Synthesis and Evaluation of Diguanosine Cap Analogs Modified at the C8-Position by Suzuki-Miyaura Cross-Coupling: Discovery of 7-Methylguanosine-Based Molecular Rotors.

Chemical modifications of the mRNA cap structure can enhance the stability, translational properties, and half-life of mRNAs, thereby altering the therapeutic properties of synthetic mRNA. However, cap structure modification is challenging because of the instability of the 5'-5'-triphosphate bridge and N7-methylguanosine. The Suzuki-Miyaura cross-coupling reaction between boronic acid and halogen compound is a mild, convenient, and potentially applicable approach for modifying biomolecules. Herein, we describe two methods to synthesize C8-modified cap structures using the Suzuki-Miyaura cross-coupling reaction. Both methods employed phosphorimidazolide chemistry to form the 5',5'-triphosphate bridge. However, in the first method, the introduction of the modification via the Suzuki-Miyaura cross-coupling reaction at the C8 position occurs postsynthetically, at the dinucleotide level, whereas in the second method, the modification was introduced at the level of the nucleoside 5'-monophosphate, and later, the triphosphate bridge was formed. Both methods were successfully applied to incorporate six different groups (methyl, cyclopropyl, phenyl, 4-dimethylaminophenyl, 4-cyanophenyl, and 1-pyrene) into either the m7G or G moieties of the cap structure. Aromatic substituents at the C8-position of guanosine form a push-pull system that exhibits environment-sensitive fluorescence. We demonstrated that this phenomenon can be harnessed to study the interaction with cap-binding proteins, e.g., eIF4E, DcpS, Nudt16, and snurportin.

Novel N7-Arylmethyl Substituted Dinucleotide mRNA 5' cap Analogs: Synthesis and Evaluation as Modulators of Translation.

Dinucleotide analogs of the messenger RNA cap (m7GpppN) are useful research tools and have potential applications as translational inhibitors or reagents for modification of in vitro transcribed mRNAs. It has been previously reported that replacing the methyl group at the N7-position with benzyl (Bn) produces a dinucleotide cap with superior properties. Here, we followed up on this finding by synthesizing 17 novel Bn7GpppG analogs and determining their structure-activity relationship regarding translation and translational inhibition. The compounds were prepared in two steps, including selective N7-alkylation of guanosine 5'-monophosphate by arylmethyl bromide followed by coupling with imidazole-activated GDP, with total yields varying from 22% to 62%. The compounds were then evaluated by determining their affinity for eukaryotic translation initiation factor 4E (eIF4E), testing their susceptibility to decapping pyrophosphatase, DcpS-which is most likely the major cellular enzyme targeting this type of compound-and determining their translation inhibitory properties in vitro. We also synthesized mRNAs capped with the evaluated compounds and tested their translational properties in A549 cells. Our studies identified N7-(4-halogenbenzyl) substituents as promising modifications in the contexts of either mRNA translation or translational inhibition. Finally, to gain more insight into the consequences at the molecular level of N7-benzylation of the mRNA cap, we determined the crystal structures of three compounds with eIF4E.

Evaluation of carboxyfluorescein-labeled 7-methylguanine nucleotides as probes for studying cap-binding proteins by fluorescence anisotropy.

Fluorescence anisotropy (FA) is a powerful technique for the discovery of protein inhibitors in a high-throughput manner. In this study, we sought to develop new universal FA-based assays for the evaluation of compounds targeting mRNA 5' cap-binding proteins of therapeutic interest, including eukaryotic translation initiation factor 4E and scavenger decapping enzyme. For this purpose, a library of 19 carboxyfluorescein probes based on 7-methylguanine nucleotides was evaluated as FA probes for these proteins. Optimal probe:protein systems were further investigated in competitive binding experiments and adapted for high-throughput screening. Using a small in-house library of compounds, we verified and confirmed the accuracy of the developed FA assay to study cap-binding protein binders. The applications of the most promising probes were then extended to include evaluation of allosteric inhibitors as well as RNA ligands. From this analysis, we confirmed the utility of the method to study small molecule ligands and evaluate differently 5' capped RNAs.

The impact of the CYP2D6 gene polymorphism on the risk of pemphigoid.

BACKGROUND: Studies concerning the etiopathogenesis of numerous diseases emphasize the involvement of genetically determined impairments of xenobiotic metabolism. Nowadays, more attention has been drawn to the role of cytochrome P450 and its isoenzymes in the course of dermatological diseases, including pemphigoid, the most frequently occurring autoimmune bullous disease, whose etiopathogenesis has not been completely elucidated. AIM: The aim of the study was to find out whether there was any relationship between the CYP2D6 gene polymorphism and the development of bullous pemphigoid (BP). MATERIAL AND METHODS: The study included 221 subjects, 71 patients with BP, and 150 healthy volunteers constituting a control group. The identification of CYP2D6 (CYP2D6*1, CYP2D6*3, CYP2D6*4) gene alleles was performed using the polymerase chain reaction-fragment length polymorphism method. RESULTS: A higher frequency of the CYP2D6*3/CYP2D6*4 genotype was observed in patients with BP (P = 0.0033) than in controls. The relative risk of developing BP expressed with the odds ratio was nearly four times higher in subjects in whom the presence of the CYP2D6*3 allele was detected (odds ratio 3.8; P = 0.0234). CONCLUSION: The study results may suggest the impact of CYP2D6 gene polymorphism (A2637 deletion) on a higher prevalence of bullous pemphigoid.

[Hypersensitivity to acetylsalicylic acid]. / Nadwrazliwosc na kwas acetylosalicylowy.

Hypersensitivity to acetylsalicylic acid (ASA) is characterized by the co-occurrence of symptoms so-called aspirin triad, which include bronchial asthma, chronic rhinitis and sinusitis and the nasal mucosa polyps. The disease affects about 1% of the general population and in patients with bronchial asthma incidence may be as high as 10%. Hypersensitivity to aspirin is a difficult diagnostic problem, so the increased knowledge on this subject is a very important for the physicians of many specialties.

The relationship between plasma concentration of metoprolol and CYP2D6 genotype in patients with ischemic heart disease.

BACKGROUND: Metoprolol is the one of the most commonly used ß-blockers in the treatment of ischemic heart disease and it is extensively metabolized in the liver undergoing oxidation by CYP2D6 isoenzyme of cytochrome P450. Gene encoding the CYP2D6 enzyme is characterized by genetic polymorphism. The CYP2D6 oxidation polymorphism has a major impact on the effectiveness and safety of the treatment. The aim of the study was to evaluate the relationship between plasma concentration of metoprolol and the CYP2D6 genotype in patients with ischemic heart disease. METHODS: Fifty patients were interviewed and subsequently enrolled into the study. The patients received metoprolol twice daily at a dose of 50mg. The blood samples were analyzed for two major defective alleles for CYP2D6 - CYP2D6*4 and CYP2D6*3--by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Metoprolol concentration in plasma was determined by using the new and unique high-performance liquid chromatography (HPLC) method in the author's own modification with Corona CAD detector (Charged Aerosol Detection). RESULTS: In the test group, genotypes conditioning poor oxidation (PM) occurred in 3 patients (6%), while 47 patients (94%) had genotypes coding for extensive metabolism (EM). Patients with PM genotypes had significantly higher plasma concentrations of metoprolol than the patients with EM genotype (mean 92.25 ± SD 36.78 ng/ml vs. mean 168.22 ± SD 5.61 ng/ml, respectively). Established relationships were statistically significant (NIR test, p=0.0009). CONCLUSIONS: This study demonstrated that the CYP2D6 genotype remains a major determinant of the metoprolol plasma concentrations. The pharmacogenetic effect is likely to have consequences on both, the clinical benefit of metoprolol treatment and adverse drug reactions. The use of Corona CAD detector seems to be a very good alternative method for the determination of metoprolol concentration in plasma.

Significance of the genetic polymorphism of CYP2D6 and NAT2 in patients with inflammatory bowel diseases.

BACKGROUND: The main types of inflammatory bowel diseases (IBD) are ulcerative colitis (UC) and Crohn's disease (CD). There is evidence that, in addition to immunological and environmental factors, genetic factors also play an important role in the pathogenesis of IBD. Determination of polymorphism of CYP2D6 and NAT2 genes encoding I and II phase enzymes of xenobiotic biotransformation may have clinical value as an indicator of individual predisposition to diseases, and also contribute to effective and safe pharmacotherapy. The aim of this study was to investigate the association between genetic polymorphism of CYP2D6 and NAT2 and the incidence of IBD, including UC and CD, among inhabitants of central Poland. METHODS: The study was performed in 258 individuals from central Poland (115 patients with IBD, including 65 patients with UC and 50 with CD; and in 143 healthy controls). The CYP2D6 genotypes of oxidation and NAT2 genotypes of acetylation were analyzed using the PCR-RFLP method. RESULTS: There were no statistically significant differences in the frequency of the CYP2D6 genotypes and alleles in patients with IBD, UC and CD in comparison with the control group. The relative risk (OR) of IBD, UC and CD was higher in carriers of the allele NAT2*7 and was OR=3.49 (p=0.0019), OR=3.86 (p=0.0019), and OR=3.02 (p=0.0247), respectively. CONCLUSIONS: Polymorphism of the gene encoding CYP2D6 does not affect the incidence of inflammatory bowel diseases. The carriers of the NAT2*7 allele which determines slow acetylation may be more predisposed to inflammatory bowel diseases, including ulcerative colitis and Crohn's disease.

[Clinical significance of some genetic polymorphisms of cytochrome P-450 subfamilies CYP2D, CYP2E and CYP3A--part II]. / Kliniczne znaczenie polimorfizmu wybranych genów cytochromu P-450 podrodzin CYP2D, CYP2E i CYP3A--czesc II.

Polymorphism of drugs biotransformation is clinically significant from the point of view of the effectiveness and safety of pharmacotherapy and an increased risk of some illnesses. The paper presents molecular mechanisms and clinical implication of several genetic polymorphisms of cytochrome P-450 (CYP) xenobiotics metabolizing enzymes and genetic predisposition to the occurrence of some diseases.

[Clinical significance of some genetic polymorphisms of cytochrome P-450: family CYP1 and subfamilies CYP2A, CYP2B and CYP2C]. / Kliniczne znaczenie polimorfizmu wybranych genów cytochromu P-450: rodziny CYP1, podrodziny CYP2A, CYP2B oraz CYP2C.

The essence of pharmacogenetic studies is to provide a scientific explanation for personal differences in the process of drugs metabolism. Polymorphism of drugs biotransformation is clinically significant from the point of view of the effectiveness and safety of pharmacotherapy and an increased risk of some illnesses. The paper presents molecular mechanisms and clinical implication of several genetic polymorphisms of cytochrome P-450 (CYP) xenobiotics metabolizing enzymes and genetic predisposition to the occurrence of some diseases.

Genetic determinants in ischemic heart disease.

The present paper is a review of current knowledge about the relations between gene polymorphism and predisposition for ischemic heart disease. Many studies investigate polymorphic variants of genes whose protein products contribute to the genesis and development of atherosclerosis of coronary arteries, thrombogenesis and fibrinolysis and other processes significant for the progression of ischemic heart disease. In ischemic heart disease the most often analyzed genes are those connected with metabolism of lipids, the coagulation and fibrinolytic system and the renin-angiotensin-aldosterone system. Factors of inflammation (cytokines, TNF), proliferation of smooth muscles cells and vasoactivation are also important. Manifestation of the illness is connected with accumulation of several genetic determinants, while the clinical picture is additionally modified by environmental factors. Studies of genetic etiopathogenesis of ischemic heart disease may result in effective prevention and treatment in particular patients.

Polymorphism of dextromethorphan oxidation in Polish population.

Determination of oxidation phenotype is used to obtain the best results of pharmacotherapy and to explain a lower efficiency of some drugs in particular patients. The purpose of the present study was to determine the distribution of CYP2D6 metabolizer status in subjects from central Poland, using dextromethorphan as the probe drug. The study included 104 healthy Polish volunteers. All subjects received a single oral dose of 40 mg of dextromethorphan and the complete urine output was collected over a period of 10 h. DM and the metabolite dextrorphan (DT) were analyzed by HPLC method. The results of a Polish population study revealed a bimodal distribution of the dextromethorphan metabolic ratio and showed the existence of two oxidation phenotypes as extensive (EM) and poor (PM) metabolizers. In our population, the frequency of the PM phenotype was 9.6%. The results obtained (9.6% of PM phenotype) were in accordance with other results obtained in Poland and other Caucasian populations.

[Genetics in ischaemic heart disease]. / Genetyczne uwarunkowanie choroby niedokrwiennej serca.

The role of genetic factors in the pathogenesis of ischaemic heart disease has recently raised a considerable interest among researchers. Numerous investigations aim at finding variants of genes which might be responsible for increasing the risk of this illness. Many studies investigate polymorphic variants of genes whose protein products contribute to the genesis and development of atherosclerosis of coronary arteries, thrombogenesis and fibrinolysis and other processes significant for the progression of ischaemic heart disease. Genes whose polymorphisms are potentially connected with a given illness are called genes candidates. In ischaemic heart disease the most often analyzed genes are those connected with metabolism of lipids, the coagulation and fibrinolytic system and the renin-angiotensin-aldosterone system. Factors of inflammation (cytokines, TNF), proliferation of smooth muscles cells and vasoactivation are also important. The analysis of genetic multifunctional basis of the disease is rather difficult. Manifestation of the illness is connected with accumulation of several genetic determinants, while the clinical picture is additionally modified by environmental factors. Studies of genetic etiopathogenesis of ischaemic heart disease may result in effective prevention and treatment in particular patients.

CYP2D6 phenotyping with dextromethorphan.

Genetically determined individual differences in the ability to oxidize certain drugs have raised recently a considerable interest because of clinical importance of this problem. Determination of CYP2D6 oxidation phenotype is used to obtain more efficient pharmacotherapy and to explain lower efficacy of some drugs and presentation of adverse effects in particular patients. The aim of this study was to identify the CYP2D6 oxidation phenotype with dextromethorphan (DM) as a probe drug. The study included 85 healthy volunteers of Polish origin. DM (40 mg) was given orally to healthy adults and 10-h urine samples were collected. DM and the metabolite dextrorphan (DX) were analyzed by the HPLC method. Phenotyping was performed using the metabolic ratio (MR) calculated as the urinary DM/DX output. Based on the metabolic ratio, we can distinguish extensive (EM) and poor (PM) metabolizers in human population. Individuals with a dextromethorphan MR greater than 0.3 (log > -0.5) were classified as PMs. In our study, the frequency of the PM phenotype was 9.4%, which is in the range found in other Caucasian populations (3-10%).

Selenium as an anticancerogenic agent.

Selenium is an essential component of the intracellular antioxidant system, which defends the cell and the whole organism against oxidative damage and so from oxidative disease, especially from cancer. Selenium may be considered as an anticancerogenic agent, and a higher concentration of selenium in plasma may be a protective factor against human malignancies. It is also interesting to note that higher doses of selenium, applied to patients simultaneously with chemotherapy, may lower the toxicity of antineoplasm drugs without weakening the therapeutic effect.

Effects of six months melatonin treatment on sleep quality and serum concentrations of estradiol, cortisol, dehydroepiandrosterone sulfate, and somatomedin C in elderly women.

OBJECTIVES: The role of melatonin is aging is still under debate. Therefore, an open pilot study on the effects of melatonin administration on some sleep parameters, routine hematological and biochemical parameters, and concentrations of hormones was performed in elderly women. SUBJECTS AND METHODS: The study was performed on 14 women (volunteers), aged from 64 to 80 years (mean age 71+/-4.6 years). Melatonin (2 mg daily at 19:00 h) was administered during 6 months. Before and after melatonin treatment the peripheral venous blood samples were taken in the morning (approx. at 08:00 h) after the overnight fast. The total blood count, glucose, total cholesterol, LDL, HDL, and triglycerides were estimated by routine laboratory methods. The serum concentrations of the following hormones were determined: 17-beta-estradiol, dehydroepiandrosterone sulfate (DHEAS), cortisol, and somatomedin C (IGF-I). Additionally, before and after 6 months of melatonin therapy the investigated subjects answered to a questionnaire dealing with sleep parameters and self-estimation of general health status. RESULTS: In 35.7% of investigated subjects an improvement in general sleep quality and in such sleep parameters as sleep initiation, sleep latency, number of awakenings episodes, wake time after sleep onset, was observed. A significant decrease of estradiol concentrations was observed after 6 months of the melatonin treatment in comparison to initial levels. IGF-I was found to be slightly but significantly increased after the 6 months melatonin therapy. Cortisol levels did not change significantly, during the melatonin treatment. DHEAS concentrations increased after melatonin therapy. Moreover, a tendency towards a higher DHEAS/cortisol ratio was found after 6 months of treatment. Melatonin treatment did not influence significantly either the parameters of total blood count or glucose and serum lipids levels. CONCLUSIONS: On the basis of this preliminary open study it seems that melatonin administration may be beneficial for elderly subjects.