Natural Product Inhibition and Enzyme Kinetics Related to Phylogenetic Characterization for Bacterial Peptidyl-tRNA Hydrolase 1.

With the relentless development of drug resistance and re-emergence of many pathogenic bacteria, the need for new antibiotics and new antibiotic targets is urgent and growing. Bacterial peptidyl-tRNA hydrolase, Pth1, is emerging as a promising new target for antibiotic development. From the conserved core and high degree of structural similarity, broad-spectrum inhibition is postulated. However, Pth1 small-molecule inhibition is still in the earliest stages. Focusing on pathogenic bacteria, herein we report the phylogenetic classification of Pth1 and natural product inhibition spanning phylogenetic space. While broad-spectrum inhibition is found, narrow-spectrum and even potentially clade-specific inhibition is more frequently observed. Additionally reported are enzyme kinetics and general in vitro Pth1 solubility that follow phylogenetic boundaries along with identification of key residues in the gate loop region that appear to govern both. The studies presented here demonstrate the sizeable potential for small-molecule inhibition of Pth1, improve understanding of Pth enzymes, and advance Pth1 as a much-needed novel antibiotic target.

Bigger Data Approach to Analysis of Essential Oils and Their Antifungal Activity against Aspergillus niger, Candida albicans, and Cryptococcus neoformans.

With increasing drug resistance and the poor state of current antifungals, the need for new antifungals is urgent and growing. Therefore, we tested a variety of essential oils for antifungal activity. We report the minimum inhibitory concentrations (MIC) values for a common set of 82 essential oils against Aspergillus niger, Candida albicans, and Cryptococcus neoformans. Generally, narrow-spectrum activity was found. However, C. neoformans was much more susceptible to inhibition by essential oils with over one-third of those tested having MIC values below 160 ppm. GC-MS analysis showed the essential oils to be chemically diverse, yet, the potentially active major constituents typically fell into a few general categories (i.e., terpenes, terpenoids, terpenols). While essential oils remain a rich source of potential antifungals, focus should shift to prioritizing activity from novel compounds outside the commonalities reported here, instead of simply identifying antifungal activity. Further, capitalizing on bigger data approaches can provide significant returns in expediting the identification of active components.

Novel Antifungal Activity for the Lectin Scytovirin: Inhibition of Cryptococcus neoformans and Cryptococcus gattii.

Pathogenic cryptococci are encapsulated yeast that can cause severe meningoencephalitis. Existing therapeutic options are dated and there is a growing need for new alternative antifungal agents for these fungi. Here we report novel inhibition of pathogenic cryptococci by the antimicrobial lectin Scytovirin. Inhibition was most potent against Cryptococcus neoformans var neoformans and C. gattii, with MFC values of 500 nM. Scytovirin binding was localized to the cell wall and shown to affect capsule size and release. No effect was observed on melanization or with cells grown in the presence the cell wall stressor Congo red. Synergy with existing antifungals was indicated, most strongly for amphotericin B. Overall, Scytovirin serves as a much needed new avenue for anticryptococcal development.

Bacterial production of site specific 13C labeled phenylalanine and methodology for high level incorporation into bacterially expressed recombinant proteins.

Nuclear magnetic resonance spectroscopy studies of ever larger systems have benefited from many different forms of isotope labeling, in particular, site specific isotopic labeling. Site specific 13C labeling of methyl groups has become an established means of probing systems not amenable to traditional methodology. However useful, methyl reporter sites can be limited in number and/or location. Therefore, new complementary site specific isotope labeling strategies are valuable. Aromatic amino acids make excellent probes since they are often found at important interaction interfaces and play significant structural roles. Aromatic side chains have many of the same advantages as methyl containing amino acids including distinct 13C chemical shifts and multiple magnetically equivalent 1H positions. Herein we report economical bacterial production and one-step purification of phenylalanine with 13C incorporation at the Cα, Cγ and Cε positions, resulting in two isolated 1H-13C spin systems. We also present methodology to maximize incorporation of phenylalanine into recombinantly overexpressed proteins in bacteria and demonstrate compatibility with ILV-methyl labeling. Inexpensive, site specific isotope labeled phenylalanine adds another dimension to biomolecular NMR, opening new avenues of study.

Expedited isolation of natural product peptidyl-tRNA hydrolase inhibitors from a Pth1 affinity column.

New antibiotics and new antibiotic targets are needed to counter the development of bacterial drug resistance that threatens to return the human population to the pre-antibiotic era. Bacterial peptidyl-tRNA hydrolase (Pth1) is a promising new antibiotic target in the early stages of development. While inhibitory activity has been observed in a variety of natural products, bioactive fractionation has been a bottleneck for inhibitor isolation. To expedite the isolation of inhibitory compounds from complex mixtures, we constructed a Pth1 affinity column and used it to isolate inhibitory compounds from crude natural products. Recombinantly produced S. typhimurium Pth1 was covalently attached to a column matrix and the inhibitory activity isolated from ethanol extracts of Salvinia minima. The procedure reported here demonstrates that isolation of Pth1 inhibitory compounds from complex natural product extracts can be greatly expedited over traditional bioactive fractionation, decreasing time and expense. The approach is generally applicable to Pth1s from other bacterial species and opens an avenue to advance and accelerate inhibitor development against this promising antimicrobial target.

α-Pyrone derivatives, tetra/hexahydroxanthones, and cyclodepsipeptides from two freshwater fungi.

Eighteen (1-18) and seven (1, 4, 6-8, 17 and 18) compounds were isolated from organic extracts of axenic cultures of two freshwater fungi Clohesyomyces sp. and Clohesyomyces aquaticus (Dothideomycetes, Ascomycota), respectively. Compounds 1-12 belong to the α-pyrone class of natural products, compounds 13 and 14 were tetrahydroxanthones, compounds 15 and 16 were hexahydroxanthones, while compounds 17 and 18 were cyclodepsipeptides. The structures were elucidated using a set of spectroscopic and spectrometric techniques. The absolute configurations of compounds 2, 3, 6, and 7 were assigned via a modified Mosher's ester method using 1H NMR data. The relative configurations of compounds 14-16 were determined through NOE data. Compounds 1, 2, 6, 8, 13, 14, and 15 were found to inhibit the essential enzyme bacterial peptidyl-tRNA hydrolase (Pth1), with (13; secalonic acid A) being the most potent. Compounds 1 and 4-18 were also evaluated for antimicrobial activity against an array of bacteria and fungi but were found to be inactive.

Small Molecule Docking Supports Broad and Narrow Spectrum Potential for the Inhibition of the Novel Antibiotic Target Bacterial Pth1.

Peptidyl-tRNA hydrolases (Pths) play ancillary yet essential roles in protein biosynthesis by recycling peptidyl-tRNA. In E. coli, inhibition of bacterial Pth1 leads to accumulation of peptidyl-tRNA, depletion of aminoacyl-tRNA, and cell death. Eukaryotes have multiple Pths and Pth1 knock out was shown to have no effect on viability in yeast. Thereby, bacterial Pth1 is a promising target for novel antibiotic development. With the abundance of Pth1 structural data, molecular docking was used for virtual screening of existing, commercially available antibiotics to map potential interactions with Pth enzymes. Overall, 83 compounds were docked to eight different bacterial Pth1 and three different Pth2 structures. A variety of compounds demonstrated favorable docking with Pths. Whereas, some compounds interacted favorably with all Pths (potential broad spectrum inhibition), more selective interactions were observed for Pth1 or Pth2 and even specificity for individual Pth1s. While the correlation between computational docking and experimentation still remains unknown, these findings support broad spectrum inhibition, but also point to the possibility of narrow spectrum Pth1 inhibition. Also suggested is that Pth1 can be distinguished from Pth2 by small molecule inhibitors. The findings support continued development of Pth1 as an antibiotic target.

Expression, purification, and buffer solubility optimization of the putative human peptidyl-tRNA hydrolase PTRHD1.

Performing the essential function of recycling peptidyl-tRNAs, peptidyl-tRNA hydrolases are ubiquitous in all domains of life. The multicomponent eukaryotic Pth system differs greatly from the bacterial system composed predominantly of a single Pth1 enzyme. While bacterial Pth1s are structurally well characterized and promising new targets for antibiotic development, eukaryotic Pths are largely understudied. From amino acid sequence alignment and secondary structure predictions, the human gene product PTRHD1 was classified as a eukaryotic Pth. Herein, we report cloning, recombinant bacterial expression, and weak binding to peptidyl-tRNA for PTRHD1. Additionally, we report binding to tRNA but absence of peptidyl-tRNA hydrolase activity. Thus, PTRHD1 is not a Pth and the functional consequence of nucleotide binding remains undefined.

Neutron diffraction analysis of Pseudomonas aeruginosa peptidyl-tRNA hydrolase 1.

Perdeuterated peptidyl-tRNA hydrolase 1 from Pseudomonas aeruginosa was crystallized for structural analysis using neutron diffraction. Crystals of perdeuterated protein were grown to 0.15 mm(3) in size using batch crystallization in 22.5% polyethylene glycol 4000, 100 mM Tris pH 7.5, 10%(v/v) isopropyl alcohol with a 20-molar excess of trilysine as an additive. Neutron diffraction data were collected from a crystal at room temperature using the MaNDi single-crystal diffractometer at Oak Ridge National Laboratory.

Antifungal activity of plant extracts against Aspergillus niger and Rhizopus stolonifer.

Despite recent advances in antifungal development, fungi remain a devastating threat to human health and compromise viability of the food supply. Plant based antimicrobials represent a vast untapped source with tremendous potential. Herein we present the antifungal properties of more than 50 plant extracts against two important human and agricultural pathogens, Aspergillus niger and Rhizopus stolonifer. Multiple extracts exhibit promising MIC values of less than 100 µg/mL and are reported for both fungal species.

Recombinant production, crystallization and X-ray crystallographic structure determination of peptidyl-tRNA hydrolase from Salmonella typhimurium.

Peptidyl-tRNA hydrolase (Pth; EC 3.1.1.29) from the pathogenic bacterium Salmonella typhimurium has been cloned, expressed in Escherichia coli and crystallized for X-ray analysis. Crystals were grown using hanging-drop vapor diffusion against a reservoir solution consisting of 0.03 M citric acid, 0.05 M bis-tris propane, 1% glycerol, 3% sucrose, 25% PEG 6000 pH 7.6. Crystals were used to obtain the three-dimensional structure of the native protein at 1.6 Šresolution. The structure was determined by molecular replacement of the crystallographic data processed in space group P212121 with unit-cell parameters a=62.1, b=64.9, c=110.5 Å, α=ß=γ=90°. The asymmetric unit of the crystallographic lattice was composed of two copies of the enzyme molecule with a 51% solvent fraction, corresponding to a Matthews coefficient of 2.02 Å3 Da(-1). The structural coordinates reported serve as a foundation for computational and structure-guided efforts towards novel small-molecule Pth1 inhibitors and potential antibacterial development.

Expression, purification, and solubility optimization of peptidyl-tRNA hydrolase 1 from Bacillus cereus.

Peptidyl-tRNA hydrolase 1 cleaves the ester bond of peptidyl-tRNA thereby recycling peptidyl-tRNAs generated from premature termination of translation and expression of minigenes and short ORFs. Bacterial Pth1 is essential, highly conserved, and has no essential eukaryotic homolog making it a good target for antibacterial development. Herein we describe the cloning of pth1 gene from Bacillus cereus as an N-terminal hexahistidine fusion protein. Solubility was optimized for overexpression in Escherichia coli. Purity greater than 95% was achieved in one chromatography step. Yields greater than 12mg of purified Pth1 per liter of minimal media were achieved and buffer conditions for long-term solubility were determined. Enzymatic activity of Pth1 from B. cereus was confirmed and quantification of Michaelis-Menten parameters reported.

Small molecule binding, docking, and characterization of the interaction between Pth1 and peptidyl-tRNA.

Bacterial Pth1 is essential for viability. Pth1 cleaves the ester bond between the peptide and nucleotide of peptidyl-tRNA generated from aborted translation, expression of mini-genes, and short ORFs. We have determined the shape of the Pth1:peptidyl-tRNA complex using small angle neutron scattering. Binding of piperonylpiperazine, a small molecule constituent of a combinatorial synthetic library common to most compounds with inhibitory activity, was mapped to Pth1 via NMR spectroscopy. We also report computational docking results, modeling piperonylpiperazine binding based on chemical shift perturbation mapping. Overall these studies promote Pth1 as a novel antibiotic target, contribute to understanding how Pth1 interacts with its substrate, advance the current model for cleavage, and demonstrate feasibility of small molecule inhibition.

Recombinant production, crystallization and X-ray crystallographic structure determination of the peptidyl-tRNA hydrolase of Pseudomonas aeruginosa.

The peptidyl-tRNA hydrolase enzyme from the pathogenic bacterium Pseudomonas aeruginosa (Pth; EC 3.1.1.29) has been cloned, expressed in Escherichia coli and crystallized for X-ray structural analysis. Suitable crystals were grown using the sitting-drop vapour-diffusion method after one week of incubation against a reservoir solution consisting of 20% polyethylene glycol 4000, 100 mM Tris pH 7.5, 10%(v/v) isopropyl alcohol. The crystals were used to obtain the three-dimensional structure of the native protein at 1.77 Šresolution. The structure was determined by molecular replacement of the crystallographic data processed in space group P6(1)22 with unit-cell parameters a=b=63.62, c=155.20 Å, α=ß=90, γ=120°. The asymmetric unit of the crystallographic lattice was composed of a single copy of the enzyme molecule with a 43% solvent fraction, corresponding to a Matthews coefficient of 2.43 Å3 Da(-1). The crystallographic structure reported here will serve as the foundation for future structure-guided efforts towards the development of novel small-molecule inhibitors specific to bacterial Pths.

Inhibition of essential bacterial peptidyl-tRNA hydrolase activity by tropical plant extracts.

Peptidyl-tRNA Hydrolase (Pth) is a highly conserved, essential enzyme in bacteria. It removes the peptide portion from peptidyl-tRNA, returning free tRNAs to participate in translation. Build-up of peptidyl-tRNAs is toxic and defects in Pth function result in cell death. Herein we use in vitro activity of recombinant E. coli Pth to screen tropical plant extracts for inhibition. Multiple extracts were found to have inhibitory activity with some exhibiting different inhibitory effects depending on extraction conditions. IC50 values ranged from 0.02 to > 53.8 microg of extract per 1 unit of Pth, holding promise for in vivo screening. The inhibitory components in these extracts may serve as lead compounds for development of novel antibacterials.

Peptidyl-tRNA hydrolase screening combined with molecular docking reveals the antibiotic potential of Syzygium johnsonii bark extract.

With the rapid rise of antibiotic resistance in pathogenic bacteria, the need for new antibacterial agents is overwhelming. Herein we report the limited screening of tropical plant extracts for inhibitory activity against the essential enzyme peptidyl-tRNA hydrolase (Pth). Initial screening was conducted through an electrophoretic mobility assay and Northern blot detection. The ability of Pth to cleave the peptide-tRNA ester bond was assessed. The ethanol bark extract of Syzygium johnsonii showed strong inhibitory potential. Molecular docking studies point to Syzygium polyphenolics as the potential source of inhibition. This work is the forerunner of activity-directed isolation, purification, and structure elucidation of the inhibitory components from Syzygium johnsonii extracts and studies of compound interaction with Pth.