Epetraborole, a leucyl-tRNA synthetase inhibitor, demonstrates murine efficacy, enhancing the in vivo activity of ceftazidime against Burkholderia pseudomallei, the causative agent of melioidosis.

Burkholderia pseudomallei is the causative agent of melioidosis, which is increasingly being reported worldwide. Mortality rates as high as 40% have been reported based on clinical patient outcomes in the endemic areas of Australia and Thailand. Novel therapies are needed to reduce treatment duration and adverse effects and improve treatment outcomes. Epetraborole, a novel antibiotic, targets leucyl-tRNA synthetase (LeuRS), an essential enzyme that catalyzes the attachment of leucine to transfer RNA. Epetraborole was evaluated for in vitro activity and efficacy in a murine model to assess clinical relevance against Burkholderia pseudomallei infections for possible treatment of melioidosis. Epetraborole was tested against 13 clinically derived and three reference B. pseudomallei strains that have a broad spectrum of susceptibilities to the standard-of-care (SoC) drugs for melioidosis, which showed that epetraborole exhibited minimal inhibitory concentrations of 0.25-4 µg/mL. Ex vivo studies using THP-1 macrophages confirmed the potency of epetraborole and demonstrated synergy between epetraborole and ceftazidime. In the acute pulmonary murine infection model of melioidosis, epetraborole demonstrated equivalent efficacy when delivered orally or subcutaneously, which compared well with the standard-of-care drug ceftazidime. In addition, adding epetraborole to ceftazidime significantly improved antimicrobial activity in this animal model. This work warrants further exploration of epetraborole as a candidate for treating melioidosis and substantiates LeuRS as a clinically relevant drug target in B. pseudomallei.

In vivo efficacy of the boron-pleuromutilin AN11251 against Wolbachia of the rodent filarial nematode Litomosoides sigmodontis.

The elimination of filarial diseases such as onchocerciasis and lymphatic filariasis is hampered by the lack of a macrofilaricidal-adult worm killing-drug. In the present study, we tested the in vivo efficacy of AN11251, a boron-pleuromutilin that targets endosymbiotic Wolbachia bacteria from filarial nematodes and compared its efficacy to doxycycline and rifampicin. Doxycycline and rifampicin were previously shown to deplete Wolbachia endosymbionts leading to a permanent sterilization of the female adult filariae and adult worm death in human clinical studies. Twice-daily oral treatment of Litomosoides sigmodontis-infected mice with 200 mg/kg AN11251 for 10 days achieved a Wolbachia depletion > 99.9% in the adult worms, exceeding the Wolbachia reduction by 10-day treatments with bioequivalent human doses of doxycycline and a similar reduction as high-dose rifampicin (35 mg/kg). Wolbachia reductions of > 99% were also accomplished by 14 days of oral AN11251 at a lower twice-daily dose (50 mg/kg) or once-per-day 200 mg/kg AN11251 treatments. The combinations tested of AN11251 with doxycycline had no clear beneficial impact on Wolbachia depletion, achieving a > 97% Wolbachia reduction with 7 days of treatment. These results indicate that AN11251 is superior to doxycycline and comparable to high-dose rifampicin in the L. sigmodontis mouse model, allowing treatment regimens as short as 10-14 days. Therefore, AN11251 represents a promising pre-clinical candidate that was identified in the L. sigmodontis model, and could be further evaluated and developed as potential clinical candidate for human lymphatic filariasis and onchocerciasis.

Macrofilaricidal Benzimidazole-Benzoxaborole Hybrids as an Approach to the Treatment of River Blindness: Part 2. Ketone Linked Analogs.

The optimization of a series of benzimidazole-benzoxaborole hybrid molecules linked via a ketone that exhibit good activity against Onchocerca volvulus, a filarial nematode responsible for the disease onchocerciasis, also known as river blindness, is described. The lead identified in this series, 21 (AN15470), was found to have acceptable pharmacokinetic properties to enable an evaluation following oral dosing in an animal model of onchocerciasis. Compound 21was effective in killing worms implanted in Mongolian gerbils when dosed orally as a suspension at 100 mg/kg/day for 14 days but not when dosed orally at 100 mg/kg/day for 7 days.

Macrofilaricidal Benzimidazole-Benzoxaborole Hybrids as an Approach to the Treatment of River Blindness: Part 1. Amide Linked Analogs.

A series of benzimidazole-benzoxaborole hybrid molecules linked via an amide linker are described that exhibit good in vitro activity against Onchocerca volvulus, a filarial nematode responsible for the disease onchocerciasis, also known as river blindness. The lead identified in this series, 8a (AN8799), was found to have acceptable pharmacokinetic properties to enable evaluation in animal models of human filariasis. Compound 8a was effective in killing Brugia malayi, B. pahangi, and Litomosoides sigmodontis worms present in Mongolian gerbils when dosed subcutaneously as a suspension at 100 mg/kg/day for 14 days but not when dosed orally at 100 mg/kg/day for 28 days. The measurement of plasma levels of 8a at the end of the dosing period and at the time of sacrifice revealed an interesting dependence of activity on the extended exposure for both 8a and the positive control, flubendazole.

Identification of a potent benzoxaborole drug candidate for treating cryptosporidiosis.

Cryptosporidiosis is a leading cause of life-threatening diarrhea in young children and causes chronic diarrhea in AIDS patients, but the only approved treatment is ineffective in malnourished children and immunocompromised people. We here use a drug repositioning strategy and identify a promising anticryptosporidial drug candidate. Screening a library of benzoxaboroles comprised of analogs to four antiprotozoal chemical scaffolds under pre-clinical development for neglected tropical diseases for Cryptosporidium growth inhibitors identifies the 6-carboxamide benzoxaborole AN7973. AN7973 blocks intracellular parasite development, appears to be parasiticidal, and potently inhibits the two Cryptosporidium species most relevant to human health, C. parvum and C. hominis. It is efficacious in murine models of both acute and established infection, and in a neonatal dairy calf model of cryptosporidiosis. AN7973 also possesses favorable safety, stability, and PK parameters, and therefore, is an exciting drug candidate for treating cryptosporidiosis.

Boron-Pleuromutilins as Anti- Wolbachia Agents with Potential for Treatment of Onchocerciasis and Lymphatic Filariasis.

A series of pleuromutilins modified by introduction of a boron-containing heterocycle on C(14) of the polycyclic core are described. These analogs were found to be potent anti- Wolbachia antibiotics and, as such, may be useful in the treatment of filarial infections caused by Onchocerca volvulus, resulting in Onchocerciasis or river blindness, or Wuchereria bancrofti and Brugia malayi and related parasitic nematodes resulting in lymphatic filariasis. These two important neglected tropical diseases disproportionately impact patients in the developing world. The lead preclinical candidate compound containing 7-fluoro-6-oxybenzoxaborole (15, AN11251) was shown to have good in vitro anti- Wolbachia activity and physicochemical and pharmacokinetic properties providing high exposure in plasma. The lead was effective in reducing the Wolbachia load in filarial worms following oral administration to mice.

Studies on the mechanism of telavancin decreased susceptibility in a laboratory-derived mutant.

Telavancin is a novel semisynthetic lipoglycopeptide derivative of vancomycin with a dual mode of action. This study sought to understand the mechanisms of decreased telavancin susceptibility in a laboratory-derived Staphlococcus aureus mutant Tlv(DS)MED1952. There were extensive changes in the transcriptome of Tlv(DS)MED1952 compared to the susceptible parent strain MED1951. Genes upregulated included cofactor biosynthesis genes, cell wall-related genes, fatty acid biosynthesis genes, and stress genes. Downregulated genes included lysine operon biosynthesis genes and lrgB, which are induced by telavancin in susceptible strains, agr and kdpDE genes, various cell surface protein genes, phenol-soluble modulin genes, several protease genes, and genes involved in anaerobic metabolism. The decreased susceptibility mutant had somewhat thicker cell walls and a decreased autolytic activity that may be related to decreased proteolytic peptidoglycan hydrolase processing. Membrane fatty acid changes correlated with increased membrane fluidity were observed. It seems likely that there are multiple genetic changes associated with the development of decreased telavancin susceptibility. The Tlv(DS) mutant showed some similar features to vancomycin-intermediate S. aureus and decreased daptomycin susceptibility strains, but also exhibited its own unique features.

Deprotonation of D96 in bacteriorhodopsin opens the proton uptake pathway.

Despite extensive investigation, the precise mechanism controlling the opening of the cytoplasmic proton uptake pathway in bacteriorhodopsin (bR) has remained a mystery. From an analysis of the X-ray structure of the D96G/F171C/F219L triple mutant of bR and 60 independent molecular dynamics simulations of bR photointermediates, we report that the deprotonation of D96, a key residue in proton transfer reactions, serves two roles that occur sequentially. First, D96 donates a proton to the Schiff base. Subsequently, the deprotonation of D96 serves to "unlatch" the cytoplasmic side. The latching function of D96 appears to be remarkably robust, functioning to open hydration channels in all photointermediate structures. These results suggest that the protonation state of D96 may be the critical biophysical cue controlling the opening and closing of the cytoplasmic half-channel in bR. We suspect that this protonation-switch mechanism could also be utilized in other proton pumps to minimize backflow and reinforce directionality.

In vitro activity of telavancin and occurrence of vancomycin heteroresistance in isolates from patients enrolled in phase 3 clinical trials of hospital-acquired pneumonia.

In phase 3 studies of the efficacy of telavancin for the treatment of hospital-acquired pneumonia, 704 Gram-positive and 627 Gram-negative aerobic bacterial pathogens were obtained at baseline from 1503 patients. The majority of Gram-positive isolates (n = 650 [92%]) were Staphylococcus aureus, of which 410 (63%) were methicillin-resistant (MRSA). Of the MRSA isolates, 9.5% were identified as heterogeneous vancomycin-intermediate S. aureus. All Gram-positive isolates were inhibited by ≤1 µg/mL of telavancin.

Further insights into the mode of action of the lipoglycopeptide telavancin through global gene expression studies.

Telavancin is a novel semisynthetic lipoglycopeptide derivative of vancomycin with a decylaminoethyl side chain that is active against Gram-positive bacteria, including Staphylococcus aureus strains resistant to methicillin or vancomycin. A dual mechanism of action has been proposed for telavancin involving inhibition of peptidoglycan biosynthesis and membrane depolarization. Here we report the results of genome-wide transcriptional profiling of the response of S. aureus to telavancin using microarrays. Short (15-min) challenge of S. aureus with telavancin revealed strong expression of the cell wall stress stimulon, a characteristic response to inhibition of cell wall biosynthesis. In the transcriptome obtained after 60-min telavancin challenge, in addition to induction of the cell wall stress stimulon, there was induction of various genes, including lrgA and lrgB, lysine biosynthesis operon (dap) genes, vraD and vraE, and hlgC, that have been reported to be induced by known membrane-depolarizing and active agents, including carbonyl cyanide m-chlorophenylhydrazone, daptomycin, bacitracin, and other antimicrobial peptides These genes were either not induced or only weakly induced by the parent molecule vancomycin. We suggest that expression of these genes is a response of the cell to mitigate and detoxify such molecules and is diagnostic of a membrane-depolarizing or membrane-active molecule. The results indicate that telavancin causes early and significant induction of the cell wall stress stimulon due to strong inhibition of peptidoglycan biosynthesis, with evidence in support of membrane depolarization and membrane activity that is expressed after a longer duration of drug treatment.

Fluorescence microscopy demonstrates enhanced targeting of telavancin to the division septum of Staphylococcus aureus.

The cellular binding patterns of fluorescent conjugates of telavancin and vancomycin were evaluated in Staphylococcus aureus by fluorescence microscopy and ratio imaging analysis. Telavancin showed enhanced binding at the division septum compared to vancomycin. This result is consistent with observations that telavancin binds with higher affinity to lipid II than to d-Ala-d-Ala residues in the cell wall, thus demonstrating the preferential binding of telavancin to the site of active cell wall biosynthesis.

Telavancin disrupts the functional integrity of the bacterial membrane through targeted interaction with the cell wall precursor lipid II.

Telavancin is an investigational lipoglycopeptide antibiotic currently being developed for the treatment of serious infections caused by gram-positive bacteria. The bactericidal action of telavancin results from a mechanism that combines the inhibition of cell wall synthesis and the disruption of membrane barrier function. The purpose of the present study was to further elucidate the mechanism by which telavancin interacts with the bacterial membrane. A flow cytometry assay with the diethyloxacarbocyanine dye DiOC(2)(3) was used to probe the membrane potential of actively growing Staphylococcus aureus cultures. Telavancin caused pronounced membrane depolarization that was both time and concentration dependent. Membrane depolarization was demonstrated against a reference S. aureus strain as well as phenotypically diverse isolates expressing clinically important methicillin-resistant (MRSA), vancomycin-intermediate (VISA), and heterogeneous VISA (hVISA) phenotypes. The cell wall precursor lipid II was shown to play an essential role in telavancin-induced depolarization. This was demonstrated both in competition binding experiments with exogenous D-Ala-D-Ala-containing ligand and in experiments with cells expressing altered levels of lipid II. Finally, monitoring of the optical density of S. aureus cultures exposed to telavancin showed that cell lysis does not occur during the time course in which membrane depolarization and bactericidal activity are observed. Taken together, these data indicate that telavancin's membrane mechanism requires interaction with lipid II, a high-affinity target that mediates binding to the bacterial membrane. The targeted interaction with lipid II and the consequent disruption of both peptidoglycan synthesis and membrane barrier function provide a mechanistic basis for the improved antibacterial properties of telavancin relative to those of vancomycin.

Membrane-protein stability in a phospholipid-based crystallization medium.

Protein stability is a crucial factor to consider when attempting to crystallize integral membrane proteins. Cubic phase, or in meso, lipid-bilayer crystallization media are thought to provide native-like environments that should facilitate membrane protein crystallization by helping to stabilize the native protein conformation for the duration of the crystallization process. While excellent crystals of bacteriorhodopsin (bR) and other Halobacterial rhodopsins have been obtained in lipid-bilayer gels formed with monoglycerides, success remains elusive in the general application of such media to other membrane proteins. Additionally, we have noted that some mutants of bR are highly unstable in gels formed with monoolein. Phosphatidylethanolamines (PE) and derivatives of PE represent another class of lipids that can form connected-bilayer gels. When wildtype bR and a labile bR mutant were reconstituted into this phospholipid gel, spectroscopy showed that the protein is both more stable and has improved conformational homogeneity as compared to gels formed using monoolein. In addition, we demonstrate that well-diffracting crystals of bR can be grown from a PE-based crystallization medium. Since most proteins lack a stability-indicating chromophore and other structure-based analytical techniques are poorly compatible with the lipid gel, we developed a generally-applicable spectroscopic technique based on the intrinsic fluorescence of tryptophan residues. This fluorescence assay makes possible the rapid evaluation of lipid gels as media for the crystallization of membrane proteins.

Specificity of anion binding in the substrate pocket of bacteriorhodopsin.

The structure of the D85S mutant of bacteriorhodopsin with a nitrate anion bound in the Schiff base binding site and the structure of the anion-free protein have been obtained in the same crystal form. Together with the previously solved structures of this anion pump, in both the anion-free state and bromide-bound state, these new structures provide insight into how this mutant of bacteriorhodopsin is able to bind a variety of different anions in the same binding pocket. The structural analysis reveals that the main structural change that accommodates different anions is the repositioning of the polar side chain of S85. On the basis of these X-ray crystal structures, the prediction is then made that the D85S/D212N double mutant might bind similar anions and do so over a broader pH range than does the single mutant. Experimental comparison of the dissociation constants, K(d), for a variety of anions confirms this prediction and demonstrates, in addition, that the binding affinity is dramatically improved by the D212N substitution.

Modes of antifungal action of (2E)-alkenals against Saccharomyces cerevisiae.

A series of aliphatic (2E)-alkenals from C(5) to C(14) were tested for their antifungal activity against Saccharomyces cerevisiae ATCC 7754. (2E)-Undecenal (C(11)) was found to be the most effective with the minimum fungicidal concentration (MFC) of 6.25 microgram/mL, followed by (2E)-decenal (C(10)) with an MFC of 12.5 microgram/mL. The time-kill curve study showed that (2E)-undecenal was fungicidal against S. cerevisiae at any growth stage, and this activity was not influenced by pH values. The (2E)-alkenals inhibited glucose-induced acidification by inhibiting the plasma membrane H(+)-ATPase. The primary antifungal action of medium-chain (C(9)-C(12)) (2E)-alkenals against S. cerevisiae comes from their ability to function as nonionic surface-active agents (surfactants), disrupting the native membrane-associated function nonspecifically. Hence, the antifungal activity of (2E)-alkenals is mediated by biophysical processes, and the maximum activity can be obtained when the balance between the hydrophilic and hydrophobic portions becomes the most appropriate.

Crystallization of membrane proteins from media composed of connected-bilayer gels.

The use of hydrated-lipid gels in which the bilayer is an infinitely periodic (or at least continuous), three-dimensional structure offers a relatively new approach for the crystallization of membrane proteins. While excellent crystals of the Halobacterial rhodopsins have been obtained with such media, success remains poor in extending their use to other membrane proteins. Experience with crystallization of bacteriorhodopsin has led us to recognize a number of improvements that can be made in the use of such hydrated-gel media, which may now prove to be of general value for the crystallization of other membrane proteins.