Revealing antibiotic resistance's ancient roots: insights from pristine ecosystems.

The prevailing belief that antibiotic resistance mechanisms emerged with human antibiotic use has been challenged. Evidence indicates that some antibiotic resistance genes (ARGs) have a long evolutionary history, predating the advent of antibiotics in human medicine, thereby demonstrating that resistance is an ancient phenomenon. Despite extensive surveys of resistance elements in environments impacted by human activity, limited data are available from remote and pristine habitats.

Development of a luminescence-based method for measuring West Nile Virus MTase activity and its application to screen for antivirals.

West Nile virus (WNV) is a flavivirus responsible for causing febrile illness and severe neurological diseases, with an increasing impact on human health around the world. However, there is still no adequate therapeutic treatment available to struggle WNV infections. Therefore, there is an urgent need to develop new techniques to accelerate the discovery of drugs against this pathogen.

Identification of West Nile virus RNA-dependent RNA polymerase non-nucleoside inhibitors by real-time high throughput fluorescence screening.

West Nile virus (WNV) is a re-emergent mosquito-borne RNA virus that causes major outbreaks of encephalitis around the world. However, there is no therapeutic treatment to struggle against WNV, and the current treatment relies on alleviating symptoms. Therefore, due to the threat virus poses to animal and human health, there is an urgent need to come up with fast strategies to identify and assess effective antiviral compounds.

Novel Nonnucleoside Inhibitors of Zika Virus Polymerase Identified through the Screening of an Open Library of Antikinetoplastid Compounds.

Zika virus (ZIKV) is a mosquito-borne pathogen responsible for neurological disorders (Guillain-Barré syndrome) and congenital malformations (microcephaly). Its ability to cause explosive epidemics, such as that of 2015 to 2016, urges the identification of effective antiviral drugs. Viral polymerase inhibitors constitute one of the most successful fields in antiviral research.

Development of a fluorescence-based method for the rapid determination of Zika virus polymerase activity and the screening of antiviral drugs.

Zika virus (ZIKV) is an emerging pathogen that has been associated with large numbers of cases of severe neurologic disease, including Guillain-Barré syndrome and microcephaly. Despite its recent establishment as a serious global public health concern there are no licensed therapeutics to control this virus. Accordingly, there is an urgent need to develop methods for the high-throughput screening of antiviral agents.

Engineering human PrimPol into an efficient RNA-dependent-DNA primase/polymerase.

We have developed a straightforward fluorometric assay to measure primase-polymerase activity of human PrimPol (HsPrimPol). The sensitivity of this procedure uncovered a novel RNA-dependent DNA priming-polymerization activity (RdDP) of this enzyme. In an attempt to enhance HsPrimPol RdDP activity, we constructed a smart mutant library guided by prior sequence-function analysis, and tested this library in an adapted screening platform of our fluorometric assay.

Involvement of a joker mutation in a polymerase-independent lethal mutagenesis escape mechanism.

We previously characterized a foot-and-mouth disease virus (FMDV) with three amino acid replacements in its polymerase (3D) that conferred resistance to the mutagenic nucleoside analogue ribavirin. Here we show that passage of this mutant in the presence of high ribavirin concentrations resulted in selection of viruses with the additional replacement I248T in 2C. This 2C substitution alone (even in the absence of replacements in 3D) increased FMDV fitness mainly in the presence of ribavirin, prevented an incorporation bias in favor of A and U associated with ribavirin mutagenesis, and conferred the ATPase activity of 2C decreased sensitivity to ribavirin-triphosphate.

Biocatalytic route to chiral acyloins: P450-catalyzed regio- and enantioselective α-hydroxylation of ketones.

P450-BM3 and mutants of this monooxygenase generated by directed evolution are excellent catalysts for the oxidative α-hydroxylation of ketones with formation of chiral acyloins with high regioselectivity (up to 99%) and enantioselectivity (up to 99% ee). This constitutes a new route to a class of chiral compounds that are useful intermediates in the synthesis of many kinds of biologically active compounds.

CH-activating oxidative hydroxylation of 1-tetralones and related compounds with high regio- and stereoselectivity.

Mutants of P450-BM3 evolved by directed evolution are excellent catalysts in the CH-activating oxidative hydroxylation of 1-tetralone derivatives and of indanone, with unusually high regio- and enantioselectivity being observed. Similar results were achieved in the oxidative hydroxylation of tetralin and indane. The products are useful building blocks in the synthesis of a number of biologically active compounds.

Directed evolution of stereoselective enzymes based on genetic selection as opposed to screening systems.

Directed evolution of stereoselective enzymes provides a means to generate useful biocatalysts for asymmetric transformations in organic chemistry and biotechnology. Almost all of the numerous examples reported in the literature utilize high-throughput screening systems based on suitable analytical techniques. Since the screening step is the bottleneck of the overall procedure, researchers have considered the use of genetic selection systems as an alternative to screening.

Cytochrome P450 catalyzed oxidative hydroxylation of achiral organic compounds with simultaneous creation of two chirality centers in a single C-H activation step.

Regio- and stereoselective oxidative hydroxylation of achiral or chiral organic compounds mediated by synthetic reagents, catalysts, or enzymes generally leads to the formation of one new chiral center that appears in the respective enantiomeric or diastereomeric alcohols. By contrast, when subjecting appropriate achiral compounds to this type of C-H activation, the simultaneous creation of two chiral centers with a defined relative and absolute configuration may result, provided that control of the regio-, diastereo-, and enantioselectivity is ensured. The present study demonstrates that such control is possible by using wild type or mutant forms of the monooxygenase cytochrome P450 BM3 as catalysts in the oxidative hydroxylation of methylcyclohexane and seven other monosubstituted cyclohexane derivatives.

Directed evolution by using iterative saturation mutagenesis based on multiresidue sites.

Iterative saturation mutagenesis (ISM) in combination with reduced amino acid alphabets has been shown to be an efficient method for directed evolution. In order to minimize the screening effort, the number of residues in a given randomization site has thus far been restricted to two or three; this prevents oversampling from reaching astronomical numbers when 95 % library coverage is aimed for. In this study, ISM is applied for the first time by using randomization sites composed of five amino acid positions.

Designer cells for stereocomplementary de novo enzymatic cascade reactions based on laboratory evolution.

Designer cells for a synthetic cascade reaction harnessing selective redox reactions were devised, featuring two successive regioselective P450-catalyzed CH-activating oxidations of 1-cyclohexene carboxylic acid methyl ester followed by stereoselective olefin-reduction catalysed by (R)- or (S)-selective mutants of an enoate reductase.

Molecular dissection of a viral quasispecies under mutagenic treatment: positive correlation between fitness loss and mutational load.

Low fidelity replication and the absence of error-repair activities in RNA viruses result in complex and adaptable ensembles of related genomes in the viral population, termed quasispecies, with important implications for natural infections. Theoretical predictions suggested that elevated replication error rates in RNA viruses might be near to a maximum compatible with viral viability. This fact encouraged the use of mutagenic nucleosides as a new antiviral strategy to induce viral extinction through increased replication error rates.

Induced axial chirality in biocatalytic asymmetric ketone reduction.

Catalytic asymmetric reduction of prochiral ketones of type 4-alkylidene cyclohexanone with formation of the corresponding axially chiral R-configurated alcohols (up to 99% ee) was achieved using alcohol dehydrogenases, whereas chiral transition-metal catalysts fail. Reversal of enantioselectivity proved to be possible by directed evolution based on saturation mutagenesis (up to 98% ee (S)). Utilization of ketone with a vinyl bromide moiety allows respective R- and S-alcohols to be exploited as key compounds in Pd-catalyzed cascade reactions.

Achieving regio- and enantioselectivity of P450-catalyzed oxidative CH activation of small functionalized molecules by structure-guided directed evolution.

Directed evolution of the monooxygenase P450-BM3 utilizing iterative saturation mutagenesis at and near the binding site enables a high degree of both regio- and enantioselectivity in the oxidative hydroxylation of cyclohexene-1-carboxylic acid methyl ester. Wild-type P450-BM3 is 84% regioselective for the allylic 3-position with 34% enantioselectivity in favor of the R alcohol. Mutants enabling R selectivity (>95% ee) or S selectivity (>95% ee) were evolved, while reducing other oxidation products and thus maximizing regioselectivity to >93%.

Influence of mutagenesis and viral load on the sustained low-level replication of an RNA virus.

Lethal mutagenesis is an antiviral strategy that aims to extinguish viruses as a consequence of enhanced mutation rates during virus replication. The molecular mechanisms that underlie virus extinction by mutagenic nucleoside analogues are not well understood. When mutagenic agents and antiviral inhibitors are administered sequentially or in combination, interconnected and often conflicting selective constraints can influence the fate of the virus either towards survival through selection of mutagen-escape or inhibitor-escape mutants or towards extinction.

A multi-step process of viral adaptation to a mutagenic nucleoside analogue by modulation of transition types leads to extinction-escape.

Resistance of viruses to mutagenic agents is an important problem for the development of lethal mutagenesis as an antiviral strategy. Previous studies with RNA viruses have documented that resistance to the mutagenic nucleoside analogue ribavirin (1-β-D-ribofuranosyl-1-H-1,2,4-triazole-3-carboxamide) is mediated by amino acid substitutions in the viral polymerase that either increase the general template copying fidelity of the enzyme or decrease the incorporation of ribavirin into RNA. Here we describe experiments that show that replication of the important picornavirus pathogen foot-and-mouth disease virus (FMDV) in the presence of increasing concentrations of ribavirin results in the sequential incorporation of three amino acid substitutions (M296I, P44S and P169S) in the viral polymerase (3D).

Structure of foot-and-mouth disease virus mutant polymerases with reduced sensitivity to ribavirin.

Passage of poliovirus (PV) or foot-and-mouth disease virus (FMDV) in the presence of ribavirin selected for viruses with decreased sensitivity to R, which included different mutations in their polymerase (3D): G64S located in the finger subdomain in the case of PV and M296I located within loop beta9-alpha11 at the active site in the case of FMDV. To investigate why disparate substitutions were selected in two closely related 3Ds, we constructed FMDVs with a 3D that included either G62S (the equivalent replacement in FMDV of PV G64S), M296I, or both substitutions. G62S, but not M296I, inflicts upon FMDV a strong selective disadvantage which is partially compensated for by the substitution M296I.

Structural insights into replication initiation and elongation processes by the FMDV RNA-dependent RNA polymerase.

RNA-dependent RNA polymerases (RdRPs) play central roles in both transcription and viral genome replication. In picornaviruses, these functions are catalyzed by the virally encoded RdRP, termed 3D. Polymerase 3D also catalyzes the covalent linkage of UMP to a tyrosine on the small protein VPg.

Potential benefits of sequential inhibitor-mutagen treatments of RNA virus infections.

Lethal mutagenesis is an antiviral strategy consisting of virus extinction associated with enhanced mutagenesis. The use of non-mutagenic antiviral inhibitors has faced the problem of selection of inhibitor-resistant virus mutants. Quasispecies dynamics predicts, and clinical results have confirmed, that combination therapy has an advantage over monotherapy to delay or prevent selection of inhibitor-escape mutants.

5-fluorouracil in lethal mutagenesis of foot-and-mouth disease virus.

5-fluorouracil (FU) is a pyrimidine analogue extensively used in cancer chemotherapy. FU can be metabolized into 5-fluorouridine-triphosphate, which can be used as substrate for viral RNA-dependent RNA polymerases. This results in the incorporation of mutations into viral RNA.

Counteracting quasispecies adaptability: extinction of a ribavirin-resistant virus mutant by an alternative mutagenic treatment.

Background: Lethal mutagenesis, or virus extinction promoted by mutagen-induced elevation of mutation rates of viruses, may meet with the problem of selection of mutagen-resistant variants, as extensively documented for standard, non-mutagenic antiviral inhibitors. Previously, we characterized a mutant of foot-and-mouth disease virus that included in its RNA-dependent RNA polymerase replacement M296I that decreased the sensitivity of the virus to the mutagenic nucleoside analogue ribavirin.

Methodology And Principal Findings: Replacement M296I in the viral polymerase impedes the extinction of the mutant foot-and-mouth disease virus by elevated concentrations of ribavirin.

Molecular characterization of a dual inhibitory and mutagenic activity of 5-fluorouridine triphosphate on viral RNA synthesis. Implications for lethal mutagenesis.

The basis for a dual inhibitory and mutagenic activity of 5-fluorouracil (5-FU) on foot-and-mouth disease virus (FMDV) RNA replication has been investigated with purified viral RNA-dependent RNA polymerase (3D) in vitro. 5-Fluorouridine triphosphate acted as a potent competitive inhibitor of VPg uridylylation, the initial step of viral replication. Peptide analysis by mass spectrometry has identified a VPg fragment containing 5-fluorouridine monophosphate (FUMP) covalently attached to Tyr3, the amino acid target of the uridylylation reaction.

Topology of evolving, mutagenized viral populations: quasispecies expansion, compression, and operation of negative selection.

Background: The molecular events and evolutionary forces underlying lethal mutagenesis of virus (or virus extinction through an excess of mutations) are not well understood. Here we apply for the first time phylogenetic methods and Partition Analysis of Quasispecies (PAQ) to monitor genetic distances and intra-population structures of mutant spectra of foot-and-mouth disease virus (FMDV) quasispecies subjected to mutagenesis by base and nucleoside analogues.

Results: Phylogenetic and PAQ analyses have revealed a highly dynamic variation of intrapopulation diversity of FMDV quasispecies.