Screening the NCI diversity set V for anti-MRSA activity: cefoxitin synergy and LC-MS/MS confirmation of folate/thymidine biosynthesis inhibition.

IMPORTANCE: New antibacterial agents are urgently needed to counter increasingly resistant bacteria. One approach to this problem is library screening for new antibacterial agents. Library screening efforts can be improved by increasing the information content of the screening effort. In this study, we screened the National Cancer Institute diversity set V against methicillin-resistant Staphylococcus aureus (MRSA) with several enhancements. One of these is to screen the library before and after microsomal metabolism as means to identify potential active metabolites. A second enhancement is to screen the library in the absence and presence of sub-minimum inhibitory concentration levels of another antibiotic, such as cefoxitin in this study. This identified four agents with synergistic activity with cefoxitin out of 16 agents with good MRSA activity alone. Finally, active agents from this effort were counter-screened in the presence of thymidine, which quickly identified three folate/thymidine biosynthesis inhibitors, and also screened for bactericidal vs bacteriostatic activity.

High-Throughput Screening of Natural Product and Synthetic Molecule Libraries for Antibacterial Drug Discovery.

Due to the continued emergence of resistance and a lack of new and promising antibiotics, bacterial infection has become a major public threat. High-throughput screening (HTS) allows rapid screening of a large collection of molecules for bioactivity testing and holds promise in antibacterial drug discovery. More than 50% of the antibiotics that are currently available on the market are derived from natural products. However, with the easily discoverable antibiotics being found, finding new antibiotics from natural sources has seen limited success. Finding new natural sources for antibacterial activity testing has also proven to be challenging. In addition to exploring new sources of natural products and synthetic biology, omics technology helped to study the biosynthetic machinery of existing natural sources enabling the construction of unnatural synthesizers of bioactive molecules and the identification of molecular targets of antibacterial agents. On the other hand, newer and smarter strategies have been continuously pursued to screen synthetic molecule libraries for new antibiotics and new druggable targets. Biomimetic conditions are explored to mimic the real infection model to better study the ligand-target interaction to enable the designing of more effective antibacterial drugs. This narrative review describes various traditional and contemporaneous approaches of high-throughput screening of natural products and synthetic molecule libraries for antibacterial drug discovery. It further discusses critical factors for HTS assay design, makes a general recommendation, and discusses possible alternatives to traditional HTS of natural products and synthetic molecule libraries for antibacterial drug discovery.

Correlative metabologenomics of 110 fungi reveals metabolite-gene cluster pairs.

Natural products research increasingly applies -omics technologies to guide molecular discovery. While the combined analysis of genomic and metabolomic datasets has proved valuable for identifying natural products and their biosynthetic gene clusters (BGCs) in bacteria, this integrated approach lacks application to fungi. Because fungi are hyper-diverse and underexplored for new chemistry and bioactivities, we created a linked genomics-metabolomics dataset for 110 Ascomycetes, and optimized both gene cluster family (GCF) networking parameters and correlation-based scoring for pairing fungal natural products with their BGCs. Using a network of 3,007 GCFs (organized from 7,020 BGCs), we examined 25 known natural products originating from 16 known BGCs and observed statistically significant associations between 21 of these compounds and their validated BGCs. Furthermore, the scalable platform identified the BGC for the pestalamides, demystifying its biogenesis, and revealed more than 200 high-scoring natural product-GCF linkages to direct future discovery.

Self-Assembled Nanomicellar Formulation of Docetaxel as a Potential Breast Cancer Chemotherapeutic System.

Docetaxel (DTX) is classified as a class IV drug that exhibits poor aqueous solubility (6-7 µg/mL in water) and permeability (P-glycoprotein substrate). The main objective of this study was to construct, characterize, and evaluate docetaxel loaded nanomicellar formulation in vitro for oral delivery to enhance the absorption and bioavailability of DTX, as well as to circumvent P-gp efflux inhibition. Formulations were prepared with two polymeric surfactants, hydrogenated castor oil-40 (HCO-40) and D-α-Tocopherol polyethylene glycol 1000 succinate (VIT E TPGS) with solvent evaporation technique, and the resulting DTX nanomicellar formulations were characterized by proton nuclear magnetic resonance spectroscopy (1H NMR), Fourier Transform Infrared Spectroscopy (FT-IR), X-ray powder diffraction (XRD), and transmission electron microscopy (TEM). Proton NMR, FT-IR, and XRD data indicated that DTX was completely encapsulated within the hydrophobic core of the nanomicelles in its amorphous state. TEM data revealed a smooth spherical shape of the nanomicellar formulation. The optimized formulation (F-2) possessed a mean diameter of 13.42 nm, a zeta potential of -0.19 mV, with a 99.3% entrapment efficiency. Dilution stability study indicated that nanomicelles were stable up to 100-fold dilution with minimal change in size, poly dispersity index (PDI), and zeta potential. In vitro cytotoxicity study revealed higher anticancer activity of DTX nanomicelles at 5 µM compared to the native drug against breast cancer cell line (MCF-7) cells. The LC-MS data confirmed the chemical stability of DTX within the nanomicelles. In vitro drug release study demonstrated faster dissolution of DTX from the nanomicelles compared to the naked drug. Our experimental results exhibit that nanomicelles could be a drug delivery system of choice to encapsulate drugs with low aqueous solubility and permeability that can preserve the stability of the active constituents to provide anticancer activity.

Enhancing Etoposide Aqueous Solubility and Anticancer Activity with L-Arginine.

It is hypothesized that L-arginine (ARG) can improve etoposide (VP-16) water solubility while preserving its anticancer activity. Factorial design is used to identify conditions for optimum drug aqueous solubility after freeze-drying. The physicochemical properties of the optimized formulation is further analyzed by X-ray powder diffraction, scanning electron microscopy, proton nuclear magnetic resonance, and fourier transform infrared spectroscopy. Drug stability in formulation is analyzed using mass spectrometry based fragmentation analysis. Liquid chromatography tandem mass spectrometry and cell viability assay on Michigan Cancer Foundation-7 (MCF-7) cell line are performed to assess the drug cellular uptake and the anticancer activity, respectively. At the VP-16: ARG ratio of 4:10 (w/w), the drug apparent solubility increased significantly (∼65-folds) with a 3.5-fold improvement in the drug dissolution rate. The interaction between VP-16 and ARG transforms the drug from crystalline to amorphous solid state. VP-16-ARG complex in lower native drug concentration range (50-300 µM) has lower anticancer activity compared with native VP-16, due to reduced intracellular transport of the more hydrophilic complex as indicated by the cell viability assay and confirmed by cell uptake study. However, at higher drug concentrations (500 µM) the complex's higher anticancer activity is ascribed to the synergistic effect between ARG and VP-16. These data suggest that an optimal ARG concentration can have positive effects with potential benefits for chemotherapy.

Self-Assembled Nanomicelles to Enhance Solubility and Anticancer Activity of Etoposide.

It is hypothesized that etoposide/VP-16 nanomicellar formulation (VP-16 NMF) utilizing D-α-Tocopherol polyethylene glycol 1000 succinate (TPGS) can improve etoposide solubility and anticancer activity. The following four different concentrations of TPGS: 3, 6, 8, and 10 wt% were used to solubilize the drug. Among these four formulations, 10 wt% of TPGS loaded with VP-16 NMF dramatically enhanced etoposide apparent solubility by 26-folds compared with the native drug. The physicochemical properties of the optimized formulation were further analyzed by dynamic light scattering, X-ray powder diffraction, scanning electron microscopy, proton nuclear magnetic resonance (1HNMR) and Fourier transform infrared spectroscopy. Liquid chromatography tandem-mass spectrometry (LC-MS/MS) was used to assess solubility and intracellular uptake of the drug from the NMF. Cell viability assay ([3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium solution [MTS]) was performed on MCF-7 and MCF-10A cell lines to assess intracellular uptake and anticancer activity of etoposide. The MTS assay results showed that the VP-16 NMF platform provides a higher anticancer activity than the native VP-16 on the MCF-7 cells line as it integrates a dual anticancer activity of VP-16 and TPGS. LC-MS/MS data showed a threefold increase in cellular uptake of VP-16 NMF in MCF-7 cell line compared with the native etoposide. These data suggest that an optimal TPGS concentration can improve VP-16 bioavailability and efficacy with potential benefits for chemotherapy.

Positive/negative ion-switching-based LC-MS/MS method for quantification of cytosine derivatives produced by the TET-family 5-methylcytosine dioxygenases.

Cytosine methylation at carbon-5 (5mC) in DNA plays crucial roles in epigenetic transcriptional regulation during metazoan development. The iron (II), 2-oxoglutarate-dependent Ten-Eleven Translocation (TET)-family dioxygenases initiate active demethylation of 5mC. TET2 oxidizes 5mC in nucleic acids into 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine by iterative oxidation. Mutations in the TET2 gene are frequently detected in myeloid malignancies. Despite the established and emerging roles of TET oxygenases in health and diseases, in vitro characterization of these enzymes and their mutants is still in rudimentary stages. Here, we describe an improved positive/negative ion-switching-based liquid chromatography-tandem mass spectrometry (LC-MS/MS) method that can separate and quantify modified cytosine bases produced by TET-family 5-methylcytosine dioxygenases. This method will help in further elucidate the function of epigenetically important cytosine modifications. To the best of our knowledge, this is the first study reporting ion-switching-based LC-MS/MS method to analyse cytosine variants produced in TET catalysed reactions.

Dimensionally Enhanced Antibacterial Library Screening.

The emergence and spread of antimicrobial resistance is a major public health threat, and there is an urgent need to develop new strategies to address the issue. In this study, the possibility of enhancing a whole cell based antibacterial library screen by increasing the dimensionality of the screening effort is explored using methicillin-resistant Staphylococcus aureus (MRSA) as the target organism. One dimension involved generating and screening a human liver microsome metabolized FDA approved drug library. Comparative screening of the un-metabolized (UM) and pre-metabolized (PM) libraries allows identification of intrinsically active agents from the UM library screen and of agents with active metabolites from the PM library screen. To further enhance this screening effort, it was combined with a -/+ resistant to antibiotic screen (-/+ cefoxitin; Cef). This allows the identification of agents that can act synergistically with the resistant to antibiotic. This approach revealed five compounds with substantially improved activity after metabolism and four compounds with substantial synergistic activity with cefoxitin. Capecitabine in particular only had significant antibacterial activity after metabolism. Its metabolites were isolated, identified, and characterized for spectrum of activity along with several other anticancer drugs with anti-MRSA activity. Floxuridine, gemcitabine, novobiocin, and rifaximin were identified as having substantial synergy with cefoxitin from the -/+Cef screens. Checkerboard assays verified synergy for these agents. Floxuridine demonstrated a particularly high degree of synergy with cefoxitin (FIC = 0.14). This study demonstrates how a dimensionally enhanced comparative screening effort can identify new antibacterial agents and strategies for countering antibacterial agent resistance.

LC-MS/MS-Based Separation and Quantification of Marfey's Reagent Derivatized Proteinogenic Amino Acid DL-Stereoisomers.

D-Amino acids are important biological molecules. Improved analytical methods for their resolution and quantification remain of keen interest. In this study, we investigated the use of Marfey's reagent (chiral) derivatization coupled with LC-MS/MS-based separation and detection of the resulting diastereomers for quantification of the 19 common L- and D-amino acids and glycine. Standard formic acid (pH 2)-based separations on reverse phase media were unable to separate all 19 amino acid DL pairs. In contrast, a water/acetonitrile/ammonium acetate (pH 6.5) solvent system allowed all 19 amino acid DL pairs to be chromatographically resolved on a 30 min gradient, with negative mode detection at pH 6.5 giving good sensitivity. Derivatization reaction rates between amino acids varied substantially, with overnight derivatization required for some amino acids. Chromatography at pH 6.5 combined with MS/MS quantification in negative mode demonstrated good linearity over a wide concentration range for all 20 amino acids. Matrix effects, assessed with an MRSA extract, were negligible. Marfey's derivatized analytes were stable for 24 h at room temperature. This method was demonstrated by determining the levels of these analytes in mid-log phase MRSA extracts. This approach provides for the chromatographic resolution and MS/MS-based quantification of all 20 common L- and D-amino acids in complex matrices. Graphical Abstract.

Efficient Purification and LC-MS/MS-based Assay Development for Ten-Eleven Translocation-2 5-Methylcytosine Dioxygenase.

The epigenetic transcription regulation mediated by 5-methylcytosine (5mC) has played a critical role in eukaryotic development. Demethylation of these epigenetic marks is accomplished by sequential oxidation by ten-eleven translocation dioxygenases (TET1-3), followed by the thymine-DNA glycosylase-dependent base excision repair. Inactivation of the TET2 gene due to genetic mutations or by other epigenetic mechanisms is associated with a poor prognosis in patients with diverse cancers, especially hematopoietic malignancies. Here, we describe an efficient single step purification of enzymatically active untagged human TET2 dioxygenase using cation exchange chromatography. We further provide a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach that can separate and quantify the four normal DNA bases (A, T, G, and C), as well as the four modified cytosine bases (5-methyl, 5-hydroxymethyl, 5-formyl, and 5-carboxyl). This assay can be used to evaluate the activity of wild type and mutant TET2 dioxygenases.

Engineering fast dissolving sodium acetate mediated crystalline solid dispersion of docetaxel.

It is hypothesized that a novel crystalline solid dispersion (CSD) of docetaxel (C-DXT) can be engineered by dispersing native docetaxel (DXT, a BCS class II drug) in sodium acetate crystal (SA). DXT is dissolved in glacial acetic/SA solution and freeze-dried. The resulting C-DXT is characterized by differential scanning calorimetry (DSC), powder X-ray analysis (PXRD), LC-MS/MS, scanning electron microscopy (SEM), transmission electron microscopy (TEM), Quartz crystal microbalance with dissipation monitoring (QCM-D) and dynamic light scattering (DLS). Its cytotoxicity on model cancerous (MCF-7, MDA-MB-468) and normal breast cells (MCF-10A) is assessed by MTS assay. SEM/TEM data and the absence of the characteristics peaks of DXT on the DSC curve (at 193.4 °C) and the XRD scan (at 2θ = 15.31 °C and 23.04 °C) confirm the presence of C-DXT in SA. The LC-MS/MS data indicates the chemical stability of DXT. The yield and C-DXT loading are 95.2% and 6.52% w/w, respectively. The C-DXT rapidly forms an aqueous non-rigid nanosuspension with a faster drug dissolution rate compared to native DXT. Unlike, control Tween 80/ethanol, SA is noncytotoxic to normal cells. However, C-DXT's cytotoxicity is time and dose dependent for all diseased cells. This unique CSD process might be applicable to other hydrophobic bioactive agents to enhance their safety and efficacy.

Layer-by-Layer Engineered Microbicide Drug Delivery System Targeting HIV-1 gp120: Physicochemical and Biological Properties.

The purpose of this study was to engineer a model anti-HIV microbicide (tenofovir) drug delivery system targeting HIV-1 envelope glycoprotein gp120 (HIV-1 g120) for the prevention of HIV sexual transmission. HIV-1 g120 and mannose responsive particles (MRP) were prepared through the layer-by-layer coating of calcium carbonate (CaCO3) with concanavalin A (Con A) and glycogen. MRP average particle size ranged from 881.7 ± 15.45 nm to 1130 ± 15.72 nm, depending on the number of Con A layers. Tenofovir encapsulation efficiency in CaCO3 was 74.4% with drug loading of 16.3% (w/w). MRP was non-cytotoxic to Lactobacillus crispatus, human vaginal keratinocytes (VK2), and murine macrophage RAW 264.7 cells and did not induce any significant proinflammatory nitric oxide release. Overall, compared to control, no statistically significant increase in proinflammatory cytokine IL-1α, IL-1ß, IL-6, MKC, IL-7, and interferon-γ-inducible protein 10 (IP10) levels was observed. Drug release profiles in the presence of methyl α-d-mannopyranoside and recombinant HIV-1 envelope glycoprotein gp120 followed Hixson-Crowell and Hopfenberg kinetic models, indicative of a surface-eroding system. The one Con A layer containing system was found to be the most sensitive (∼2-fold increase in drug release vs control SFS:VFS) at the lowest HIV gp120 concentration tested (25 µg/mL). Percent mucoadhesion, tested ex vivo on porcine vaginal tissue, ranged from 10% to 21%, depending on the number of Con A layers in the formulation. Collectively, these data suggested that the proposed HIV-1 g120 targeting, using MRP, potentially represent a safe and effective template for vaginal microbicide drug delivery, if future preclinical studies are conclusive.

Antibiotic Effects on Methicillin-Resistant Staphylococcus aureus Cytoplasmic Peptidoglycan Intermediate Levels and Evidence for Potential Metabolite Level Regulatory Loops.

Cytoplasmic peptidoglycan (PG) precursor levels were determined in methicillin-resistant Staphylococcus aureus (MRSA) after exposure to several cell wall-targeting antibiotics. Three experiments were performed: (i) exposure to 4× MIC levels (acute); (ii) exposure to sub-MIC levels (subacute); (iii) a time course experiment of the effect of vancomycin. In acute exposure experiments, fosfomycin increased UDP-GlcNAc, as expected, and resulted in substantially lower levels of total UDP-linked metabolite accumulation relative to other pathway inhibitors, indicating reduced entry into this pathway. Upstream inhibitors (fosfomycin, d-cycloserine, or d-boroalanine) reduced UDP-MurNAc-pentapeptide levels by more than fourfold. Alanine branch inhibitors (d-cycloserine and d-boroalanine) reduced d-Ala-d-Ala levels only modestly (up to 4-fold) but increased UDP-MurNAc-tripeptide levels up to 3,000-fold. Downstream pathway inhibitors (vancomycin, bacitracin, moenomycin, and oxacillin) increased UDP-MurNAc-pentapeptide levels up to 350-fold and UDP-MurNAc-l-Ala levels up to 80-fold, suggesting reduced MurD activity by downstream inhibitor action. Sub-MIC exposures demonstrated effects even at 1/8× MIC which strongly paralleled acute exposure changes. Time course data demonstrated that UDP-linked intermediate levels respond rapidly to vancomycin exposure, with several intermediates increasing three- to sixfold within minutes. UDP-linked intermediate level changes were also multiphasic, with some increasing, some decreasing, and some increasing and then decreasing. The total (summed) UDP-linked intermediate pool increased by 1,475 µM/min during the first 10 min after vancomycin exposure, providing a revised estimate of flux in this pathway during logarithmic growth. These observations outline the complexity of PG precursor response to antibiotic exposure in MRSA and indicate likely sites of regulation (entry and MurD).

Gaussian and linear deconvolution of LC-MS/MS chromatograms of the eight aminobutyric acid isomers.

Isomeric molecules present a challenge for analytical resolution and quantification, even with MS-based detection. The eight aminobutyric acid (ABA) isomers are of interest for their various biological activities, particularly γ-aminobutyric acid (GABA) and the d- and l-isomers of ß-aminoisobutyric acid (ß-AIBA; BAIBA). This study aimed to investigate LC-MS/MS-based resolution of these ABA isomers as their Marfey's (Mar) reagent derivatives. HPLC was able to separate three Mar-ABA isomers l-ß-ABA (l-BABA), and l- and d-α-ABA (AABA) completely, with three isomers (GABA, and d/l-BAIBA) in one chromatographic cluster, and two isomers (α-AIBA (AAIBA) and d-BABA) in a second cluster. Partially separated cluster components were deconvoluted using Gaussian peak fitting except for GABA and d-BAIBA. MS/MS detection of Marfey's derivatized ABA isomers provided six MS/MS fragments, with substantially different intensity profiles between structural isomers. This allowed linear deconvolution of ABA isomer peaks. Combining HPLC separation with linear and Gaussian deconvolution allowed resolution of all eight ABA isomers. Application to human serum found a substantial level of l-AABA (13 µM), an intermediate level of l-BAIBA (0.8 µM), and low but detectable levels (<0.2 µM) of GABA, l-BABA, AAIBA, d-BAIBA, and d-AABA. This approach should be useful for LC-MS/MS deconvolution of other challenging groups of isomeric molecules.

Cytoplasmic peptidoglycan intermediate levels in Staphylococcus aureus.

Intracellular cytoplasmic peptidoglycan (PG) intermediate levels were determined in Staphylococcus aureus during log-phase growth in enriched media. Levels of UDP-linked intermediates were quantitatively determined using ion pairing LC-MS/MS in negative mode, and amine intermediates were quantitatively determined stereospecifically as their Marfey's reagent derivatives in positive mode. Levels of UDP-linked intermediates in S. aureus varied from 1.4 µM for UDP-GlcNAc-Enolpyruvyate to 1200 µM for UDP-MurNAc. Levels of amine intermediates (L-Ala, D-Ala, D-Ala-D-Ala, L-Glu, D-Glu, and L-Lys) varied over a range of from 860 µM for D-Ala-D-Ala to 30-260 mM for the others. Total PG was determined from the D-Glu content of isolated PG, and used to estimate the rate of PG synthesis (in terms of cytoplasmic metabolite flux) as 690 µM/min. The total UDP-linked intermediates pool (2490 µM) is therefore sufficient to sustain growth for 3.6 min. Comparison of UDP-linked metabolite levels with published pathway enzyme characteristics demonstrates that enzymes on the UDP-branch range from >80% saturation for MurA, Z, and C, to <5% saturation for MurB. Metabolite levels were compared with literature values for Escherichia coli, with the major difference in UDP-intermediates being the level of UDP-MurNAc, which was high in S. aureus (1200 µM) and low in E. coli (45 µM).