AG7088 is a potent, irreversible inhibitor of human rhinovirus (HRV) 3C protease {inactivation rate constant (k obs /[I]} ‫؍‬ 1,470,000 ؎ 440,000 M ؊1 s ؊1 for HRV 14) that was discovered by protein structure-based drug design methodologies. In H1-HeLa and MRC-5 cell protection assays, AG7088 inhibited the replication of all HRV serotypes (48 of 48) tested with a mean 50% effective concentration (EC 50 ) of 0.023 M (range, 0.003 to 0.081 M) and a mean EC 90 of 0.082 M (range, 0.018 to 0.261 M) as well as that of related picornaviruses including coxsackieviruses A21 and B3, enterovirus 70, and echovirus 11. No significant reductions in the antiviral activity of AG7088 were observed when assays were performed in the presence of ␣ 1 -acid glycoprotein or mucin, proteins present in nasal secretions. The 50% cytotoxic concentration of AG7088 was >1,000 M, yielding a therapeutic index of >12,346 to >333,333. In a single-cycle, time-of-addition assay, AG7088 demonstrated antiviral activity when added up to 6 h after infection. In contrast, a compound targeting viral attachment and/or uncoating was effective only when added at the initiation of virus infection. Direct inhibition of 3C proteolytic activity in infected cells treated with AG7088 was demonstrated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis analysis of radiolabeled proteins, which showed a dose-dependent accumulation of viral precursor polyproteins and reduction of processed protein products. The broad spectrum of antiviral activity of AG7088, combined with its efficacy even when added late in the virus life cycle, highlights the advantages of 3C protease as a target and suggests that AG7088 will be a promising clinical candidate. MATERIALS AND METHODSCompounds. AG7088 and pleconaril (17) were synthesized at Agouron Pharmaceuticals, Inc. Pirodavir (1) was kindly provided by Janssen Research Foundation (Beerse, Belgium), and WIN 51711 (40) was kindly provided by Sterling Winthrop Research Institute (Collegeville, Pa.). Ganciclovir (Syntex Corp., Palo Alto, Calif.) was obtained from a local pharmacy, and acyclovir was purchased from Sigma (St. Louis, Mo.).Cells and virus strains. All numbered HRV serotypes, echovirus type 11 (EV 11), enterovirus type 70 (ETV 70), coxsackievirus types A21 (CAV 21) and B3 strain Nancy (CVB 3), human cytomegalovirus (HCMV) strain AD169, and herpes simplex virus type 1 (HSV-1) strain McIntyre were purchased from the American Type Culture Collection (ATCC; Manassas, Va.). HRV Hanks and a nasal lavage from a patient challenged with HRV Hanks were kindly provided by Ronald Turner from the Medical University of South Carolina, Charleston, S.C. HRV and coxsackievirus stocks were propagated, and antiviral assays were performed, in H1-HeLa cells (ATCC) incubated at 34 and 37°C, respectively. ETV 70, EV 11, and HCMV stocks were propagated, and antiviral assays were performed, in MRC-5 (ATCC) cells at 37°C. HSV-1 stocks were propagated, and antiviral assays were performed, in Vero (ATCC) cells incubated at 37°C. Vero cells ...

With the recent increase in computing power, the molecular modeling community is now more focused on improving the accuracy and overall quality of biomolecular simulations. For the available simulation packages, force fields, and all other associated methods used, this relates to how well they describe the conformational space and thermodynamic properties of a biomolecular system. The parameter sets of GROMOS force fields have been parametrized and validated with the reaction field (RF) method using charge groups and a twin-range cutoff scheme (0.8/1.4 nm). However, the most recent versions of GROMACS (since v.2016) discontinued the support for charge groups. To take full advantage of the newer and faster versions of this software package with GROMOS 54A7 and RF, we need to evaluate the impact of using a single cutoff scheme (vs twin-range) and of using the Verlet list update method (which is atomistic) compared to the group-based cutoff scheme. Our results show that the GROMOS 54A7 force field seems consistent with a single cutoff, since the resulting conformation and protonation ensembles were indistinguishable. The GROMOS parametrization procedure was also reproduced using an atomistic cutoff scheme, and we have observed that the hydration free energy values of small amino acid side-chain analogues were similar to the ones obtained with the group-based protocol. We do observe a small impact of the atomistic cutoff scheme in the conformational space of the model systems studied (G1-PAMAM and DMPC). However, since the structural properties of these systems are well converged for the cutoff range used (1.4–2.0 nm), unlike with the group-based cutoff schemes, we are confident that the atomistic cutoff can be adopted with RF for MD and constant-pH MD biomolecular simulations using the GROMOS 54A7 force field.

Molecular simulations of nanoscale systems invariably involve assumptions and approximations to describe the electrostatic interactions, which are long-ranged in nature. One approach is the use of cutoff schemes with a reaction-field contribution to account for the medium outside the cutoff scheme. Recent reports show that macroscopic properties may depend on the exact choice of cutoff schemes in modern day simulations. In this work, a systematic analysis of the effects of different cutoff schemes was performed using a set of 52 proteins. We find no statistically significant differences between using a twin-range or a single-range cutoff scheme. Applying the cutoff based on charge groups or based on atomic positions, does lead to significant differences, which is traced to the cutoff noise for energies and forces. While group-based cutoff schemes show increased cutoff noise in the potential energy, applying an atomistic cutoff leads to artificial structure in the solvent at the cutoff distance. Carefully setting the temperature control, or using an atomistic cutoff for the solute and a group-based cutoff for the solvent significantly reduces the effects of the cutoff noise, without introducing structure in the solvent. This study aims to deepen the understanding of the implications different cutoffs have on molecular dynamics simulations.

Estragole, naturally occurring in a variety of herbs and spices, can form DNA adducts after bioactivation. Estragole DNA adduct formation and repair was studied in in vitro liver cell models, and a molecular dynamics simulation was used to investigate the conformation dependent (in)efficiency of N 2 -(trans-isoestragol-3′-yl)-2′-deoxyguanosine (E-3′-N 2 -dG) DNA adduct repair. HepG2, HepaRG cells, primary rat hepatocytes and CHO cells (including CHO wild-type and three NER-deficient mutants) were exposed to 50 μM estragole or 1′-hydroxyestragole and DNA adduct formation was quantified by LC-MS immediately following exposure and after a period of repair. Results obtained from CHO cell lines indicated that NER plays a role in repair of E-3′-N 2 -dG adducts, however, with limited efficiency since in the CHO wt cells 80% DNA adducts remained upon 24 h repair. Inefficiency of DNA repair was also found in HepaRG cells and primary rat hepatocytes. Changes in DNA structure resulting from E-3′-N 2 -dG adduct formation were investigated by molecular dynamics simulations. Results from molecular dynamics simulations revealed that conformational changes in double-stranded DNA by E-3′-N 2 -dG adduct formation are small, providing a possible explanation for the restrained repair, which may require larger distortions in the DNA structure. NER-mediated enzymatic repair of E-3′-N 2 -dG DNA adducts upon exposure to estragole will be limited, providing opportunities for accumulation of damage upon repeated daily exposure. The inability of this enzymatic repair is likely due to a limited distortion of the DNA double-stranded helix resulting in inefficient activation of nucleotide excision repair.

Recently, concerns have been voiced regarding the validity of the GROMOS force fields, being parametrized using a twin-range cutoff scheme, in which longer ranged nonbonded forces and energies are updated less frequently than shorter ranged ones. Here we demonstrate that the influence of such a scheme on the thermodynamic, structural, and dynamic properties used in the parametrization of the GROMOS force fields is minor. We find root-mean-square differences of maximally 0.5 kJ/mol for the solvation free energy and heat of vaporization and of maximally 0.4% for the density. Slightly larger differences are observed when switching from a group-based to an atom-based cutoff scheme. In cases where the twin-range cutoff scheme does result in minor differences compared to a single-range cutoff these are well within the deviation from the experimentally measured values.