Evaluation of intranasal nafamostat or camostat for SARS-CoV-2 chemoprophylaxis in Syrian golden hamsters.

Successful development of a chemoprophylaxis against SARS-CoV-2 could provide a tool for infection prevention implementable alongside vaccination programmes. Camostat and nafamostat are serine protease inhibitors that inhibit SARS-CoV-2 viral entry in vitro but have not been characterised for chemoprophylaxis in animal models. Clinically, nafamostat is limited to intravenous delivery and while camostat is orally available, both drugs have extremely short plasma half-lives. This study sought to determine whether intranasal dosing at 5 mg/kg twice daily was able to prevent airborne transmission of SARS-CoV-2 from infected to uninfected Syrian golden hamsters. SARS-CoV-2 viral RNA was above the limits of quantification in both saline- and camostat-treated hamsters 5 days after cohabitation with a SARS-CoV-2 inoculated hamster. However, intranasal nafamostat-treated hamsters remained RNA negative for the full 7 days of cohabitation. Changes in body weight over the course of the experiment were supportive of a lack of clinical symptomology in nafamostat-treated but not saline- or camostat-treated animals. These data are strongly supportive of the utility of intranasally delivered nafamostat for prevention of SARS-CoV-2 infection and further studies are underway to confirm absence of pulmonary infection and pathological changes.

Structure of a histidine-X4-histidine zinc finger domain: insights into ADR1-UAS1 protein-DNA recognition.

The solution structure for a mutant zinc finger peptide based on the sequence of the C-terminal ADR1 finger has been determined by two-dimensional NMR spectroscopy. The mutant peptide, called PAPA, has both proline residues from the wild-type sequence replaced with alanines. A nonessential cysteine was also replaced with alanine. The behavior of PAPA in solution implicates the prolines in the conformational heterogeneity reported earlier for the wild-type peptide [Xu, R. X., Horvath, S. J., & Klevit, R. E. (1991) Biochemistry 30, 3365-3371]. The solution structure of PAPA reveals several interesting features of the zinc finger motif. The residue immediately following the second cysteine ligand adopts a positive phi angle, which we propose is a common feature of this class of zinc fingers, regardless of whether this residue is a glycine. The NMR spectrum and resulting solution structure of PAPA suggest that a side-chain to side-chain hydrogen bond involving an arginine and an aspartic acid analogous to one observed in the Zif268 protein-DNA cocrystal structure exists in solution in the absence of DNA [Pavletich, N. P., & Pabo, C. O. (1991) Science 252, 809-817]. A model for the interaction between the two ADR1 zinc fingers and their DNA binding sites was built by superpositioning the refined solution structures of PAPA and ADR1b onto the Zif268 structure. This model offers structural explanations for a variety of mutations to the ADR1 zinc finger domains that have been shown to affect DNA-binding affinity or specificity.

Atomic structure of ferredoxin-NADP+ reductase: prototype for a structurally novel flavoenzyme family.

The three-dimensional structure of spinach ferredoxin-NADP+ reductase (NADP+, nicotinamide adenine dinucleotide phosphate) has been determined by x-ray diffraction at 2.6 angstroms (A) resolution and initially refined to an R factor of 0.226 at 2.2 A resolution. The model includes the flavin-adenine dinucleotide (FAD) prosthetic group and the protein chain from residue 19 through the carboxyl terminus at residue 314 and is composed of two domains. The FAD binding domain (residues 19 to 161) has an antiparallel beta barrel core and a single alpha helix for binding the pyrophosphate of FAD. The NADP binding domain (residues 162 to 314) has a central five-strand parallel beta sheet and six surrounding helices. Binding of the competitive inhibitor 2'-phospho-AMP (AMP, adenosine monophosphate) places the NADP binding site at the carboxyl-terminal edge of the sheet in a manner similar to the nucleotide binding of the dehydrogenase family. The structures reveal the key residues that function in cofactor binding and the catalytic center. With these key residues as a guide, conclusive evidence is presented that the ferredoxin reductase structure is a prototype for the nicotinamide dinucleotide and FAD binding domains of the enzymes NADPH-cytochrome P450 reductase, NADPH-sulfite reductase, NADH-cytochrome b5 reductase, and NADH-nitrate reductase. Thus this structure provides a structural framework for the NADH- or NADPH-dependent flavoenzyme parts of five distinct enzymes involved in photosynthesis, in the assimilation of inorganic nitrogen and sulfur, in fatty-acid oxidation, in the reduction of methemoglobin, and in the metabolism of many pesticides, drugs, and carcinogens.

Solution structure of a zinc finger domain of yeast ADR1.

The "zinc finger" is a 30-residue repeating motif that has been identified in a variety of eukaryotic transcription factors. Each domain is capable of binding a Zn2+ ion through invariant Cys and His residues. We have determined the three-dimensional structure of a synthetic peptide that corresponds to one of the two zinc finger domains in the yeast transcription factor ADR1, using two-dimensional nuclear magnetic resonance spectroscopy. The Zn2(+)-bound structure of the peptide consists of a loop containing the two Cys residues, a "fingertip," a 12- to 13-residue alpha-helix containing the two His residues, and a C-terminal tail. A majority of the interresidue contacts observed involve the seven conserved residues of the prototypic zinc finger (i.e., the four zinc ligands and the three hydrophobic residues), indicating that these residues are largely responsible for the three-dimensional structure of the domain and that all the zinc finger domains of the TFIIIA class will have similar structures. Potential DNA-binding residues are found throughout the structure, with the highest concentration of such residues on the external face of the alpha-helix.

Amino acid sequence of spinach ferredoxin:NADP+ oxidoreductase.

The amino acid sequence of spinach ferredoxin: NADP+ oxidoreductase was determined by using overlapping sets of peptides derived by cleavage at arginyl or methionyl residues. The protein from different preparations varied in its length at the amino terminus. In the longest form the amino terminus is blocked with a pyroglutamyl residue, as determined by NMR. A single disulfide bond was placed between cysteine residues 132 and 137. The 314-residue sequence corresponds to a molecular weight of 35 317. The carboxyl-terminal half of the sequence has been fit to the electron density map of the NADP binding domain, revealing that this portion of the chain forms a typical nucleotide binding fold.