Bozeman, MT, United States
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Bharate S.B.,University of Montana | Thompson C.M.,University of Montana | Thompson C.M.,Ateris Technologies, Llc
Chemical Biology and Drug Design | Year: 2010

Pyridinium-based oxime compounds have been utilized worldwide as antidotes following exposure to anticholinesterase agents. In the event of combined chemical and biological incident, it is of vital importance to know the ability of antidotes to provide additional protection against biological threats. This paper reports results of in vitro antimicrobial and antiprotozoal activities of a series of quaternary pyridinium oximes against a number of lower pathogenicity BSL-1 and 2 agents. In general, our compound panel had little to no antimicrobial action except for thiophene- and benzothiophene-substituted monoquaternary pyridinium compounds 21 and 24 that showed moderate antibacterial activity against Staphylococus aureus and methicillin-resistant S. aureus with IC50 values ranging from 12.2 to 17.7 μg/mL. Compounds 21 and 24 also exhibited antileishmanial activity against Leishmania donovani with IC50 values of 19 and 18 μg/mL, respectively. Another monoquaternary pyridinium compound with a bromobutyl side chain 17 showed antimalarial activity against both a chloroquine sensitive and resistant strains of Plasmodium falciparum with IC50 values of 3.7 and 4.0 μg/mL, respectively. None of the bisquaternary pyridinium compounds showed antimicrobial or antiprotozoal activity. None of the compounds showed cytotoxic effects toward mammalian kidney fibroblasts. Results of this study indicate that the pyridinium compounds, some of which are already in use as antidotes, do not have significant antimicrobial and antiprotozoal activities and cannot be relied upon for additional protection in the event of combined chemical-biological incident. © 2010 John Wiley & Sons A/S.

Bharate S.B.,University of Montana | Prins J.M.,University of Montana | George K.M.,University of Montana | Thompson C.M.,University of Montana | Thompson C.M.,Ateris Technologies, Llc
Journal of Agricultural and Food Chemistry | Year: 2010

The stability, hydrolysis, and uptake of the organophosphates methyl parathion and methyl paraoxon were investigated in SH-SY5Y cells. The stabilities of (14CH3O)2-methyl parathion (14C-MPS) and (14CH3O)2-methyl paraoxon (14C-MPO) at 1 μM in culture media had similar half-lives of 91.7 and 101.9 h, respectively. However, 100 μM MPO caused >95% cytotoxicity at 24 h, whereas 100 μM MPS caused 4-5% cytotoxicity at 24 h (∼60% cytotoxicity at 48 h). Greater radioactivity was detected inside cells treated with MPO as compared to MPS, although >80% of the total MPO uptake was primarily dimethyl phosphate (DMP). Maximum uptake was reached after 48 h of 14C-MPS or 14C-MPO exposure with total uptakes of 1.19 and 1.76 nM/106 cells for MPS and MPO, respectively. The amounts of MPS and MPO detected in the cytosol after 48 h of exposure time were 0.54 and 0.37 nM/106 cells, respectively. © 2010 American Chemical Society.

Guo L.,University of Montana | Guo L.,Ateris Technologies, Llc | Suarez A.I.,Central University of Venezuela | Braden M.R.,University of Montana | And 3 more authors.
Bioorganic and Medicinal Chemistry Letters | Year: 2010

Fluorophosphonate (FP) head groups were tethered to a variety of chromophores (C) via a triazole group and tested as FPC inhibitors of recombinant mouse (rMoAChE) and electric eel (EEAChE) acetylcholinesterase. The inhibitors showed bimolecular inhibition constants (ki) ranging from 0.3 × 105 M-1 min-1 to 10.4 × 105 M-1 min-1. When tested against rMoAChE, the dansyl FPC was 12.5-fold more potent than the corresponding inhibitor bearing a Texas Red as chromophore, whereas the Lissamine and dabsyl chromophores led to better anti-EEAChE inhibitors. Most inhibitors were equal or better inhibitors of rMoAChE than EEAChE. 3-Azidopropyl fluorophosphonate, which served as one of the FP head groups, showed excellent inhibitory potency against both AChE's (≅ 1 × 107 M-1 min-1) indicating, in general, that addition of the chromophore reduced the overall anti-AChE activity. Covalent attachment of the dabsyl-FPC analog to rMoAChE was demonstrated using size exclusion chromatography and spectroscopic analysis, and visualized using molecular modeling. © 2009 Elsevier Ltd. All rights reserved.

Etoga J.-L.G.,University of Montana | Ahmed S.K.,University of Montana | Patel S.,University of Montana | Bridges R.J.,University of Montana | And 2 more authors.
Bioorganic and Medicinal Chemistry Letters | Year: 2010

A panel of amino acid analogs and conformationally-restricted amino acids bearing a sulfonic acid were synthesized and tested for their ability to preferentially inhibit the obligate cysteine-glutamate transporter system xc - versus the vesicular glutamate transporter (VGLUT). Several promising candidate molecules were identified: R/S-4-[4′-carboxyphenyl]-phenylglycine, a biphenyl substituted analog of 4-carboxyphenylglycine and 2-thiopheneglycine-5-sulfonic acid both of which reduced glutamate uptake at system xc - by 70-75% while having modest to no effect on glutamate uptake at VGLUT. © 2009 Elsevier Ltd. All rights reserved.

Ahmed S.K.,University of Montana | Etoga J.-L.G.,University of Montana | Patel S.A.,University of Montana | Bridges R.J.,University of Montana | And 2 more authors.
Bioorganic and Medicinal Chemistry Letters | Year: 2011

Evidence was acquired prior to suggest that the vesicular glutamate transporter (VGLUT) but not other glutamate transporters were inhibited by structures containing a weakly basic α-amino group. To test this hypothesis, a series of analogs using a hydantoin (pKa ∼ 9.1) isostere were synthesized and analyzed as inhibitors of VGLUT and the obligate cystine-glutamate transporter (system xc-). Of the hydantoin analogs tested, a thiophene-5-carboxaldehyde analog 2l and a bis-hydantoin 4b were relatively strong inhibitors of VGLUT reducing uptake to less than 6% of control at 5 mM but few inhibited system xc- greater than 50% of control. The benzene-2,4-disulfonic acid analog 2b and p-diaminobenzene analog 2e were also good hydantoin-based inhibitors of VGLUT reducing uptake by 11% and 23% of control, respectively, but neither analog was effective as a system xc- inhibitor. In sum, a hydantoin isostere adds the requisite chemical properties needed to produce selective inhibitors of VGLUT. © 2011 Elsevier Ltd. All rights reserved.

Bharate S.B.,University of Montana | Guo L.,Ateris Technologies, Llc | Reeves T.E.,Research Division | Cerasoli D.M.,Research Division | And 2 more authors.
Bioorganic and Medicinal Chemistry | Year: 2010

Oxime reactivators are the drugs of choice for the post-treatment of OP (organophosphorus) intoxication and used widely for mechanistic and kinetic studies of OP-inhibited cholinesterases. The purpose of the present study was to evaluate new oxime compounds to reactivate acetylcholinesterase (AChE) inhibited by the OP paraoxon. Several new bisquaternary pyridinium oximes with heterocyclic linkers along with some known bisquaternary pyridinium oximes bearing aliphatic linkers were synthesized and evaluated for their in vitro reactivation potency against paraoxon-inhibited electric eel acetylcholinesterase (EeAChE) and recombinant human acetylcholinesterase (rHuAChE). Results herein indicate that most of the compounds are better reactivators of EeAChE than of rHuAChE. The reactivation potency of two different classes of compounds with varying linker chains was compared and observed that the structure of the connecting chain is an important factor for the activity of the reactivators. At a higher concentration (10-3 M), compounds bearing aliphatic linker showed better reactivation than compounds with heterocyclic linkers. Interestingly, oximes with a heterocyclic linker inhibited AChE at higher concentration (10-3 M), whereas their ability to reactivate was increased at lower concentrations (10-4 M and 10-5 M). Compounds bearing either a thiophene linker 26, 46 or a furan linker 31 showed 59%, 49% and 52% reactivation of EeAChE, respectively, at 10-5 M. These compounds showed 14%, 6% and 15% reactivation of rHuAChE at 10-4 M. Amongst newly synthesized analogs with heterocyclic linkers (26-35 and 45-46), compound 31, bearing furan linker chain, was found to be the most effective reactivator with a kr 0.042 min-1, which is better than obidoxime (3) for paraoxon-inhibited EeAChE. Compound 31 showed a kr 0.0041 min-1 that is near equal to pralidoxime (1) for paraoxon-inhibited rHuAChE. © 2009 Elsevier Ltd.

PubMed | University of Montana and Ateris Technologies, Llc
Type: | Journal: Chemico-biological interactions | Year: 2016

In this study, the first mechanism-based monoclonal antibodies have been produced that recognize and differentiate diethoxy- and monoethoxyphosphorylated serine residues. Haptens were synthesized as the stable phosphonate form of phosphoserine esters to improve the immunoresponse. Following condensation with a glutaric anhydride to link the phosphoserine moieties to carrier protein, the hapten densities attached to bovine serum albumin and keyhole limpet henocyanin were determined by partial trypsin digestion and MALDI mass spectrometry, and confirmed using a fluorescent assay (FITC) to quantify unmodified lysine residues. The conjugation reactions were pH optimized to improve hapten density. Screening of subclones led to the identification of two monoclonal antibodies: (a) N257/25.11 that specifically recognizes (EtO)2P(O)-Ser as the phosphylated or inhibited form, and (b) N262/16 that recognizes (EtO)(HO)P(O)-Ser as the aged form. Analysis of blood samples treated with paraoxon (EtO)2P(O)-OPhNO2 showed a concentration dependent recognition of the phosphylated form.

Hitt D.M.,Ateris Technologies, Llc | Hitt D.M.,Carroll College | Belabassi Y.,Ateris Technologies, Llc | Suhy J.,Synarc | And 2 more authors.
Tetrahedron Asymmetry | Year: 2014

Malathion, diethyl 2-[(dimethoxyphosphorothioyl)sulfanyl]butanedioate, is an organophosphate used to control insect pests. Malathion contains a diethyl succinate moiety that is a known functional group susceptible to desymmetrizing enzymes such as esterases that selectively react with a single enantiomer. Purified rac-malathion was subjected to hydrolysis at the diethyl succinate moiety of malathion under various conditions using wild type pig liver esterase to form (S)-malathion (12% ee) and ∼3:2 mixture of α- and β-monoacids of (R)-malathion. Technical malathion could not be enriched due to the presence of esterase inhibitors. Further investigation of this resolution using a panel of six PLE isoenzymes also demonstrated the formation of (S)-malathion, however, an improvement of up to 56% ee was obtained. © 2014 Elsevier Ltd. All rights reserved.

Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 158.92K | Year: 2012

DESCRIPTION (provided by applicant): This Phase I SBIR project seeks to develop specialized protein-polymer nanoparticles that are engineered to a therapeutic drug to counter the ill effects following exposure to organophosphate (OP) insecticides. The concept for these nanoparticles is based on 'customized, triggered-release' in which the membrane of a polymerized liposome nanoparticle (PLN) displays functional acetylcholinesterase, the primary target for OPs, while a therapeutic cargo is encapsulated (e.g., oxime). During an OP exposure, the AChE is inhibited (the same as exogenous ChE) to cause protein changes that later the polymeric nanoparticle membrane allowing the oxime cargo (various mechanisms possible) to be released and immediately available to restore exogenous ChE. Because the PLNs are customized and specific to OPs, PLN-AChE nanoparticles will not produce unwanted, high concentrations of the therapeutic agents in the body without OP exposure. In this application, we will develop and subsequentlyshow that AChE-PLNs can be generated with functional enzyme and are selectively inhibited by organophosphates (OP). We will further demonstrate that small molecule cargo can be loaded and stored in functional AChE-PLNs that reaction with OPs releases theentrapped cargo. We will produce 25 mg each of three functional, intact ChE-PLNs in readiness for the Phase II portion of this RandD project. PUBLIC HEALTH RELEVANCE: Citizens can be exposed to organophosphate (OP) insecticides via domestic application, aerial spraying, crops, as a pediculicide, and through the food chain. Accidental exposure to OP- containing pesticides can cause many tiers of toxicity, injury or be fatal to humans. For decades, only high-level exposures to OPs receive medical attention because low, chronic exposures have gone unchecked largely because they can asymptomatic, usually manifesting illnesses through accumulated exposures. If therapeutic measures could be available through a preventative process and highly specific toward OP exposures, a reduction in short- and long- term ill health effects could be expected.

Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 106.00K | Year: 2010

DESCRIPTION (provided by applicant): This Phase I project will test the idea that mechanostress-sensitive, fluorescent polymers can detect structural changes induced in proteins following reaction of the protein with small molecule inhibitors. This concept will be tested and proven using recombinant acetylcholinesterase (AChE) an enzyme that is known to undergo catastrophic denaturation when it reacts with organophosphate (OP) inhibitors. The scientific and technical merits of the idea will be tested in this Phase I period, and optimized to define a course of action for a Phase II project. The long-term goal is to develop and commercialize an entirely new class of polymeric sensors based on catastrophic denaturation. In Phase I, we will attach or place rAChE onto structurally reactive polymer film sensor elements in a manner that retains the enzyme's activity. The rAChE-film will be exposed to OP inhibitors causing the protein to respond structurally. This enzyme-inhibitor denaturation process causes a corresponding structural change in the attached reactive thin film, which results in a fluorescent/color change in the polymer that can be detected visually or though instrumental measurements. The principle challenges for this Phase I project are to determine: (a) if rAChE can be attached or inserted onto a biological matrix or polymeric surface containing a structural response element, and (b) if rAChE retains function when attached to the film and is inhibited when exposed to OP compounds. Project advantages are that ATERIS Technologies has developed polymeric response elements, has over three decades of experience with AChE and OP-AChE interactions including the production of recombinant protein. ATERIS will use these combined areas of expertise to accomplish the following milestones: AIM 1. Attach rAChE (test species) and natural AChE (control) to a model surface membrane (liposome) and ATERIS' polydiacetylene (PDA) thin film and optimize the attachment to retain enzyme function. AIM 2. Visualize a color or fluorescence change resulting from the reaction of PDA thin film coated- rAChE or liposome embedded rAChE with an OP insecticide oxon. PUBLIC HEALTH RELEVANCE: There are 150,000 and 300,000 toxicity incidences reported yearly in the US for exposure to organophosphate (OP) insecticides and millions treated worldwide. Structurally similar OP chemical nerve agents continue as a threat to civilian and military personnel and can compromise public health, injure or fatally harm humans. Realization of the proposed sensor device will allow for rapid assessment of environmental or military OP exposure.

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