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Lipopolysaccharide (LPS) resides in the outer membrane of Gram-negative bacteria where it is responsible for barrier function1,2. LPS can cause death as a result of septic shock, and its lipid A core is the target of polymyxin antibiotics3,4. Despite the clinical importance of polymyxins and the emergence of multidrug resistant strains5, our understanding of the bacterial factors that regulate LPS biogenesis is incomplete. Here we characterize the inner membrane protein PbgA and report that its depletion attenuates the virulence of Escherichia coli by reducing levels of LPS and outer membrane integrity. In contrast to previous claims that PbgA functions as a cardiolipin transporter6–9, our structural analyses and physiological studies identify a lipid A-binding motif along the periplasmic leaflet of the inner membrane. Synthetic PbgA-derived peptides selectively bind to LPS in vitro and inhibit the growth of diverse Gram-negative bacteria, including polymyxin-resistant strains. Proteomic, genetic and pharmacological experiments uncover a model in which direct periplasmic sensing of LPS by PbgA coordinates the biosynthesis of lipid A by regulating the stability of LpxC, a key cytoplasmic biosynthetic enzyme10–12. In summary, we find that PbgA has an unexpected but essential role in the regulation of LPS biogenesis, presents a new structural basis for the selective recognition of lipids, and provides opportunities for future antibiotic discovery. Structural and physiological studies show that the inner membrane protein PbgA is a crucial sensor of lipopolysaccharide (LPS) and regulates the activity of the LPS biosynthesis enzyme LpxC.

To better understand regional brain glycosphingolipid (GSL) accumulation in Gaucher disease (GD) and its relationship to neuropathology, a feasibility study using mass spectrometry and immunohistochemistry was conducted using brains derived from a GD mouse model (4L/PS/NA) homozygous for a mutant GCase (V394L [4L]) and expressing a prosaposin hypomorphic (PS-NA) transgene. Whole brains from GD and control animals were collected using one hemisphere for MALDI FTICR IMS analysis and the other for quantitation by LC-ESI-MS/MS. MALDI IMS detected several HexCers across the brains. Comparison with the brain hematoxylin and eosin (H&E) revealed differential signal distributions in the midbrain, brain stem, and CB of the GD brain versus the control. Quantitation of serial brain sections with LC-ESI-MS/MS supported the imaging results, finding the overall HexCer levels in the 4L/PS-NA brains to be four times higher than the control. LC-ESI-MS/MS also confirmed that the elevated hexosyl isomers were glucosylceramides rather than galactosylceramides. MALDI imaging also detected differential analyte distributions of lactosylceramide species and gangliosides in the 4L/PS-NA brain, which was validated by LC-ESI-MS/MS. Immunohistochemistry revealed regional inflammation, altered autophagy, and defective protein degradation correlating with regions of GSL accumulation, suggesting that specific GSLs may have distinct neuropathological effects.

The objectives of the present study were to characterize GNE-947 for its phosphoinositide 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) inhibitory activities, in vitro anti-cell migration activity in Human Umbilical Vein Endothelial Cells (HUVEC), in vivo anti-neovascularization activity in laser induced rat choroidal neovascular (CNV) eyes, pharmacokinetics in rabbit plasma and eyes, and ocular distribution using Matrix-Assisted Laser Desorption Ionization Imaging mass spectrometry (MALDI-IMS) and autoradioluminography. Its PI3K and mTOR Ki were 0.0005 µM and 0.045 µM, respectively, and its HUVEC IC50 was 0.093µM. GNE-947 prevented neovascularization in the rat CNV model at 50 or 100 µg/eye with repeat dosing. Following a single intravenous injection at 2.5 and 500 μg/kg in rabbits, its plasma terminal half-lives (t1/2) were 9.11 and 9.59 hours, respectively. Following a single intravitreal injection of a solution at 2.5 μg/eye in rabbits, its apparent t1/2 values were 14.4, 16.3, and 23.2 hours in the plasma, vitreous humor, and aqueous humor, respectively. Following a single intravitreal injection of a suspension at 33.5, 100, 200 μg/eye in rabbits, the t1/2 were 29, 74, and 219 days in the plasma; and 46, 143, and 191 days in the eyes, respectively. MALDI-IMS and autoradioluminography images show that GNE-947 did not homogenously distribute in the vitreous humor and aggregated at the injection sites following injection of the suspension, which was responsible for the long t1/2 of the suspension due to slow dissolution process. This hypothesis was supported by pharmacokinetic modeling analyses. In conclusion, PI3K/mTOR inhibitor GNE-947 prevented neovascularization in a rat CNV model with t1/2 up to approximately six months following a single intravitreal injection of the suspension in rabbit eyes. SIGNIFICANCE STATEMENT GNE-947 is a potent PI3K/mTOR inhibitor and exhibits anti-CNV activity in rat eyes. The duration of GNE-947 in the rabbit eyes following intravitreal injection in a solution is short with t1/2 less than a day. However, the duration following intravitreal dose of a suspension is very long with t1/2 up to six months due to its low solubility and slow dissolution process. These results indicate that intravitreal injection of a suspension formulation for low solubility drugs can be used to achieve long-term drug exposure potentially useful for the treatment of wet AMD and other retinal diseases.

Aim: Objective of this study is to develop a robust multi-matrix LC–MS/MS for the quantitation of endogenous short-chain fatty acids (SCFA) biomarkers in human plasma and urine. Methods: Developed method utilizes stable isotope-labeled internal standards, high-throughput derivatization procedure for sample preparation and LC–MS/MS analysis using multiple reaction monitoring transitions in positive electrospray ionization mode. Results: Surrogate matrix method was used for quantitation. Accuracy, precision, parallelism, curve linearity, derivatization efficiency, stability and recovery were all evaluated, and the results were well within the acceptable criteria. Conclusion: SCFA levels in human plasma and urine of inflammatory bowel disease patients versus non-disease subjects were quantified and compared by LC–MS/MS.

Cobimetinib is a MEK inhibitor currently in clinical trials as an anticancer agent. The objectives of this study were to determine in vitro and in vivo if cobimetinib is a substrate of P-glycoprotein (P-gp) and/or breast cancer resistance protein (Bcrp1) and to assess the implications of efflux on cobimetinib pharmacokinetics (PK), brain penetration, and target modulation. Cell lines transfected with P-gp or Bcrp1 established that cobimetinib was a substrate of P-gp but not a substrate of Bcrp1. In vivo, after intravenous and oral administration of cobimetinib to FVB (wild-type; WT), Mdr1a/b(-/-), Bcrp1 (-/-), and Mdr1a/b(-/-)/Bcrp(-/-) knockout (KO) mice, clearance was similar in WT (35.5 ± 16.7 mL/min/kg) and KO animals (22.0 ± 3.6 to 27.6 ± 5.2 mL/min/kg); oral exposure was also similar between WT and KO animals. After an oral 10 mg/kg dose of cobimetinib, the mean total brain to plasma ratio (Kp) at 6 h postdose was 0.3 and 0.2 in WT and Bcrp1(-/-) mice, respectively. In Mdr1a/b(-/-) and Mdr1a/1b/Bcrp1(-/-) KO mice and WT mice treated with elacridar (a P-gp and BCRP inhibitor), Kp increased to 11, 6, and 7, respectively. Increased brain exposure in Mdr1a/b(-/-) and Mdr1a/1b/Bcrp1(-/-) KO and elacridar treated mice was accompanied by up to ∼65% suppression of the target (pErk) in brain tissue, compared to WT mice. By MALDI imaging, the cobimetinib signal intensity was relatively high and was dispersed throughout the brain of Mdr1a/1b/Bcrp1(-/-) KO mice compared to low/undetectable signal intensity in WT mice. The efflux of cobimetinib by P-gp may have implications for the treatment of patients with brain tumors/metastases.

In drug discovery and development, in vitro absorption and metabolism assays along with in vivo pharmacokinetic (PK), pharmacodynamic (PD), and toxicokinetic (TK) studies are used to evaluate a potential drug candidate. More recently, imaging mass spectrometry approaches have been successfully reported to aid in the preclinical assessment of drug candidates, resulting in the rapid and noteworthy acceptance of the technique in pharmaceutical research. Traditionally, drug distribution studies via mass spectrometric imaging (MSI) are performed as targeted MS/MS analyses, where the analytes of interest, drug and/or metabolite, are known before the imaging experiment is performed. The study presented here describes a whole-body mass spectrometric imaging (WB-MSI) approach using a hybrid MALDI-LTQ-Orbitrap-MS to detect the distribution of reserpine at 2 h post a 20 mg/kg oral dose. This study effectively demonstrates the utility of obtaining accurate mass measurements across a wide mass range combined with postprocessing tools to efficiently identify drug and metabolite distributions without the need for any a priori knowledge.

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults, and the limited available treatment options have not meaningfully impacted patient survival in the past decades. Such poor outcomes can be at least partly attributed to the inability of most drugs tested to cross the blood-brain barrier and reach all areas of the glioma. The objectives of these studies were to visualize and compare by matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry the brain and tumor distribution of the phosphatidylinositol 3-kinase (PI3K) inhibitors pictilisib (GDC-0941, 2-(1H-indazol-4-yl)-6-(4-methanesulfonyl-piperazin-1-ylmethyl)-4-morpholin-4-yl-thieno[3,2-d]pyrimidine) and GNE-317 [5-(6-(3-methoxyoxetan-3-yl)-7-methyl-4-morpholinothieno[3,2-d]pyrimidin-2-yl)pyrimidin-2-amine] in U87 and GS2 orthotopic models of GBM, models that exhibit differing blood-brain barrier characteristics. Following administration to tumor-bearing mice, pictilisib was readily detected within tumors of the contrast-enhancing U87 model whereas it was not located in tumors of the nonenhancing GS2 model. In both GBM models, pictilisib was not detected in the healthy brain. In contrast, GNE-317 was uniformly distributed throughout the brain in the U87 and GS2 models. MALDI imaging revealed also that the pictilisib signal varied regionally by up to 6-fold within the U87 tumors whereas GNE-317 intratumor levels were more homogeneous. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analyses of the nontumored half of the brain showed pictilisib had brain-to-plasma ratios lower than 0.03 whereas they were greater than 1 for GNE-317, in agreement with their brain penetration properties. These results in orthotopic models representing either the contrast-enhancing or invasive areas of GBM clearly demonstrate the need for whole-brain distribution to potentially achieve long-term efficacy in GBM.

The discovery and optimization of a series of tetrahydropyridopyrimidine based extracellular signal-regulated kinase (Erks) inhibitors discovered via HTS and structure based drug design is reported. The compounds demonstrate potent and selective inhibition of Erk2 and knockdown of phospho-RSK levels in HepG2 cells and tumor xenografts.

DNA glycosylases catalyze scission of the N-glycosylic bond linking a damaged base to the DNA sugar phosphate backbone. Some of these enzymes carry out a concomitant abasic (apyrimidinic/apurinic (AP)) lyase reaction at a rate approximately equal to that of the glycosylase step. As a generalization of the mechanism described for T4 endonuclease V, a repair glycosylase/AP lyase that is specific for ultraviolet light-induced cis-syn pyrimidine dimers, a hypothesis concerning the mechanism of these repair glycosylases has been proposed. This hypothesis describes the initial action of all DNA glycosylases as a nucleophilic attack at the sugar C-1′ of the damaged base nucleoside, resulting in scission of the N-glycosylic bond. It is proposed that the enzymes that are only glycosylases differ in the chemical nature of the attacking nucleophile from the glycosylase/AP lyases. Those DNA glycosylases, which carry out the AP lyase reaction at a rate approximately equal to the glycosylase step, are proposed to use an amino group as the nucleophile, resulting in an imino enzyme-DNA intermediate. The simple glycosylases, lacking the concomitant AP lyase activity, are proposed to use some nucleophile from the medium, e.g. an activated water molecule. This paper reports experimental tests of this hypothesis using five representative enzymes, and these data are consistent with this hypothesis.

Endonuclease V, a N-glycosylase/lyase from T4 bacteriophage that initiates the repair of cyclobutane pyrimidine dimers in DNA, has been reported to form a monomer-dimer equilibrium in solution [Nickell and Lloyd (1991) Biochemistry 30, 8638], although the enzyme has only been crystallized in the absence of substrate as a monomer [Morikawa et al. (1992) Science 256, 523]. In this study, analytical gel filtration and sedimentation equilibrium techniques were used to rigorously characterize the association state of the enzyme in solution. In contrast to the previous report, at 100 mM KCl endonuclease V was found to exist predominantly as a monomer in solution by both of these techniques; no evidence for dimerization was seen. To characterize the oligomeric state of the enzyme at its target sites on DNA, the enzyme was bound to oligonucleotides containing a single site specific pyrimidine dimer or tetrahydrofuran residue. These complexes were analyzed by nondenaturing gel electrophoresis at various acrylamide concentrations in order to determine the molecular weights of the enzyme-DNA complexes. The results from these experiments demonstrate that endonuclease V binds to cyclobutane pyrimidine dimer and tetrahydrofuran site containing DNA as a monomer.

The interaction between endonuclease V, the cyclobutane pyrimidine dimer-specific N-glycosylase/abasic lyase from bacteriophage T4, and DNA was investigated by DNase I footprinting methods. The catalytically inactive mutant E23Q was found to interact with a smaller region of DNA at the abasic site analog, tetrahydrofuran, than at a thymine dimer site. Like the wild-type enzyme, the mutant contacted the DNA substrates primarily on the strand opposite the damage. The various complexes examined by footprinting techniques represent distinct points along the catalytic pathway of endonuclease V: before catalysis at a dimer, after N-glycosylase action but before abasic lyase action, and before catalysis at an abasic site. The differences between the footprints of the mutant and wild-type enzymes on both DNA substrates likely represent subtly different conformations within these complexes.

Bacteriophage T4 displays enhanced ultraviolet light (UV) resistance over other T-even bacteriophages. This enhanced resistance was initially ascribed to the v gene, now known as denV. The product of the denV gene, endonuclease V, has been shown to be responsible for initiating the removal of pyrimidine dimers from DNA. Initial characterization of purified endonuclease V revealed that the enzyme produces single-strand breaks in DNA at the site of UV-induced pyrimidine dimers, yet shows no activity towards unirradiated or heat-denatured DNA. The enzyme seems almost completely specific for cis-syn cyclobutane pyrimidine dimers, although recently it has been demonstrated to react with trans-syn dimers at approximately 1/100th the rate of the cis-syn dimer. DenV has been cloned and sequenced, allowing for expression of endonuclease V within Escherichia coli. The gene encodes a 138-amino acid protein with a predicted molecular mass of 16 kDa. Extensive biochemical and genetic studies have shown endonuclease V to possess four distinct activities: (1) a salt-dependent scanning or looping mechanism allowing for nontarget double-stranded DNA interactions; (2) a pyrimidine dimer-specific binding activity; (3) a pyrimidine dimer-specific DNA glycosylase activity; and (4) an apurinic/apyrimidinic (AP) lyase activity.

Certain cellular responses to thyroid hormones appear to be mediated by non-histone chromatin protein receptors. Purification of these proteins is important for an investigation of the detailed mechanisms of their regulatory role. In the present studies, we report the development of an affinity chromatographic system that can be used to purify thyroid hormone receptors solubilized from nuclei. Amine-substituted hormone analogs were prepared with D and L isomers of T3; these bind to the receptor. This finding supports the hypothesis that thyroid hormones fit into the receptor with the amino groups accessible from outside the binding site. Although L-triiodothyronine (LT3) (the naturally occurring isomer) binds more tightly (relative Kd = 1.0 nM) to the nuclear receptor than D-triiodothyronine (DT3) (relative Kd = 2.0 nM), the amine-substituted analog of DT3 binds more tightly than the LT3 analog (DT3 analog, relative Kd = 40 nM; LT3 analog, relative Kd = 1500 nM). Agarose-based gels containing DT3 and LT3 covalently coupled by their amino groups were also prepared. Binding of receptor to these gels was biospecific in that it could be inhibited by prior incubation of the receptors with LT3. In addition, as predicted by the analog studies, the DT3 affinity gels were more effective than LT3 gels in adsorbing receptor. Elution of receptor from the LT3-derived gels was achieved in a predicted volume and concentration of counter-ligand in elution buffer. These results suggest that affinity chromatography can be applied to the purification of thyroid hormone receptors.