Hindered nucleoside analogs as antiflaviviridae agents

Abstract Flaviviridae are an important family of viruses, responsible for widely spread diseases such as dengue and West Nile fever and hepatitis C. Despite the severity of the related diseases, no effective antiviral treatments for infection are available. Following our discovery of adenosine-hindered analogs as potent antiflaviviridae agents, we have continued our investigation on guanosine and inosine derivatives, which were evaluated for activity against BVDV, YFV, DENV, and WNV viruses in cell-based assays. The present study allowed us to identify some newer features that led to improve the antiviral potency (down to the µM range) and to selectively inhibit BVDV and YFV viruses. The molecular modeling results were consistent with the hypothesis that test analogs act as RNA-dependent RNA polymerase (RdRp) inhibitors by interacting with a surface allosteric binding pocket

A non-invasive method for the in vivo determination of skin antioxidant capacity

Skin antioxidant network protects cells against oxidative injury and prevents the production of oxidation products. When oxidative stress overwhelms the skin antioxidant capacity, the subsequent modification of the cellular redox apparatus leads to an alteration of cell homeostasis leading to degenerative processes. In the dermocosmetic field, the topical application of antioxidants is often suggested as a possible strategy to prevent and modulate oxidative skin damages. Continuing our studies addressed to set-up new bio-engineering protocols for the claim substantiation of antioxidant cosmetic products, we have developed a new non-invasive methodology for the evaluation of antioxidants cosmetics ingredients and finished products. METHODS: The effects of a pre-treatment on forearm skin with an antioxidant ingredient were investigated on 15 volunteers, in a double-blind randomised fashion. A non-invasive method was devised that comprises the collection of forehead SC layers of the pre-treated area and control and the next evaluation of skin antioxidant capacity (IAC-S) by a luminescence-based method. RESULTS: The results showed that the antioxidant preparation was able to increase, to a statistically significant extent (P<0.01), the IAC-S in comparison with the control area. The data were confirmed (P<0.05) by comparison with a method, previously developed by us, based on DermAnalyzer. CONCLUSIONS: In view of the simplicity and reliability of the method here presented, this new technique is proposed as a possible tool for the routine evaluation of in vivo efficacy of antioxidant functional ingredients and finished products

Two novel mutations in thymidine kinase-2 cause early onset fatal encephalomyopathy and severe mtDNA depletion

Deficiency of thymidine kinase-2 (TK2) has been described in children with early onset fatal skeletal myopathy. TK2 is a mitochondrial deoxyribonucleoside kinase required for the phosphorylation of deoxycytidine and deoxythymidine and hence is vital for the maintenance of a balanced mitochondrial dNTP pool in post-mitotic tissues. We describe a patient with two novel TK2 mutations, which caused disease onset shortly after birth and death at the age of three months. One mutation (219insCG) generated an early stop codon, thus preventing the synthesis of a functional protein. The second mutation (R130W) resulted in an amino acid substitution, which caused a severe reduction (<3%) of TK2 enzyme activity. These two novel TK2 mutations cause an extremely severe phenotype with overwhelming central nervous system symptoms not commonly seen in patients with TK2-deficiency. We conclude that the severe clinical presentation in this patient was due to a virtual lack of mitochondrial TK2 activity

Engineering of a single conserved amino acid residue of herpes simplex virus type 1 thymidine kinase allows a predominant shift from pyrimidine to purine nucleoside phosphorylation.

Studies of herpes simplex virus type 1 (HSV-1) thymidine (dThd) kinase (TK) crystal structures show that purine and pyrimidine bases occupy distinct positions in the active site but approximately the same geometric plane. The presence of a bulky side chain, such as tyrosine at position 167, would not be sterically favorable for pyrimidine or pyrimidine nucleoside analogue binding, whereas purine nucleoside analogues would be less affected because they are located further away from the phenylalanine side chain. Site-directed mutagenesis of the conserved Ala-167 and Ala-168 residues in HSV-1 TK resulted in a wide variety of differential affinities and catalytic activities in the presence of the natural substrate dThd and the purine nucleoside analogue drug ganciclovir (GCV), depending on the nature of the amino acid mutation. A168H- and A167F-mutated HSV-1 TK enzymes turned out to have a virtually complete knock-out of dThd kinase activity (at least approximately 4-5 orders of magnitude lower) presumably due to a steric clash between the mutated amino acid and the dThd ring. In contrast, a full preservation of the GCV (and other purine nucleoside analogues) kinase activity was achieved for A168H TK. The enzyme mutants also markedly lost their binding capacity for dThd and showed a substantially diminished feedback inhibition by thymidine 5'-triphosphate. The side chain size at position 168 seems to play a less important role regarding GCV or dThd selectivity than at position 167. Instead, the nitrogen-containing side chains from A168H and A168K seem necessary for efficient ligand discrimination. This explains why A168H-mutated HSV-1 TK fully preserves its GCV kinase activity (Vmax/Km 4-fold higher than wild-type HSV-1 TK), although still showing a severely compromised dThd kinase activity (Vmax/Km 3-4 orders of magnitude lower than wild-type HSV-1 TK)

Engineering of a single conserved amino acid residue of herpes simplex virus type 1 thymidine kinase allows a predominant shift from pyrimidine to purine nucleoside phosphorylation.

Studies of herpes simplex virus type 1 (HSV-1) thymidine (dThd) kinase (TK) crystal structures show that purine and pyrimidine bases occupy distinct positions in the active site but approximately the same geometric plane. The presence of a bulky side chain, such as tyrosine at position 167, would not be sterically favorable for pyrimidine or pyrimidine nucleoside analogue binding, whereas purine nucleoside analogues would be less affected because they are located further away from the phenylalanine side chain. Site-directed mutagenesis of the conserved Ala-167 and Ala-168 residues in HSV-1 TK resulted in a wide variety of differential affinities and catalytic activities in the presence of the natural substrate dThd and the purine nucleoside analogue drug ganciclovir (GCV), depending on the nature of the amino acid mutation. A168H- and A167F-mutated HSV-1 TK enzymes turned out to have a virtually complete knock-out of dThd kinase activity (at least approximately 4-5 orders of magnitude lower) presumably due to a steric clash between the mutated amino acid and the dThd ring. In contrast, a full preservation of the GCV (and other purine nucleoside analogues) kinase activity was achieved for A168H TK. The enzyme mutants also markedly lost their binding capacity for dThd and showed a substantially diminished feedback inhibition by thymidine 5'-triphosphate. The side chain size at position 168 seems to play a less important role regarding GCV or dThd selectivity than at position 167. Instead, the nitrogen-containing side chains from A168H and A168K seem necessary for efficient ligand discrimination. This explains why A168H-mutated HSV-1 TK fully preserves its GCV kinase activity (Vmax/Km 4-fold higher than wild-type HSV-1 TK), although still showing a severely compromised dThd kinase activity (Vmax/Km 3-4 orders of magnitude lower than wild-type HSV-1 TK)

Progressive loss of mitochondrial DNA in thymidine kinase 2-deficient mice

Deficient enzymatic activity of the mitochondrial deoxyribonucleoside kinases deoxyguanosine kinase (DGUOK) or thymidine kinase 2 (TK2) cause mitochondrial DNA (mtDNA)-depletion syndromes in humans. Here we report the generation of a Tk2-deficient mouse strain and show that the mice develop essentially normally for the first week but from then on exhibit growth retardation and die within 2-4 weeks of life. Several organs including skeletal muscle, heart, liver and spleen showed progressive loss of mtDNA without increased mtDNA mutations or structural alterations. There were no major histological changes in skeletal muscle, but heart muscle showed disorganized and damaged muscle fibers. Electron microscopy showed mitochondria with distorted cristae. The Tk2-deficient mice exhibited pronounced hypothermia and showed loss of hypodermal fat and abnormal brown adipose tissue. We conclude that Tk2 has a major role in supplying deoxyribonucleotides for mtDNA replication and that other pathways of deoxyribonucleotide synthesis cannot compensate for loss of this enzyme

AZITROMICINA VERSUS DOXICICLINA NELLE URETRITI MASCHILI DA CHLAMYDIA TRACHOMATIS

The efficacy of a short-term therapy with azithromycin in the treatment of urethral Chlamydia trachomatis infections in males was compared to that of a standard seven-days course of treatment with doxycycline. Urethral samples from 60 male patients reacted positively in a DNA-probe method for detection of Chlamydia trachomatis. Thirty of the 60 patients were treated with azithromycin for two days in a dosage of 1 g the first day and 0.5 g the second day, and thirty with doxycyclin in a dosage of 200 mg daily for seven days. Twenty-seven patients treated with azithromycin and 18 patients treated with doxycycline returned to follow-up, which was performed one week arter the end of the therapy. The control urethral samples were negative, with the exception of one from a patient in the azithromycin group. However, a second control after one week gave negative results. We found no differences between the two treatment groups with respect to the clinical and laboratoristic parameters and conclude that both treatments are equally efficacious. However, it should be also stressed that due to a frequent lack of patient compliance a short-term therapy with azithromycin is much more reliable than a course of several days with doxycycline