mRNA vaccines against H10N8 and H7N9 influenza viruses of pandemic potential are immunogenic and well tolerated in healthy adults in phase 1 randomized clinical trials.

BACKGROUND: We evaluated safety and immunogenicity of the first mRNA vaccines against potentially pandemic avian H10N8 and H7N9 influenza viruses. METHODS: Two randomized, placebo-controlled, double-blind, phase 1 clinical trials enrolled participants between December 2015 and August 2017 at single centers in Germany (H10N8) and USA (H7N9). Healthy adults (ages 18-64 years for H10N8 study; 18-49 years for H7N9 study) participated. Participants received vaccine or placebo in a 2-dose vaccination series 3 weeks apart. H10N8 intramuscular (IM) dose levels of 25, 50, 75, 100, and 400 µg and intradermal dose levels of 25 and 50 µg were evaluated. H7N9 IM 10-, 25-, and 50-µg dose levels were evaluated; 2-dose series 6 months apart was also evaluated. Primary endpoints were safety (adverse events) and tolerability. Secondary immunogenicity outcomes included humoral (hemagglutination inhibition [HAI], microneutralization [MN] assays) and cell-mediated responses (ELISPOT assay). RESULTS: H10N8 and H7N9 mRNA IM vaccines demonstrated favorable safety and reactogenicity profiles. No vaccine-related serious adverse event was reported. For H10N8 (N = 201), 100-µg IM dose induced HAI titers ≥ 1:40 in 100% and MN titers ≥ 1:20 in 87.0% of participants. The 25-µg intradermal dose induced HAI titers > 1:40 in 64.7% of participants compared to 34.5% of participants receiving the IM dose. For H7N9 (N = 156), IM doses of 10, 25, and 50 µg achieved HAI titers ≥ 1:40 in 36.0%, 96.3%, and 89.7% of participants, respectively. MN titers ≥ 1:20 were achieved by 100% in the 10- and 25-µg groups and 96.6% in the 50-µg group. Seroconversion rates were 78.3% (HAI) and 87.0% (MN) for H10N8 (100 µg IM) and 96.3% (HAI) and 100% (MN) in H7N9 (50 µg). Significant cell-mediated responses were not detected in either study. CONCLUSIONS: The first mRNA vaccines against H10N8 and H7N9 influenza viruses were well tolerated and elicited robust humoral immune responses. ClinicalTrials.gov NCT03076385 and NCT03345043.

Identification of oxidative degradates of the thrombin inhibitor, 3-(2-phenethylamino)-6-methyl-1-(2-amino-6-methyl-5-methyleneca rboxamidomethylpyridinyl)pyrazinone, using liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry.

Liquid chromatography/mass spectrometry was used to identify reaction products from a solution of 3-(2-phenethylamino)-6-methyl-1-(2-amino-6-methyl-5-methyleneca rboxamidomethylpyridinyl)pyrazinone (L-375,378) and hydrogen peroxide, a system that generates high levels of the oxidative degradates which form in the tablets and intravenous (i.v.) solutions of L-375,378. Two major hydrogen peroxide reaction products of L-375,378 (m/z 407) with m/z values of 369 and 370 were separated and identified. Both compounds were products of ring opening with elimination of three carbon atoms from the center pyrazinone ring. The structural assignments for these two products were alpha-amidinoamide and alpha-diamide compounds, respectively. In addition, five products (m/z 423) with a molecular weight 16 Da greater than that for L-375,378 were separated. Further liquid chromatography/tandem mass spectrometry experiments indicated that three of these M + 16 products were phenolic derivatives of L-375,378. Among them, the para-hydroxy compound has been verified using an authentic standard. The other two phenolic compounds were believed to be the meta- and ortho-hydroxy derivatives of L-375,378. The fourth M + 16 product was derived from hydroxylation of the methyl group on the center pyrazinone ring. The fifth M + 16 product was derived from oxidation on the aminopyridine moiety, most likely N-oxide of the pyridine ring. Other minor hydrogen peroxide reaction products were not studied in detail because they did not appear in tablets or i.v. formulations.

Meropenem exists in equilibrium with a carbon dioxide adduct in bicarbonate solution.

Evidence is provided for the existence of a second discrete chemical form of meropenem at clinically relevant concentrations prepared from the marketed formulation of the drug. Proton and carbon-13 NMR spectra in D2O, coupled with tandem mass spectroscopy (MS/MS) and cross polarization/magic angle spinning (CP-MAS) NMR experiments, allow structural assignment of the compound as a covalent carbon dioxide adduct of meropenem. This carbon dioxide adduct exists in equilibrium with the free drug in solution and can be observed in the solid state following lyophilization. The equilibrium constant of formation of the adduct (Keq = 20 +/- 8 M-1) was estimated in D2O at 25 degreesC.

The influence of the mode of enzyme preparation on enzymatic enantioselectivity in organic solvents and its temperature dependence.

The enantioselectivities of subtilisin Carlsberg and Rhizomucor miehei lipase in organic solvents are found to strongly depend on the method by which the enzymes are prepared. For the transesterification between sec-phenethyl alcohol and vinyl butyrate in dioxane at 7 degrees C, the enantioselectivity of subtilisin precipitated with isopropanol is more than twice that of the enzyme prepared by lyophilization from aqueous buffer. Furthermore, the temperature dependence of the enantioselectivity is influenced by the mode of enzyme preparation. For example, in the aforementioned process the enantioselectivities of subtilisin lyophilized from aqueous buffer and crosslinked subtilisin crystals increase when the temperature is raised from 7 to 45 degrees C. In contrast, the enantioselectivities decrease with temperature for the enzyme precipitated from aqueous solution with acetone or isopropanol and for the enzymatic hydrolysis in water. The temperature dependence of the enantioselectivity of subtilisin lyophilized from buffer is markedly affected by the solvent: In acetonitrile and nitromethane the enzyme is more enantioselective at higher temperatures, while negligible temperature effects have been found in tetrahydrofuran and pyridine. Lyophilized lipase exhibits striking temperature dependencies of its enantioselectivity in dioxane, acetonitrile, and nitromethane, while showing almost none in pyridine, triethylamine, and tetrahydrofuran. The results underscore the importance of the mode of enzyme recovery on enantioselectivity and its temperature dependence in enzymatic reactions in organic solvents (in contrast to those in water). (c) 1996 John Wiley & Sons, Inc.

Molecular mechanics calculations of the riboacetal internucleotide linkage in double and triple helices.

Structures of Watson-Crick base paired 15-nucleobase oligomer strands in A-type or B-type conformation in which one strand [a strand of alternating nucleotide and riboacetal thymidine nucleoside (RT) units, RP] is DNA and the other is composed of alternating nucleotides and riboacetal nucleosides have been studied by molecular mechanics. Analogously, oligomer strands of RNA in place of DNA have been modeled. The calculations indicate that the RP strand is more stable when complexed in an A-type duplex relative to a B-type form and that this conformational preference is presumably due to the more uniform nature of the former. Nearly planar ribose rings were more commonly observed in the minimized structures of the B-type DNA.RP duplexes as compared with A-type duplexes, despite the fact that planar ribofuranose rings are known to be energetically unfavorable in oligonucleotides. Computed relative stabilities of all duplexes containing the RP strand suggest that such heteroduplexes are less stable than the corresponding double-stranded DNA and double-stranded RNA species. These findings are in agreement with experimental results which show, when equivalent sequences were compared, that a DNA.RNA control forms a more stable duplex than RP hound to a complementary single-stranded RNA strand. In contrast, molecular mechanics studies of complementary triple-helical (DNA)2.RP, (DNA)2.DNA, and (DNA)2.RNA structures indicate that the binding of RP as a Hoogsteen strand stabilizes the underlying duplex to a greater extent compared with native oligonucleotides. These calculations suggest that puckering of the ribose ring in the riboacetal linkage leads to a more favorable interaction with a complementary nucleic acid target than the proposed planar geometry and that this puckering may account for the enhanced binding of RP to a double-stranded target.

Mechanism of aldehyde oxidation catalyzed by horse liver alcohol dehydrogenase.

The mechanism of oxidation of benzaldehyde to benzoic acid catalyzed by horse liver alcohol dehydrogenase (HLADH) has been investigated using the HLADH structure at 2.1 A resolution with NAD+ and pentafluorobenzyl alcohol in the active site [Ramaswamy et al. (1994) Biochemistry 33,5230-5237]. Constructs for molecular dynamics (MD) investigations with HLADH were obtained by a best-fit superimposition of benzaldehyde or its hydrate on the pentafluorobenzyl alcohol bound to the active site Zn(II)ion. Equilibrium bond lengths, angles, and dihedral parameters for Zn(II) bonding residues His67, Cys46, and Cys174 were obtained from small-molecule X-ray crystal structures and an ab initio-derived parameterization of zinc in HLADH [Ryde, U. (1995) Proteins: Struct., Funct., Genet. 21,40-56]. Dynamic simulations in CHARMM were carried out on the following three constructs to 100 ps: (MD1) enzyme with NAD+, benzaldehyde, and zinc-ligated HO-in the active site; (MD2) enzyme with NAD+ and hydrated benzaldehyde monoanion bound to zinc via the pro-R oxygen, with a proton residing on the pro-S oxygen; and (MD3) enzyme with NAD+ and hydrated benzaldehyde monoanion bound to zinc via the pro-S oxygen, with a proton residing on the pro-R oxygen. Analyses were done of 800 sample conformations taken in the last 40 ps of dynamics. Structures from MD1 and MD3 were used to define the initial spatial arrangements of reactive functionalities for semiempirical PM3 calculations. Using PM3, model systems were calculated of ground states and some transition states for aldehyde hydration, hydride transfer, and subsequent proton shuttling. With benzaldehyde and zinc-bound hydroxide ion in the active site, the oxygen of Zn(II)-OH resided at a distance of 2.8-5.5 A from the aldehyde carbonyl carbon during the dynamics simulation. This may be compared to the PM3 transition state for attack of the Zn(II)-OH oxygen on the benzaldehyde carbonyl carbon, which has an O...C distance of 1.877 A. HLADH catalysis of the aldehyde hydration would require very little motion aside from that in the ground state. Two simulations of benzaldehyde hydrate ligated to zinc (MD2 and MD3) both showed close approach of the aldehyde hydrate hydrogen to NAD+C4, varying from 2.3 to 3.3 A, seemingly favorable for the hydride transfer reaction. The MD2 configuration does not allow proton shuttling. On the other hand, when the pro-S oxygen is ligated to zinc (MD3), the proton on the pro-R oxygen averages 2.09 A from the hydroxyl oxygen of Ser48 such that initiation of shuttling of protons via Ser48 to the ribose 2'-hydroxyl oxygen to the 3'-hydroxyl oxygen to His51 nitrogen is sterically favorable. PM3 calculations suggest that this proton shuttle represents a stepwise reaction which occurs subsequent to hydride transfer. The PM3 transition state for hydride transfer based on the MD3 configuration has the transferring hydride 1.476 A from C4 of NAD+ and 1.433 A from the aldehyde alpha-carbon.

Remarkable activation of enzymes in nonaqueous media by denaturing organic cosolvents.

The rates of transesterification reactions catalyzed by the protease subtilisin Carlsberg suspended in various anhydrous solvents at 30 degrees C can be increased more than 100-fold by the addition of denaturing organic cosolvents (dimethyl sulfoxide or formamide); in water, the same cosolvents exert no enzyme activation. At 4 degrees C, the activation effect on the lyophilized protease is even higher, reaching 1000-fold. Marked enhancement of enzymatic activity in anhydrous solvents by formamide is also observed for two other enzymes, alpha-chymotrypsin and Rhizomucor miehei lipase, and is manifested in two transesterification reactions. In addition to lyophilized subtilisin, crosslinked crystals of subtilisin are also amenable to the dramatic activation by the denaturing cosolvents. In contrast, subtilisin solubilized in anhydrous media by covalent modification with poly(ethylene glycol) exhibits only modest activation. These observations are rationalized in terms of a mechanistic hypothesis based on an enhanced protein flexibility in anhydrous millieu brought about by the denaturing organic cosolvents. The latter exert their lubricating effect largely at the interfaces between enzyme molecules in a solid preparation, thus easing the flexibility constraints imposed by protein-protein contacts.

Design and synthesis of deoxynucleic guanidine: a polycation analogue of DNA.

The basic strategy is described for the connection of nucleosides by guanidinium (g) linkers to provide the positively charged deoxynucleic guanidine putative antigene agents. The synthetic procedures are provided for d(gT)n. Molecular modeling of double-stranded [d(gT)10.d(Ap)10] and the triple-helical hybrids [d(Tp)10.d(Ap)10.d(gT)10] and [d(gT)10.d(Ap)10.d(gT)10] suggest modes of interaction and anticipated structural features.

Peptide nucleic acid (PNA) conformation and polymorphism in PNA-DNA and PNA-RNA hybrids.

Two hydrogen-bonding motifs have been proposed to account for the extraordinary stability of polyamide "peptide" nucleic acid (PNA) hybrids with nucleic acids. These interresidue- and intraresidue-hydrogen-bond motifs were investigated by molecular mechanics calculations. Energy-minimized structures of Watson-Crick base-paired decameric duplexes of PNA with A-, B-, and Z-DNA and A-RNA polymorphs indicate that the inherent stability of the complementary PNA helical structures is derived from interresidue, rather than from intraresidue, hydrogen bonds in all hybrids studied. Intraresidue-hydrogen-bond lengths are consistently longer than interresidue hydrogen bonds. Helical strand stability with interresidue hydrogen bond stabilization follows the order: B-(DNA.PNA) > A-(DNA.PNA) congruent to A-RNA.PNA > Z-(DNA.PNA). In the triplex hybrids A-(RNA.PNA2) and B-(DNA.PNA2), differences between stabilities of the two decamers of thyminyl PNA with lysine amide attached to the C terminus (pnaT)10 strands are small. The Hoogsteen (pnaT)10 strands are of slightly higher potential energy than are the Watson-Crick (pnaT)10 strands. Antiparallel arrangement of PNAs in the triplex is slightly favored over the parallel arrangement based on the calculations. Examination by molecular mechanics of the PNA.DNA analogue of the NMR-derived structure for the B-double-stranded DNA dodecamer d(CG-CAAATTTGCG)2 in solution suggests that use of all bases of the genetic alphabet should be possible without loss of the specific interresidue-hydrogen-bonding pattern within the PNA strand.

Molecular mechanics calculations of the structures of polyamide nucleic acid DNA duplexes and triple helical hybrids.

Polyamide nucleic acids (PNAs) have emerged as useful agents for recognition of single- and double-stranded nucleic acids. Interresidue hydrogen bonds between the amide carbonyl nearest the nucleobase and chain NH moieties provide inherent stability to the helical conformation of PNA 1. Moving the amide carbonyl away from the nucleobase to the backbone, and replacing it with a methylene group, results in 2 lacking the stabilizing hydrogen bond. Oligomers of 2 do not interact with DNA. Modeling suggests that 2 displays a more extended conformation than 1, and nucleobase orientation is disrupted in 2 in the absence of a complementary DNA strand. This is in contrast to 1, which retains a centrosymmetric arrangement of nucleobases. Structures for 1-T10.DNA and (1-T10)2.DNA species spanned by a pyrimidine strand (D-loop) were constructed. In the triple helical (1-T10)2.DNA structure, the two PNA strands form the complementary Watson-Crick paired strand and the Hoogsteen base-paired strand in the major groove of the 1.DNA duplex. The PNA strands are proposed to bind antiparallel to one another in (1-T10)2.DNA structure. The factors suggested to account for the stability of this 2:1 complex are (i) a hydrophobic attraction between two PNA backbones and (ii) a favorable electrostatic effect resulting from replacement of a phosphodiester backbone by a neutral peptide backbone.

Systematic inflation of buckminsterfillerene c60: synthesis of diphenyl fulleroids c61 to c66.

The synthesis of a new family of spheroidal carbon molecules derived from the fullerenes is described. The fulleroids are produced by incremental addition of a divalent carbon equivalent that has two phenyl (Ph) rings to fullerene C(60). The fulleroids Ph(2)C(61), Ph(4)C(62), Ph(6)C(63), Ph(8)C(64), Ph(10)C(65), and Ph(12)C(66) have been prepared and characterized.