Small Molecule Benzothiophene with In Vivo Efficacy in a Mouse Model of Drug-Resistant Enterococcus faecium Infection.

Hospital-acquired infections, caused by ESKAPE bacteria, are a challenging global public health concern, in part due to the emergence of drug-resistant strains. While profiling a diverse set of compounds for in vitro activity versus this class of bacteria, we noted that the benzothiophene JSF-2827 exhibited promising antibacterial activity against Enterococcus faecium. A hit evolution campaign ensued, involving the design, synthesis, and biological assay of analogues designed to address early issues such as a short mouse liver microsome half-life and a modest mouse pharmacokinetic profile. Among these derivatives, JSF-3269 was found to exhibit an enhanced profile and in vivo efficacy in an immunocompetent mouse model of acute, drug-resistant E. faecium infection. The findings suggest a rationale for the further evolution of this promising series to afford a novel therapeutic strategy to treat drug-resistant E. faecium infection.

Bayesian Modeling and Intrabacterial Drug Metabolism Applied to Drug-Resistant Staphylococcus aureus.

We present the application of Bayesian modeling to identify chemical tools and/or drug discovery entities pertinent to drug-resistant Staphylococcus aureus infections. The quinoline JSF-3151 was predicted by modeling and then empirically demonstrated to be active against in vitro cultured clinical methicillin- and vancomycin-resistant strains while also exhibiting efficacy in a mouse peritonitis model of methicillin-resistant S. aureus infection. We highlight the utility of an intrabacterial drug metabolism (IBDM) approach to probe the mechanism by which JSF-3151 is transformed within the bacteria. We also identify and then validate two mechanisms of resistance in S. aureus: one mechanism involves increased expression of a lipocalin protein, and the other arises from the loss of function of an azoreductase. The computational and experimental approaches, discovery of an antibacterial agent, and elucidated resistance mechanisms collectively hold promise to advance our understanding of therapeutic regimens for drug-resistant S. aureus.

Antitubercular Triazines: Optimization and Intrabacterial Metabolism.

The triazine antitubercular JSF-2019 was of interest due to its in vitro efficacy and the nitro group shared with the clinically relevant delamanid and pretomanid. JSF-2019 undergoes activation requiring F420H2 and one or more nitroreductases in addition to Ddn. An intrabacterial drug metabolism (IBDM) platform was leveraged to demonstrate the system kinetics, evidencing formation of NO⋅ and a des-nitro metabolite. Structure-activity relationship studies focused on improving the solubility and mouse pharmacokinetic profile of JSF-2019 and culminated in JSF-2513, relying on the key introduction of a morpholine. Mechanistic studies with JSF-2019, JSF-2513, and other triazines stressed the significance of achieving potent in vitro efficacy via release of intrabacterial NO⋅ along with inhibition of InhA and, more generally, the FAS-II pathway. This study highlights the importance of probing IBDM and its potential to clarify mechanism of action, which in this case is a combination of NO⋅ release and InhA inhibition.

Optimization of N-benzyl-5-nitrofuran-2-carboxamide as an antitubercular agent.

The optimization campaign for a nitrofuran antitubercular hit (N-benzyl-5-nitrofuran-2-carboxamide; JSF-3449) led to the design, synthesis, and biological profiling of a family of analogs. These compounds exhibited potent in vitro antitubercular activity (MIC = 0.019-0.20 µM) against the Mycobacterium tuberculosis H37Rv strain and low in vitro cytotoxicity (CC50 = 40->120 µM) towards Vero cells. Significant improvements in mouse liver microsomal stability and mouse pharmacokinetic profile were realized by introduction of an α, α-dimethylbenzyl moiety. Among these compounds, JSF-4088 is highlighted due to its in vitro antitubercular potency (MIC = 0.019 µM) and Vero cell cytotoxicity (CC50 > 120 µM). The findings suggest a rationale for the continued evolution of this promising series of antitubercular small molecules.

Quantification of desmosine and isodesmosine using MALDI-ion trap tandem mass spectrometry.

Desmosine (Des) and isodesmosine (Isodes), cross-linking amino acids in the biomolecule elastin, may be used as biomarkers for various pathological conditions associated with elastin degradation. The current study presents a novel approach to quantify Des and Isodes using matrix-assisted laser desorption ionization (MALDI)-tandem mass spectrometry (MS2) in a linear ion trap coupled to a vacuum MALDI source. MALDI-MS2 analyses of Des and Isodes are performed using stable-isotope-labeled desmosine d4 (labeled-Des) as an internal standard in different biological fluids, such as urine and serum. The method demonstrated linearity over two orders of magnitude with a detection limit of 0.02 ng/µL in both urine and serum without enrichment prior to mass spectrometry, and relative standard deviation of < 5%. The method is used to evaluate the time-dependent degradation of Des upon UV irradiation (254 nm) and found to be consistent with quantification by 1H NMR. This is the first characterized MALDI-MS2 method for quantification of Des and Isodes and illustrates the potential of MALDI-ion trap MS2 for effective quantification of biomolecules. The reported method represents improvement over current liquid chromatography-based methods with respect to analysis time and solvent consumption, while maintaining similar analytical characteristics. Graphical abstract ᅟ.

Targeting protein biotinylation enhances tuberculosis chemotherapy.

Successful drug treatment for tuberculosis (TB) depends on the unique contributions of its component drugs. Drug resistance poses a threat to the efficacy of individual drugs and the regimens to which they contribute. Biologically and chemically validated targets capable of replacing individual components of current TB chemotherapy are a major unmet need in TB drug development. We demonstrate that chemical inhibition of the bacterial biotin protein ligase (BPL) with the inhibitor Bio-AMS (5'-[N-(d-biotinoyl)sulfamoyl]amino-5'-deoxyadenosine) killed Mycobacterium tuberculosis (Mtb), the bacterial pathogen causing TB. We also show that genetic silencing of BPL eliminated the pathogen efficiently from mice during acute and chronic infection with Mtb Partial chemical inactivation of BPL increased the potency of two first-line drugs, rifampicin and ethambutol, and genetic interference with protein biotinylation accelerated clearance of Mtb from mouse lungs and spleens by rifampicin. These studies validate BPL as a potential drug target that could serve as an alternate frontline target in the development of new drugs against Mtb.

Novel Pyrimidines as Antitubercular Agents.

Mycobacterium tuberculosis infection is responsible for a global pandemic. New drugs are needed that do not show cross-resistance with the existing front-line therapeutics. A triazine antitubercular hit led to the design of a related pyrimidine family. The synthesis of a focused series of these analogs facilitated exploration of their in vitro activity, in vitro cytotoxicity, and physiochemical and absorption-distribution-metabolism-excretion properties. Select pyrimidines were then evaluated for their pharmacokinetic profiles in mice. The findings suggest a rationale for the further evolution of this promising series of antitubercular small molecules, which appear to share some similarities with the clinical compound PA-824 in terms of activation, while highlighting more general guidelines for the optimization of small-molecule antitubercular agents.

Subcellular Parkinson's Disease-Specific Alpha-Synuclein Species Show Altered Behavior in Neurodegeneration.

Parkinson's disease and other synucleinopathies are characterized by the presence of intra-neuronal protein aggregates enriched in the presynaptic protein α-synuclein. α-synuclein is considered an intrinsically disordered 14 kDa monomer, and although poorly understood, its transition to higher-order multimeric species may play central roles in healthy neurons and during Parkinson's disease pathogenesis. In this study, we demonstrate that α-synuclein exists as defined, subcellular-specific species that change characteristics in response to oxidative stress in neuroblastoma cells and in response to Parkinson's disease pathogenesis in human cerebellum and frontal cortex. We further show that the phosphorylation patterns of different α-synuclein species are subcellular specific and dependent on the oxidative environment. Using high-performance liquid chromatography and mass spectrometry, we identify a Parkinson's disease enriched, cytosolic ~36-kDa α-synuclein species which can be recapitulated in Parkinson's disease model neuroblastoma cells. The characterization of subcellular-specific α-synuclein features in neurodegeneration will allow for the identification of neurotoxic α-synuclein species, which represent prime targets to reduce α-synuclein pathogenicity.

A Proteomics Approach to Investigate miR-153-3p and miR-205-5p Targets in Neuroblastoma Cells.

MicroRNAs are key regulators associated with numerous diseases. In HEK293 cells, miR-153-3p and miR-205-5p down-regulate alpha-synuclein (SNCA) and Leucine-rich repeat kinase 2 (LRRK2), two key proteins involved in Parkinson's disease (PD). We have used two-dimensional gel electrophoresis (2D-PAGE) coupled to mass spectrometry (MS) to identify a spectrum of miR-153-3p and miR-205-5p targets in neuronal SH-SY5Y cells. We overexpressed and inhibited both microRNAs in SH-SY5Y cells and through comparative proteomics profiling we quantified ~240 protein spots from each analysis. Combined, thirty-three protein spots were identified showing significant (p-value < 0.05) changes in abundance. Modulation of miR-153-3p resulted in seven up-regulated proteins and eight down-regulated proteins. miR-205 modulation resulted in twelve up-regulated proteins and six down-regulated proteins. Several of the proteins are associated with neuronal processes, including peroxiredoxin-2 and -4, cofilin-1, prefoldin 2, alpha-enolase, human nucleoside diphosphate kinase B (Nm23) and 14-3-3 protein epsilon. Many of the differentially expressed proteins are involved in diverse pathways including metabolism, neurotrophin signaling, actin cytoskeletal regulation, HIF-1 signaling and the proteasome indicating that miR-153-3p and miR-205-5p are involved in the regulation of a wide variety of biological processes in neuroblastoma cells.

Studies on quantitative phosphopeptide analysis by matrix-assisted laser desorption/ionization mass spectrometry without label, chromatography or calibration curves.

RATIONALE: Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry combined with isotope labeling methods are effective for protein and peptide quantification, but limited in their multiplexing capacity, cost-effectiveness and dynamic range. This study investigates MALDI-MS-based quantification of peptide phosphorylation without labeling, and aims to overcome the shot-to-shot variability of MALDI using a mathematical transformation and extended data acquisition times. METHODS: A linear relationship between the reciprocal of phosphopeptide mole fraction and the reciprocal of phosphorylated-to-unphosphorylated signal ratio is derived, and evaluated experimentally using three separate phosphopeptide systems containing phosphorylated serine, threonine and tyrosine residues: mixtures of phosphopeptide and its des-phospho-analog with known stoichiometry measured by vacuum MALDI-linear ion trap mass spectrometry and fit to the linear model. The model is validated for quantifying in vitro phosphorylation assays with inhibition studies on Cdk2/cyclinA. RESULTS: Dynamic range of picomoles to femtomoles, good accuracy (deviations of 1.5-3.0% from expected values) and reproducibility (relative standard deviation (RSD) = 4.3-6.3%) are achieved. Inhibition of cyclin-dependent kinase phosphorylation by the classical inhibitors olomoucine and r-roscovitine was evaluated and IC50 values found to be in agreement with reported literature values. These results, achieved with single-point calibration, without isotope or chromatography, compare favorably to those arrived at using isotope dilution (p > 0.5 for accuracy). CONCLUSIONS: The mathematical relationship derived here can be applied to a method that we term Double Reciprocal Isotope-free Phosphopeptide Quantification (DRIP-Q), as a strategy for quantification of in vitro phosphorylation assays, the first MALDI-based, isotope- and calibration curve-free method of its type. These results also pave the way for further systematic studies investigating the effect of peptide composition and experimental conditions on quantitative, label-free MALDI.

A cell-intrinsic inhibitor of HIV-1 reverse transcription in CD4(+) T cells from elite controllers.

HIV-1 reverse transcription represents the predominant target for pharmacological inhibition of viral replication, but cell-intrinsic mechanisms that can block HIV-1 reverse transcription in a clinically significant way are poorly defined. We find that effective HIV-1 reverse transcription depends on the phosphorylation of viral reverse transcriptase by host cyclin-dependent kinase (CDK) 2 at a highly conserved Threonine residue. CDK2-dependent phosphorylation increased the efficacy and stability of viral reverse transcriptase and enhanced viral fitness. Interestingly, p21, a cell-intrinsic CDK inhibitor that is upregulated in CD4(+) T cells from "elite controllers," potently inhibited CDK2-dependent phosphorylation of HIV-1 reverse transcriptase and significantly reduced the efficacy of viral reverse transcription. These data suggest that p21 can indirectly block HIV-1 reverse transcription by inhibiting host cofactors supporting HIV-1 replication and identify sites of viral vulnerability that are effectively targeted in persons with natural control of HIV-1 replication.

Arabidopsis AtPARK13, which confers thermotolerance, targets misfolded proteins.

Mutations in HTRA2/Omi/PARK13 have been implicated in Parkinson disease (PD). PARK13 is a neuroprotective serine protease; however, little is known about how PARK13 confers stress protection and which protein targets are directly affected by PARK13. We have reported that Arabidopsis thaliana represents a complementary PD model, and here we demonstrate that AtPARK13, similar to human PARK13 (hPARK13), is a mitochondrial protease. We show that the expression/accumulation of AtPARK13 transcripts are induced by heat stress but not by other stress conditions, including oxidative stress and metals. Our data show that elevated levels of AtPARK13 confer thermotolerance in A. thaliana. Increased temperatures accelerate protein unfolding, and we demonstrate that although AtPARK13 can act on native protein substrates, unfolded proteins represent better AtPARK13 substrates. The results further show that AtPARK13 and hPARK13 can degrade the PD proteins α-synuclein (SNCA) and DJ-1/PARK7 directly, without autophagy involvement, and that misfolded SNCA and DJ-1 represent better substrates than their native counterparts. Comparative proteomic profiling revealed AtPARK13-mediated proteome changes, and we identified four proteins that show altered abundance in response to AtPARK13 overexpression and elevated temperatures. Our study not only suggests that AtPARK13 confers thermotolerance by degrading misfolded protein targets, but it also provides new insight into possible roles of this protease in neurodegeneration.

Purge-assisted headspace solid-phase microextraction combined with gas chromatography/mass spectrometry for the determination of trace nitrated polycyclic aromatic hydrocarbons in aqueous samples.

This study describes a new procedure, namely, purge-assisted headspace solid phase microextraction combined with gas chromatography/negative ion chemical ionization mass spectrometry (PA/HS-SPME-GC/NICI-MS), which is used to determine seven nitrated polycyclic aromatic hydrocarbons (NPAHs) in aqueous samples. High extraction efficiency was obtained with PA/HS-SPME with polydimethylsiloxane (PDMS) fiber coating. A programmable temperature vaporizing (PTV) inlet was used in the desorption process. Selected ion monitoring (SIM) was used for quantitative and qualitative purposes. The linear range of detection of the proposed method was 5-5000 pg/mL with coefficients of determination between 0.995 and 0.999. Limits of detection (LODs) for seven NPAHs were 0.01-0.06 pg/mL. The relative standard deviation was below 12.7% at a concentration of 50 pg/mL. Compared with headspace-solid phase microextraction (HS-SPME), the purge procedure enhanced the extraction efficiency for high boiling point analytes, such as 7-nitrobenz[a]anthracene (7-NBA) and 6-nitrochrysene (6-NC). The proposed method provides a sensitive method for NPAH analysis at the pg/mL level. The application of the proposed method for the determination of trace NPAHs in real samples was investigated by analyzing aqueous samples from rivers. The concentrations of NPAHs detected from the samples ranged from 5.2 to 7.5 pg/mL. This method was applied successfully in the analysis of trace NPAHs in river samples.

Supercritical carbon dioxide micronization of zeaxanthin from moderately thermophilic bacteria Muricauda lutaonensis CC-HSB-11T.

Moderately thermophilic bacterial strain CC-HSB-11(T) (Muricauda lutaonensis), which was described recently from a coastal hot spring of Green Island, Taiwan, has been identified to produce zeaxanthin as a predominant xanthophyll by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Cell culture in bioreactor produced 3.12 ± 0.18 mg zeaxanthin L(-1) of culture. Micronization of zeaxanthin was achieved through supercritical carbon dioxide antisolvent precipitation method. Yield of zeaxanthin after the process was 53.4%. Dynamic light scattering assay determined the polydisperse existence of micronized particles of size 3 nm to 2 µm. Field emission scanning electron microscopy revealed distinct morphology and size distribution heterogeneity of particles. Integrity of zeaxanthin after the antisolvent process was assessed by LC-MS/MS. The technique capitalizes on the inherent ability of CC-HSB-11(T) to synthesize zeaxanthin and the work demonstrated feasibility of antisolvent precipitation method to produce microparticles exploiting a bacterial strain.

Identification of new minor metabolites of penicillin G in human serum by multiple-stage tandem mass spectrometry.

Liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) were applied to characterize drug metabolites. Although these two methods have overcome the identification and structural characterization of metabolites analysis, they remain time-consuming processes. In this study, a novel multiple-stage tandem mass spectrometric method (MS(n) ) was evaluated for identification and characterization of new minor metabolism profiling of penicillin G, one of the ß-lactam antibiotics, in human serum. Seven minor metabolites including five phase I metabolites and two phase II metabolites of penicillin G were identified by using data-dependent LC/MS(n) screening in one chromatographic run. The accuracy masses of seven identified metabolites of penicillin G were also confirmed by mass spectral calibration software (MassWorks™). The proposed data-dependent LC/MS(n) method is a powerful tool to provide large amounts of the necessary structural information to characterize minor metabolite in metabolism profiling.

Purge-assisted headspace solid-phase microextraction combined with gas chromatography-mass spectrometry for determination of chlorophenols in aqueous samples.

A simple, economical and very effective method is demonstrated for simultaneous determination of 2,4-dichlorophenol, 2,4,6-trichlorophenol, 2,3,4,6-tetrachlorophenol and pentachlorophenol, in aqueous samples, by using purge-assisted headspace solid-phase microextraction (PA/HS-SPME) coupled to gas chromatography-mass spectrometry (GC-MS). In the new method, purging the sample enhances the removal of the trace chlorophenols without derivatization from the matrices to the headspace. Extraction parameters including extraction temperature, purge gas flow rate and extraction time were systematically investigated. Under optimal conditions, the relative standard deviations (RSDs) were 4-11% at 50 pg/mL and 5-14% at 5 pg/mL, respectively. The recoveries were in the range of 83-114%. Detection limits were determined at the fg level. These results indicate that PA/HS-SPME provides a significant contribution to highly efficient extraction of semi-volatile CPs, especially for pentachlorophenol, which has the smallest Henry's constant and large octanol-water partitioning coefficient. In addition, the proposed method was successfully applied to the analysis of chlorophenols in landfill leachate. New perspectives are opened for headspace extraction of relatively low vapor pressure compounds in complex matrices.