Charles M. Thompson

Mass spectrometry identifies covalent binding of soman, sarin, chlorpyrifos oxon, diisopropyl fluorophosphate, and FP-biotin to tyrosines on tubulin : A potential mechanism of long term toxicity by organophosphorus agents

Chronic low dose exposure to organophosphorus poisons (OP) results in cognitive impairment. Studies in rats have shown that OP interfere with microtubule polymerization. Since microtubules are required for transport of nutrients from the nerve cell body to the nerve synapse, it has been suggested that disruption of microtubule function could explain the learning and memory deficits associated with OP exposure. Tubulin is a major constituent of microtubules. We tested the hypothesis that OP bind to tubulin by treating purified bovine tubulin with sarin, soman, chlorpyrifos oxon, diisopropylfluorophosphate, and 10-fluoroethoxyphosphinyl-N-biotinamidopentyldecanamide (FP-biotin). Tryptic peptides were isolated and analyzed by mass spectrometry. It was found that OP bound to tyrosine 83 of alpha tubulin in peptide TGTYR, tyrosine 59 in beta tubulin peptide YVPR, tyrosine 281 in beta tubulin peptide GSQQYR, and tyrosine 159 in beta tubulin peptide EEYPDR. The OP reactive tyrosines are located either near the GTP binding site or within loops that interact laterally with protofilaments. It is concluded that OP bind covalently to tubulin, and that this binding could explain cognitive impairment associated with OP exposure.

Inhibitors of the Glutamate Vesicular Transporter (VGLUT)

The vesicular glutamate transporter (VGLUT) is responsible for the uptake of the excitatory amino acid, L-glutamate, into synaptic vesicles. VGLUT activity is coupled to an electrochemical gradient driven by a vacuolar ATPase and stimulated by low Cl-. VGLUT has relatively low affinity (K(m) = 1-3 mM) for glutamate and is pharmacologically and structurally distinct from the Na+-dependent, excitatory amino acid transporters (EAATs) found on the plasma membrane. Because glutamatergic neurotransmission begins with vesicular release, compounds that block the uptake of glutamate into the vesicle may reduce excitotoxic events. Several classes of competitive VGLUT inhibitors have emerged including amino acids and amino acid analogs, fatty acids, azo dyes, quinolines and alkaloids. The potency with which these agents inhibit VGLUT varies from millimolar (amino acids) to nanomolar (azo dyes) concentrations. These inhibitors represent highly diverse structures and have collectively begun to reveal key pharmacophore elements that may elucidate the key interactions important to binding VGLUT. Using known inhibitor structures and preliminary molecular modeling, a VGLUT pharmacophore is presented that will aid in the design of new, highly potent and selective agents.

Five Tyrosines and Two Serines in Human Albumin Are Labeled by the Organophosphorus Agent FP-Biotin

Tyrosine 411 of human albumin is an established site for covalent attachment of 10-fluoroethoxyphosphinyl-N-biotinamidopentyldecanamide (FP-biotin), diisopropylfluorophosphate, chlorpyrifos oxon, soman, sarin, and dichlorvos. This work investigated the hypothesis that other residues in albumin could be modified by organophosphorus agents (OP). Human plasma was aggressively treated with FP-biotin; plasma proteins were separated into high and low abundant portions using a proteome partitioning antibody kit, and the proteins were digested with trypsin. The FP-biotinylated tryptic peptides were isolated by binding to monomeric avidin beads. The major sites of covalent attachment identified by mass spectrometry were Y138, Y148, Y401, Y411, Y452, S232, and S287 of human albumin. Prolonged treatment of pure human albumin with chlorpyrifos oxon labeled Y138, Y150, Y161, Y401, Y411, and Y452. To identify the most reactive residue, albumin was treated for 2 h with DFP, FP-biotin, chlorpyrifos oxon, or soman, digested with trypsin or pepsin, and analyzed by mass spectrometry. The most reactive residue was always Tyr 411. Diethoxyphosphate-labeled Tyr 411 was stable for months at pH 7.4. These results will be useful in the development of specific antibodies to detect OP exposure and to engineer albumin for use as an OP scavenger.

2,3-Dihydro-1-Benzofuran Derivatives as a Series of Potent Selective Cannabinoid Receptor 2 Agonists: Design, Synthesis, and Binding Mode Prediction through Ligand-Steered Modeling

We recently discovered and reported a series of N‐alkyl‐isatin acylhydrazone derivatives that are potent cannabinoid receptor 2 (CB2) agonists. In an effort to improve the druglike properties of these compounds and to better understand and improve the treatment of neuropathic pain, we designed and synthesized a new series of 2,3‐dihydro‐1‐benzofuran derivatives bearing an asymmetric carbon atom that behave as potent selective CB2 agonists. We used a multidisciplinary medicinal chemistry approach with binding mode prediction through ligand‐steered modeling. Enantiomer separation and configuration assignment were carried out for the racemic mixture for the most selective compound, MDA7 (compound 18). It appeared that the S enantiomer, compound MDA104 (compound 33), was the active enantiomer. Compounds MDA42 (compound 19) and MDA39 (compound 30) were the most potent at CB2. MDA42 was tested in a model of neuropathic pain and exhibited activity in the same range as that of MDA7. Preliminary ADMET studies for MDA7 were performed and did not reveal any problems.

Inhibitors of the α-ketoglutarate dehydrogenase complex alter [1-13C]glucose and [U-13C]glutamate metabolism in cerebellar granule neurons

Diminished activity of the α‐ketoglutarate dehydrogenase complex (KGDHC), an important component of the tricarboxylic acid (TCA) cycle, occurs in several neurological diseases. The effect of specific KGDHC inhibitors [phosphonoethyl ester of succinyl phosphonate (PESP) and the carboxy ethyl ester of succinyl phosphonate (CESP)] on [1‐13C]glucose and [U‐13C]glutamate metabolism in intact cerebellar granule neurons was investigated. Both inhibitors decreased formation of [4‐13C]glutamate from [1‐13C]glucose, a reduction in label in glutamate derived from [1‐13C]glucose/[U‐13C]glutamate through a second turn of the TCA cycle and a decline in the amounts of γ‐aminobutyric acid (GABA), aspartate, and alanine. PESP decreased formation of [U‐13C]aspartate and total glutathione, whereas CESP decreased concentrations of valine and leucine. The findings are consistent with decreased KGDHC activity; increased α‐ketoglutarate formation; increased transamination of α‐ketoglutarate with valine, leucine, and GABA; and new equilibrium position of the aspartate aminotransferase reaction. Overall, the findings also suggest that some carbon derived from α‐ketoglutarate may bypass the block in the TCA cycle at KGDHC by means of the GABA shunt and/or conversion of valine to succinate. The results suggest the potential of succinyl phosphonate esters for modeling the biochemical and pathophysiological consequences of reduced KGDHC activity in brain diseases.

Substituted quinolines as inhibitors of l-glutamate transport into synaptic vesicles

This study investigated the structure-activity relationships and kinetic properties of a library of kynurenate analogues as inhibitors of 3H-L-glutamate transport into rat forebrain synaptic vesicles. The lack of inhibitory activity observed with the majority of the monocyclic pyridine derivatives suggested that the second aromatic ring of the quinoline-based compounds played a significant role in binding to the transporter. A total of two kynurenate derivatives, xanthurenate and 7-chloro-kynurenate, differing only in the carbocyclic ring substituents, were identified as potent competitive inhibitors, exhibiting Ki values of 0.19 and 0.59 mM, respectively. The Km value for L-glutamate was found to be 2.46 mM. Parallel experiments demonstrated that while none of the kynurenate analogues tested effectively inhibited the synaptosomal transport of 3H-D-aspartate, some cross-reactivity was observed with the EAA ionotropic receptors. Molecular modeling studies were carried out with the identified inhibitors and glutamate in an attempt to preliminarily define the pharmacophore of the vesicular transporter. It is hypothesized that the ability of the kynurenate analogues to bind to the transporter may be tied to the capacity of the quinoline carbocyclic ring to mimic the negative charge of the gamma-carboxylate of glutamate. A total of two low energy solution conformers of glutamate were identified that exhibited marked functional group overlap with the most potent inhibitor, xanthurenate. These results help to further refine the pharmacological specificity of the glutamate binding site on the vesicular transporter and identify a series of inhibitors with which to investigate transporter function.

Mass spectrometry identifies multiple organophosphorylated sites on tubulin.

Acute toxicity of organophosphorus poisons (OP) is explained by inhibition of acetylcholinesterase in nerve synapses. Low-dose effects are hypothesized to result from modification of other proteins, whose identity is not yet established. The goal of the present work was to obtain information that would make it possible to identify tubulin as a target of OP exposure. Tubulin was selected for study because live mice injected with a nontoxic dose of a biotinylated organophosphorus agent appeared to have OP-labeled tubulin in brain as determined by binding to avidin beads and mass spectrometry. The experiments with live mice were not conclusive because binding to avidin beads could be nonspecific. To be convincing, it is necessary to find and characterize the OP-labeled tubulin peptide. The search for OP-labeled tubulin peptides was begun by identifying residues capable of making a covalent bond with OP. Pure bovine tubulin (0.012 mM) was treated with 0.01-0.5 mM chlorpyrifos oxon for 24 h at 37 degrees C in pH 8.3 buffer. The identity of labeled amino acids and percent labeling was determined by mass spectrometry. Chlorpyrifos oxon bound covalently to tyrosines 83, 103, 108, 161, 224, 262, 272, 357, and 399 in bovine alpha tubulin, and to tyrosines 50, 51, 59, 106, 159, 281, 310, and 340 in bovine beta tubulin. The most reactive were tyrosine 83 in alpha and tyrosine 281 in beta tubulin. In the presence of 1 mM GTP, percent labeling increased 2-fold. Based on the crystal structure of the tubulin heterodimer (PDB 1jff) tyrosines 83 and 281 are well exposed to solvent. In conclusion seventeen tyrosines in tubulin have the potential to covalently bind chlorpyrifos oxon. These results will be useful when searching for OP-labeled tubulin in live animals.

Signaling by the cysteinyl-leukotriene receptor 2. Involvement in chemokine gene transcription.

Cysteinyl-leukotrienes are involved in inflammation and act on at least two G-protein-coupled receptors, CysLT1 and CysLT2. However, the role of the CysLT2 receptor as well as its signaling remain poorly understood. Here we show that leukotriene (LT)C4 induced the production of the chemokine interleukin (IL)-8 in endothelial cells. To further study the signaling cascade involved, HEK293 cells were stably transfected with CysLT2 and used to study the transcriptional regulation of the IL-8 promoter. Stimulation of the cells with increasing concentrations of LTC4 resulted in a time- and concentration-dependent induction of IL-8 transcription and protein synthesis. Use of IL-8 promoter mutants with substitutions in their NF-κB, AP-1, or NF-IL-6 binding elements revealed an almost total requirement for NF-κB and AP-1 elements, and a lesser requirement for the NF-IL-6 element. Overexpression of dominant-negative IκBα prevented the IL-8 transactivation induced by LTC4. LTC4 stimulation induced NF-κB and AP-1 DNA binding, which involved the formation of a p50/p65 and a c-JUN·c-FOS complex, respectively. Transfection of the cells with a dominant negative (dn) form of PKCϵ prevented p65 phosphorylation, whereas dnPKCδ prevented AP-1 binding. Moreover, dnPKCδ, dnPKCϵ, and dnPKCζ prevented LTC4-induced IL-8 transcription in response to LTC4. Our data show for the first time that LTC4 can act via the CysLT2 receptor to transcriptionally activate chemokine production through induction of NF-κB and AP-1 transcription factors. These findings suggest the potential implication of CysLT2 in the inflammatory response through the modulation of chemokine gene transcription.

Brain immune interactions and air pollution: macrophage inhibitory factor (MIF), prion cellular protein (PrPC), Interleukin-6 (IL-6), interleukin 1 receptor antagonist (IL-1Ra), and interleukin-2 (IL-2) in cerebrospinal fluid and MIF in serum differentiate urban children exposed to severe vs. low air pollution

Mexico City Metropolitan Area children chronically exposed to high concentrations of air pollutants exhibit an early brain imbalance in genes involved in oxidative stress, inflammation, innate and adaptive immune responses along with accumulation of misfolded proteins observed in the early stages of Alzheimer and Parkinsons diseases. A complex modulation of serum cytokines and chemokines influences childrens brain structural and gray/white matter volumetric responses to air pollution. The search for biomarkers associating systemic and CNS inflammation to brain growth and cognitive deficits in the short term and neurodegeneration in the long-term is our principal aim. We explored and compared a profile of cytokines, chemokines (Multiplexing LASER Bead Technology) and Cellular prion protein (PrPC) in normal cerebro-spinal-fluid (CSF) of urban children with high vs. low air pollution exposures. PrPC and macrophage inhibitory factor (MIF) were also measured in serum. Samples from 139 children ages 11.91 ± 4.2 years were measured. Highly exposed children exhibited significant increases in CSF MIF (p = 0.002), IL6 (p = 0.006), IL1ra (p = 0.014), IL-2 (p = 0.04), and PrPC (p = 0.039) vs. controls. MIF serum concentrations were higher in exposed children (p = 0.009). Our results suggest CSF as a MIF, IL6, IL1Ra, IL-2, and PrPC compartment that can possibly differentiate air pollution exposures in children. MIF, a key neuro-immune mediator, is a potential biomarker bridge to identify children with CNS inflammation. Fine tuning of immune-to-brain communication is crucial to neural networks appropriate functioning, thus the short and long term effects of systemic inflammation and dysregulated neural immune responses are of deep concern for millions of exposed children. Defining the linkage and the health consequences of the brain / immune system interactions in the developing brain chronically exposed to air pollutants ought to be of pressing importance for public health.

Quinoline-2,4-dicarboxylic acids: Synthesis and evaluation as inhibitors of the glutamate vesicular transport system

Twenty-six quinoline-2,4-dicarboxylic acids (QDCs) were synthesized by a modified Doebner-von Miller pathway and tested as inhibitors against the glutamate vesicular transport (GVT) protein. The QDCs were active as inhibitors with the most potent QDCs found to contain halogens at the 6-/8-position, a hydroxyl at the 8-position, or a tethered aromatic moiety at the 6- or 7-position of the quinoline.