSION Our results suggest that the gN genotype may be a virological marker of symptomatic HCMV infection in newborns.

TLR9 2848 GA Heterozygotic Status Possibly Predisposes Fetuses and Newborns to Congenital Infection with Human Cytomegalovirus

Background Some single nucleotide polymorphisms (SNP), located in Toll-like receptor (TLR) genes, were reported to be associated with human cytomegalovirus (HCMV) infections. The study was aimed to assess the correlation of SNPs at TLR4 and TLR9 genes with the occurrence of congenital cytomegaly, based on available samples. Methods Reported case-control study included both HCMV infected and non-infected fetuses and newborns. The specimens were classified to the molecular analyses, based on serological features of the recent infection and HCMV DNAemia in body fluids. TLR SNPs were studied, using multiplex nested PCR-RFLP assay, and determined genotypes were confirmed by sequencing. Hardy-Weinberg equilibrium was assessed for the identified genotypes. The linkage disequilibrium was also estimated for TLR4 SNPs. A relationship between the status of TLR genotypes and congenital cytomegaly development was estimated, using a logistic regression model. Results Hardy Weinberg equilibrium was observed for almost all SNPs, both infected and non-infected patients, with exception of TLR4 896 A>G polymorphism in the control group (P≤0.050). TLR4 896 A>G and 1196 C>T SNPs were found in linkage disequilibrium in both study groups (P≤0.050). The CC genotype at TLR4 1196 SNP and the GA variant at TLR9 2848 G>A SNP were significantly associated with HCMV infection (P≤0.050). The risk of congenital cytomegaly was higher in heterozygotes at TLR9 SNP than in the carriers of other genotypic variants at the reported locus (OR 4.81; P≤0.050). The GC haplotype at TLR4 SNPs and GCA variants at TLR4 and TLR9 SNPs were significantly associated with HCMV infection (P≤0.0001). The ACA variants were more frequent among fetuses and neonates with symptomatic, rather than asymptomatic cytomegaly (P≤0.0001). Conclusions TLR4 and TLR9 polymorphisms may contribute to the development of congenital infection with HCMV in fetuses and neonates. The TLR9 2848 GA heterozygotic status possibly predisposes to HCMV infection, increasing the risk of congenital cytomegaly development.

Mixed infections with distinct cytomegalovirus glycoprotein B genotypes in Polish pregnant women, fetuses, and newborns

The purpose of this investigation was to describe a distribution of cytomegalovirus (CMV) single and multiple genotypes among infected pregnant women, their fetuses, and newborns coming from Central Poland, as well as congenital cytomegaly outcome. The study involved 278 CMV-seropositive pregnant women, of whom 192 were tested for viral DNAemia. Human cytomegalovirus (HCMV) genotyping was performed for 18 of 34 pregnant women carrying the viral DNA and for 12 of their 15 offspring with confirmed HCMV infections. Anti-HCMV antibodies levels were assessed by chemiluminescence immunoassay (CLIA) and enzyme-linked fluorescence assay (ELFA) tests. Viral DNA loads and genotypes were determined by real-time polymerase chain reaction (PCR) assays for the UL55 gene. In the pregnant women, we identified HCMV gB1, gB2, gB3, and gB4 genotypes. Single gB2, gB3, or gB4 genotypes were observed in 14 (77.8 %) women, while multiple gB1–gB2 or gB2–gB3 genotypes were observed in four (22.2 %). Maternal HCMV genotypes determined the genotypes identified in their fetuses and newborns (p ≤ 0.050). Half of them were infected with single HCMV gB1, gB2, or gB3 genotypes and the other half with multiple gB1–gB2 or gB2–gB3 genotypes. Single and multiple genotypes were observed in both asymptomatic and symptomatic congenital cytomegaly, although no gB3 genotype was identified among asymptomatic cases. In Central Poland, infections with single and multiple HCMV strains occur in pregnant women, as well as in their fetuses and neonates, with both asymptomatic and symptomatic infections. HCMV infections identified in mothers seem to be associated with the viral genotypes in their children.