Efficient incorporation and template-dependent polymerase inhibition are major determinants for the broad-spectrum antiviral activity of remdesivir

Remdesivir (RDV) is a direct-acting antiviral agent that is approved in several countries for the treatment of coronavirus disease 2019 caused by the severe acute respiratory syndrome coronavirus 2. RDV exhibits broad-spectrum antiviral activity against positive-sense RNA viruses, for example, severe acute respiratory syndrome coronavirus and hepatitis C virus, and nonsegmented negative-sense RNA viruses, for example, Nipah virus, whereas segmented negative-sense RNA viruses such as influenza virus or Crimean-Congo hemorrhagic fever virus are not sensitive to the drug. The reasons for this apparent efficacy pattern are unknown. Here, we expressed and purified representative RNA-dependent RNA polymerases and studied three biochemical parameters that have been associated with the inhibitory effects of RDV-triphosphate (TP): (i) selective incorporation of the nucleotide substrate RDV-TP, (ii) the effect of the incorporated RDV-monophosphate (MP) on primer extension, and (iii) the effect of RDV-MP in the template during incorporation of the complementary UTP. We found a strong correlation between antiviral effects and efficient incorporation of RDV-TP. Inhibition in primer extension reactions was heterogeneous and usually inefficient at higher NTP concentrations. In contrast, template-dependent inhibition of UTP incorporation opposite the embedded RDV-MP was seen with all polymerases. Molecular modeling suggests a steric conflict between the 1′-cyano group of the inhibitor and residues of the structurally conserved RNA-dependent RNA polymerase motif F. We conclude that future efforts in the development of nucleotide analogs with a broader spectrum of antiviral activities should focus on improving rates of incorporation while capitalizing on the inhibitory effects of a bulky 1′-modification.     

Type I interferons and MAVS signaling are necessary for tissue resident memory CD8+ T cell responses to RSV infection

Respiratory syncytial virus (RSV) can cause bronchiolitis and viral pneumonia in young children and the elderly. Lack of vaccines and recurrence of RSV infection indicate the difficulty in eliciting protective memory immune responses. Tissue resident memory T cells (T_RM) can confer protection from pathogen re-infection and, in human experimental RSV infection, the presence of lung CD8⁺ T_RM cells correlates with a better outcome. However, the requirements for generating and maintaining lung T_RM cells during RSV infection are not fully understood. Here, we use mouse models to assess the impact of innate immune response determinants in the generation and subsequent expansion of the T_RM cell pool during RSV infection. We show that CD8⁺ T_RM cells expand independently from systemic CD8⁺ T cells after RSV re-infection. Re-infected MAVS and MyD88/TRIF deficient mice, lacking key components involved in innate immune recognition of RSV and induction of type I interferons (IFN-α/β), display impaired expansion of CD8⁺ T_RM cells and reduction in antigen specific production of granzyme B and IFN-γ. IFN-α treatment of MAVS deficient mice during primary RSV infection restored T_RM cell expansion upon re-challenge but failed to recover T_RM cell functionality. Our data reveal how innate immunity, including the axis controlling type I IFN induction, instructs and regulates CD8⁺ T_RM cell responses to RSV infection, suggesting possible mechanisms for therapeutic intervention.     

Resonance Raman and EPR investigations of the D251N oxycytochrome P450cam/putidaredoxin complex

We have performed resonance Raman and electron paramagnetic resonance (EPR) studies on the dioxygen bound state of the D251N mutant of cytochrome P450cam (oxy-P450cam) and its complex with reduced putidaredoxin (Pd). The D251N oxy-P450cam/Pd complex has a perturbed proton delivery mechanism and shows a significantly red-shifted UV-visible spectrum as observed in Benson et al. [Benson, D. E., Suslick, K. S., and Sligar, S. G. (1997) Biochemistry 36, 5104-5107]. The red shift has been interpreted to indicate a major perturbation of the electronic structure of the oxy-heme complex. However, we find no evidence that electron transfer has occurred from Pd to the heme active site of D251N oxy-P450cam. This suggests that both electron and proton transfer are perturbed by the D251N mutation and that these processes may be coupled. Three oxygen isotope sensitive Raman features are identified in the Pd complex, and occur at 1137, 536, and 399 cm(-1). These values are not significantly different from those for WT or D251N oxy-P450cam. However, a careful examination of the oxygen stretching feature near 1137 cm(-1) reveals the presence of three peaks at 1131, 1138, and 1146 cm(-1), which we attribute to the presence of conformational substates in oxy-P450cam. A significant change in the conformational substate population is observed for the D251N oxy-P450cam when the Pd complex is formed. We suggest that the conformational population redistribution of oxy-P450cam, along with the red-shifted electronic spectra, reflects a structural equilibrium of the oxy-heme that is perturbed upon Pd binding. We propose that this structural perturbation is connected to the effector function of Pd and may involve changes in the electron donation properties of the thiolate ligand.     

Effects of ovine LH, GH and prolactin, and testosterone on serum testosterone and estradiol-17 beta levels, and seminal vesicle and testicular activity in the catfish Clarias batrachus (L.)

Intraperitoneal administrations of testosterone (0.5 microgram/g body wt), and ovine LH (1.0 microgram/g body wt), GH (5 micrograms/g body wt) and prolactin (10 micrograms/g body wt) daily for 7 days during early prespawning phase (May) in C. batrachus produced varied effects on seminal vesicle (SVSI) and testicular (GSI) weights and biochemical correlates. Testosterone and LH treatments significantly increased serum testosterone level and concentrations of total proteins, fructose, hexosamines and sialic acid in both seminal vesicles and testis. Serum E2 levels increased significantly only after testosterone treatment. GH treatment increased significantly serum testosterone level and only the concentrations of SV hexosamines and testicular protein. Prolactin, however, significantly lowered serum testosterone level and concentrations of total protein, hexosamines in both SV and testis, and testicular fructose and sialic acid levels. The results show that the stimulating effect of LH and GH on SV and testicular activity is mediated through the increased secretion of testosterone and the inhibitory effect of prolactin by decreased testosterone secretion.     

Circular permutation of granulocyte colony-stimulating factor

The sequence of granulocyte colony-stimulating factor (G-CSF) has been circularly permuted by introducing new chain termini into interhelical loops and by constraining the N- and C-terminal helices, either by direct linkage of the termini (L0) or by substitution of the amino-terminal 10-residue segment with a seven-residue linker composed of glycines and serines (L1). All the circularly permuted G-CSFs (cpG-CSFs) were able to fold into biologically active structures that could recognize the G-CSF receptor. CD and NMR spectroscopy demonstrated that all of the cpG-CSFs adopted a fold similar to that of the native molecule, except for one [cpG-CSF(L1)[142/141]] which has the new termini at the end of loop 34 with the shorter L1 linker. All of the cpG-CSFs underwent cooperative unfolding by urea, and a systematically lower free energy change (DeltaGurea) was observed for molecules with the shorter L1 linker than for those molecules in which the original termini were directly linked (the L0 linker). The thermodynamic stability of the cpG-CSFs toward urea was found to correlate with their relative ability to stimulate proliferation of G-CSF responsive cells. Taken together, these results indicate that the G-CSF sequence is robust in its ability to undergo linear rearrangement and adopt a biologically active conformation. The choice of linker, with its effect on stability, seems to be important for realizing the full biological activity of the three-dimensional structure. The breakpoint and linker together are the ultimate determinants of the structural and biological profiles of these circularly permuted cytokines. In the following paper [McWherter, C. A., et al. (1999) Biochemistry 38, 4564-4571], McWherter and co-workers have used circularly permuted G-CSF sequences to engineer chimeric dual IL-3 and G-CSF receptor agonists in which the relative spatial orientation of the receptor agonist domains is varied. Interpreting the differences in activity for the chimeric molecules in terms of the connectivity between domains depends critically on the results reported here for the isolated cpG-CSF domains.     

Antioxidant and anticarcinogenic activity of Lycovin--an indigenous herbal preparation

Aqueous extract of Lycovin has been found to be a potent inhibitor of lipid peroxide formation, (IC50 = 500 micrograms/ml) and scavenger of hydroxyl radical (IC50 = 44 micrograms/ml) and superoxide radical (IC50 = 30 micrograms/ml) in vitro. Lycovin syrup 1.5 ml and 7.5 ml/kg body wt administered orally, reduced the development of sarcoma induced by 20 MC by 35% and 70% respectively. Lycovin syrup was also found to inhibit the hepatocarcinogenesis induced by NDEA. The tumour incidence was 100% in the control group, while none of the drug treated animals developed tumour. Liver weight, gamma-glutamyl transpeptidase (GGT), GSH-S-transferase (GST), reduced glutathione, (GSH) and aniline-4-hydroxylase in liver were elevated in NDEA alone treated animals. The serum parameters indicative of liver injury such as bilirubin, lipid peroxides, alkaline phosphatase and glutamate pyruvate transaminase were also elevated by NDEA administration. These elevated parameters were significantly reduced in animals treated with Lycovin syrup along with NDEA in a dose dependent manner. Even though the exact mechanism of action is not known at present, the observed anticarcinogenic activity may be due to the inhibition of P.450 enzyme activity and subsequent inhibition of the production of the ultimate carcinogen as well as scavenging of oxygen free radicals during promotion of the transformed cell.     

The carbonate radical anion-induced covalent aggregation of human copper, zinc superoxide dismutase, and alpha-synuclein: intermediacy of tryptophan- and tyrosine-derived oxidation products

In this review, we describe the free radical mechanism of covalent aggregation of human copper, zinc superoxide dismutase (hSOD1). Bicarbonate anion (HCO3-) enhances the covalent aggregation of hSOD1 mediated by the SOD1 peroxidase-dependent formation of carbonate radical anion (CO3*-), a potent and selective oxidant. This species presumably diffuses out the active site of hSOD1 and reacts with tryptophan residue located on the surface of hSOD1. The oxidative degradation of tryptophan to kynurenine and N-formyl kynurenine results in the covalent crosslinking and aggregation of hSOD1. Implications of oxidant-mediated aggregation of hSOD1 in the increased cytotoxicity of motor neurons in amyotrophic lateral sclerosis are discussed.     

Regulation of C. elegans longevity by specific gustatory and olfactory neurons

The life span of C. elegans is extended by mutations that inhibit the function of sensory neurons. In this study, we show that specific subsets of sensory neurons influence longevity. We find that certain gustatory neurons inhibit longevity, whereas others promote longevity, most likely by influencing insulin/IGF-1 signaling. Olfactory neurons also influence life span, and they act in a distinct pathway that involves the reproductive system. In addition, we find that a putative chemosensory G protein-coupled receptor that is expressed in some of these sensory neurons inhibits longevity. Together our findings imply that the life span of C. elegans is regulated by environmental cues and that these cues are perceived and integrated in a complex and sophisticated fashion by specific chemosensory neurons.