Author Report for: Marek J

Benzoxonium chloride belongs to the group of quaternary ammonium salts, which have been widely used for decades as disinfectants because of their high efficacy, low toxicity, and thermal stability. In this study, we have prepared the C(10)-C(18) set of benzoxonium-like salts to evaluate the effect of their chemical and biological decontamination capabilities. In particular, biocidal activity against a panel of bacterial strains including Staphylococcus aureus in biofilm form was screened. In addition, the most promising compounds were successfully tested against Francisella tularensis as a representative of potential biological warfare agents. From a point of view of chemical warfare protection, the efficiency of BOC-like compounds to degrade the organophosphate simulant fenitrothion was examined. Notwithstanding that no single compound with universal effectiveness was identified, a mixture of only two compounds from this group would be able to satisfactorily cover the proposed decontamination spectrum. In addition, the compounds were evaluated for their cytotoxicity as a basic safety parameter for potential use in practice. In summary, the dual effect on chemical and biological agents of benzoxonium-like salts offer attractive potential as active components of decontamination mixtures in the case of a terrorist threat or chemical or biological accidents.

In this study, we have carried out a combined experimental and computational investigation to elucidate several bred-in-the-bone ideas standing out in rational design of novel cationic surfactants as antibacterial agents. Five 3-hydroxypyridinium salts differing in the length of N-alkyl side chain have been synthesized, analyzed by high performance liquid chromatography, tested for in vitro activity against a panel of pathogenic bacterial and fungal strains, computationally modeled in water by a SCRF B3LYP/6-311++G(d,p) method, and evaluated by a systematic QSAR analysis. Given the results of this work, the hypothesis suggesting that higher positive charge of the quaternary nitrogen should increase antimicrobial efficacy can be rejected since 3-hydroxyl group does increase the positive charge on the nitrogen but, simultaneously, it significantly derogates the antimicrobial activity by lowering the lipophilicity and by escalating the desolvation energy of the compounds in comparison with non-hydroxylated analogues. Herein, the majority of the prepared 3-hydroxylated substances showed notably lower potency than the parent pyridinium structures, although compound 8 with C12 alkyl chain proved a distinctly better antimicrobial activity in submicromolar range. Focusing on this anomaly, we have made an effort to reveal the reason of the observed activity through a molecular dynamics simulation of the interaction between the bacterial membrane and compound 8 in GROMACS software.

Aggregation and kinetic studies have been performed to understand the hydrolytic potencies of the series of oxime-functionalized surfactants, viz., 3- hydroxyiminomethyl-1-alkylpyridinium bromide (alkyl = CnH2n+1, n = 10, 12, 14, 16, 18) in the cleavage of phosphate esters, p-nitrophenyl diphenyl phosphate (PNPDPP) and bis(2,4-dinitrophenyl) phosphate (BNDPP), in mixed micelles with cetylpyridinium bromide (CPB). Micellization and surface properties of mixed micelles functional surfactants with CPB were studied by conductivity and surface tension measurements. Acid dissociation constants (pKa) were determined, the effect of functional surfactant alkyl chain length and pH on the observed rate constant (kobs) for phosphate ester cleavage has been discussed, and the effect of substrate on the supernucleophilicities of the studied oximes was monitored. Functionalized oxime-based surfactants were proved to be supernucleophiles to attack on the P horizontal lineO center of tri- and diphosphate esters. Oximes with hexadecyl alkyl chain length (3-C16) showed maximum micellar effect on the rate constants toward PNPDPP. Micellar effects were analyzed in terms of the pseudophase model.

Carbamate inhibitors (e.g. pyridostigmine bromide) are used as a pre-treatment for the prevention of organophosphorus poisoning. They work by blocking the native function of acetylcholinesterases (AChE) and thus protect AChE against irreversible inhibition by organophosphorus compounds. However, carbamate inhibitors are known for their many undesirable side effects related to the carbamylation of AChE. In this paper, we describe 17 novel bisquaternary compounds and have analysed their effect on AChE inhibition. The newly prepared compounds were evaluated in vitro using both human erythrocyte AChE and human plasmatic butyrylcholinesterase. Their inhibitory ability was expressed as the half maximal inhibitory concentration (IC(5)(0)) and then compared to the standard carbamate drugs and two AChE reactivators. One of these novel compounds showed promising AChE inhibition in vitro (nM range) and was better than the currently used standards. Additionally, a kinetic assay confirmed the non-competitive inhibition of hAChE by this novel compound. Consequently, the docking results confirmed the apparent pi-pi or pi-cationic interactions with the key amino acid residues of hAChE and the binding of the chosen compound at the enzyme catalytic site.

All approved drugs for Alzheimer disease (AD) in clinical practice ameliorate the symptoms of the disease. Among them, acetylcholinesterase inhibitors (AChEIs) are used to increase the cholinergic activity. Among new AChEI, tacrine compounds were found to be more toxic compared to 7-MEOTA (9-amino-7-methoxy-1,2,3,4-tetrahydroacridine). In this Letter, series of 7-MEOTA analogues (N-alkyl-7-methoxytacrine) were synthesized. Their inhibitory ability was evaluated on recombinant human acetylcholinesterase (AChE) and plasmatic human butyrylcholinesterase (BChE). Three novel compounds showed promising results towards hAChE better to THA or 7-MEOTA. Three compounds resulted as potent inhibitors of hBChE. The SAR findings highlighted the C(6)-C(7)N-alkyl chains for cholinesterase inhibition.

The prophylactic antidotes are pharmaceutics which can prevent the intoxication of the human body or may improve its prognosis if administered before the exposure to a toxic substance. Pyridostigmine chloride, a reversible acetylcholinesterase(AChE) inhibitor, is used as a prophylactic antidote which may prevent the organism from organophosphorus poisoning (OP). This paper is focused on searching new AChE inhibitors that might be used in prophylactic treatment of OP intoxications. The aim of this study was to prepare 15 symmetric bisquaternary inhibitors and to evaluate their inhibition ability (IC50) via a standard in vitro method. The but-(2E)-en-1,4-diyl-1,1'-bis(4-(4-nitrobenzyl)-pyridinium) dibromide was selected, on the basis of IC50 value, the most potent AChE inhibitor.

The oxime reactivator K112 is a member of the new group of xylene linker-containing AChE reactivators. Its cholinergic properties could be of importance at OP poisoning and are not related to the AChE reactivation that has been studied. It has been found that, despite of reactivating potency, this compound has additional effects. These cholinergic effects include a weak inhibition of AChE (IC(50)=43.8 +/- 4.88 muM), inhibition of binding to the porcine muscarinic M2 receptor (IC(50)=4.36 muM) and finally, the inhibition of HACU (68.4 +/- 9.9%), a key regulatory step in the synthesis of ACh. The inhibition of the binding of (3H)-HC-3 (64.7 +/- 4.7%) and the influence on the membrane fluidity have also been observed. Blocking properties of K112 on the muscarinic receptors have been revealed in the in vitro experiment (rat urinary bladder) and in the in vivo experiment (rat heart BPM) as well. All these cholinergic properties could significantly contribute to the antidotal effect of K112 at the poisoning by the organophosphates.

Class of monoquaternary pyridinium oximes was in vitro tested as potential reactivators of acetylcholinesterase (AChE; EC 3.1.1.7) inhibited by nerve agent sarin. Human brain homogenate was used as an appropriate source of cholinesterases. Reactivation potency of novel oximes was compared with currently available reactivators - pralidoxime, obidoxime, and HI-6. According to the obtained results, only five reactivators were able to satisfactorily renew cholinesterase potency (pralidoxime, obidoxime, HI-6, 4-PAM, and K119). Unfortunately, none of the novel tested reactivators surpassed the reactivation potency of the currently most promising reactivator, HI-6. This study shows that monoquaternary reactivators are unable to reactivate nerve agent-inhibited AChE. Due to this, in future, only bisquaternary compounds derived from HI-6 or obidoxime should be designed as new potential cholinesterase reactivators.

OBJECTIVES: The current standard treatment of organophosphate poisoning consists of an administration of anticholinergic drugs and cholinesterase reactivators (oximes). Oximes can react - except their reactivating effect on cholinesterases - directly with cholinoreceptors. HI-6 is an oxime that may have an inhibitory effect on the muscarinic receptors, too. METHODS: In our work, we have investigated an influence of HI-6 on the acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and on the muscarinic receptors in vitro. The study was conducted using biosensor technique and on the rat bladder using in vitro test (tissue bath; methacholine as muscarinic agonist). IC50 for BChE from human serum was determined to be 1.01x10-6 M and for human erythrocytes AChE 3.31x10-6 M, respectively. CONCLUSION: We assume that the demonstrated contractile response can be attributed to the inhibition of the AChE at the lower concentration and to a predominant inhibition of muscarinic receptor at higher concentration of compound tested.

Preparation of 1-(4-hydroxy-iminomethylpyridinium)-3-pyridiniumpropane dibromide is described. This compound represents a new acetylcholinesterase (AChE) reactivator, which has no substituents on the second pyridinium ring as found in other commonly used AChE reactivators. The reactivation ability of this reactivator was tested on tabun- and cyclosarin-inhibited AChE. According to the results obtained, the new compound (without substitution and with decreased molecule size) showed increased reactivation potency in case of cyclosarin inhibited AChE. A potent oxime for treatment of tabun and cyclosarin-caused intoxications was thus obtained via slight modification of the reactivator structure (compared to trimedoxime and K027).

The newly developed and very promising acetylcholinesterase reactivator (E)-1-(2-hydroxyiminomethylpyridinium)-4-(4-hydroxyiminomethylpyridinium)-but-2-e ne dibromide was prepared using two different pathways via a two-step synthesis involving the appropriate (E)-1-(4-bromobut-2-enyl)-2- or 4-hydroxyiminomethyl-pyridinium bromides. Afterwards, purities and yields of the desired product prepared by both routes were compared. Finally, its potency to reactivate several nerve agent-inhibited acetylcholinesterases was tested.

Alterations in dipeptidyl peptidase-IV (DPP-IV) enzymatic activity are characteristic of malignant transformation. Through its well-characterized functionality in regulating the activity of bioactive peptides by removal of the N-terminal dipeptide, DPP-IV activity may have profound effects upon metastatic potential and cell growth. Although DPP-IV/CD26 (EC 3.4.14.5) is the canonical representative of the group, a number of other proteins including DPP-7, 8, 9, and seprase/fibroblast activation protein-alpha (FAP-alpha) have been shown to have similar enzymatic activity. This study was set up to address the relative representation and enzymatic activity of plasma membrane localized DPP-IV/CD26 and FAP-alpha in human brain and astrocytic tumours. In parallel, expression of CXCR4, receptor for glioma cell growth stimulator chemokine SDF-1alpha known to be a DPP-IV substrate, was investigated. This is the first report showing that non-malignant brain tissue contains a DPP-IV-like enzymatic activity attributable mostly to DPP-8/9, while the substantial part of the activity in glioma is due to increased DPP-IV/CD26, localized in both the vascular and parenchymal compartments. DPP-IV enzymatic activity increased dramatically with tumour grade severity. A grade-related increase in CXCR4 receptor paralleled the rise in DPP-IV expression and activity. These data might support a role for DPP-IV regulation of the CXCR4-SDF-1alpha axis in glioma development.

Zm-p60.1, a cytokinin glucoside specific beta-glucosidase from maize, is a key enzyme involved in plant development and growth. It has been overexpressed in soluble form from Escherichia coli with a His tag at its N-terminus. The recombinant protein has been purified and crystallized at room temperature using PEG 4000 as the main precipitant. At least three crystal forms have been observed from very similar growth conditions. A flash-annealed monoclinic crystal diffracted to high resolution (beyond 2 A) with space group P2(1) and unit-cell parameters a = 55.66, b = 110.72, c = 72.94 A, beta = 92.10 degrees. The asymmetric unit is estimated and confirmed by molecular-replacement solution to contain one Zm-p60.1 dimer, giving a crystal volume per protein mass (V(M)) of 1.89 A(3) Da(-1) and a solvent content of 35%.

The haloalkane dehalogenase from Sphingomonas paucimobilis UT26 (LinB) is the enzyme involved in the degradation of the important environmental pollutant gamma-hexachlorocyclohexane. The enzyme hydrolyzes a broad range of halogenated cyclic and aliphatic compounds. Here, we present the 1.58 A crystal structure of LinB and the 2.0 A structure of LinB with 1,3-propanediol, a product of debromination of 1,3-dibromopropane, in the active site of the enzyme. The enzyme belongs to the alpha/beta hydrolase family and contains a catalytic triad (Asp108, His272, and Glu132) in the lipase-like topological arrangement previously proposed from mutagenesis experiments. The LinB structure was compared with the structures of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 and from Rhodococcus sp. and the structural features involved in the adaptation toward xenobiotic substrates were identified. The arrangement and composition of the alpha-helices in the cap domain results in the differences in the size and shape of the active-site cavity and the entrance tunnel. This is the major determinant of the substrate specificity of this haloalkane dehalogenase.

Haloalkane hydrolytic dehalogenase LinB from Sphingomonas paucimobilis UT26, an enzyme which releases chloride or bromide anion from n-halogenated alkanes and has a broad range of substrate specificity, was crystallized using the hanging-drop vapour-diffusion method at 278 K. The best crystals were obtained by microseeding with a precipitant containing 18-20%(w/v) PEG 6000, 0.2 M calcium acetate and 0.1 M Tris-HCl pH 8.9. The crystals diffract to at least 1.60 A using synchrotron X-ray under cryogenic (100 K) conditions. They belong to the orthorhombic space group P21212 with unit-cell parameters a = 50.29, b = 71.70, c = 72.73 A. The asymmetric unit contains one molecule of the enzyme.