In Vitro Evaluation of Neutral Aryloximes as Reactivators for Electrophorus eel Acetylcholinesterase Inhibited by Paraoxon.

Casualties caused by organophosphorus pesticides are a burden for health systems in developing and poor countries. Such compounds are potent acetylcholinesterase irreversible inhibitors, and share the toxic profile with nerve agents. Pyridinium oximes are the only clinically available antidotes against poisoning by these substances, but their poor penetration into the blood-brain barrier hampers the efficient enzyme reactivation at the central nervous system. In searching for structural factors that may be explored in future SAR studies, we evaluated neutral aryloximes as reactivators for paraoxon-inhibited Electrophorus eel acetylcholinesterase. Our findings may result into lead compounds, useful for development of more active compounds for emergencies and supportive care.

BIBR1532, a Selective Telomerase Inhibitor, Enhances Radiosensitivity of Non-Small Cell Lung Cancer Through Increasing Telomere Dysfunction and ATM/CHK1 Inhibition.

PURPOSE: Telomerase is reactivated in non-small cell lung cancer (NSCLC), and it increases cell resistance to irradiation through protecting damaged telomeres and enhancing DNA damage repair. We investigated the radiosensitizing effect of BIBR1532, a highly selective telomerase inhibitor, and its corresponding mechanism in NSCLC. METHODS AND MATERIALS: Cell proliferation, telomerase activity, and telomere dysfunction-induced foci were measured with CCK-8 assay, real-time fluorescent quantitative polymerase chain reaction, and immunofluorescence. The effect of BIBR1532 on the response of NSCLC cells to radiation was analyzed using clonogenic survival and xenograft tumor assays. Cell death and cell senescence induced by BIBR1532 or ionizing radiation (IR), or both, were detected with western blotting, flow cytometry, and senescence-association ß-galactosidase staining assay. RESULTS: We observed dose-dependent direct cytotoxicity of BIBR1532 at relatively high concentrations in NSCLC cells. Low concentrations of BIBR1532 did not appear toxic to NSCLC cells; however, they substantially increased the therapeutic efficacy of IR in vitro by enhancing IR-induced apoptosis, senescence, and mitotic catastrophe. Moreover, in a mouse xenograft model, BIBR1532 treatment synergized with IR at nontoxic dose levels promoted the antitumor efficacy of IR without toxicity to hematologic and internal organs. Mechanistically, lower concentrations of BIBR1532 effectively inhibited telomerase activity and increased IR-induced telomere dysfunction, resulting in disruption of chromosomal stability and inhibition of the ATM/CHK1 (ataxia-telangiectasia-mutated/Checkpoint kinase 1) pathway, which impaired DNA damage repair. CONCLUSIONS: Our findings demonstrate that disturbances in telomerase function by nontoxic dose levels of BIBR1532 effectively enhance the radiosensitivity of NSCLC cells. This finding provides a rationale for the clinical assessment of BIBR1532 as a radiosensitizer.

The active-site cysteine residue of Ca2+/calmodulin-dependent protein kinase I is protected from irreversible modification via generation of polysulfidation.

Ca2+/calmodulin (CaM)-dependent protein kinase (CaMK) I is activated by the phosphorylation of a crucial activation loop Thr177 by upstream kinases, CaMK kinase (CaMKK), and regulates axonal or dendritic extension and branching. Reactive sulfur species (RSS) modulate protein functions via polysulfidation of the reactive Cys residues. Here, we report that the activity of CaMKI was reversibly inhibited via its polysulfidation at Cys179 by RSS. In vitro incubation of CaMKI with the exogenous RSS donor Na2S3 resulted in a dose-dependent inhibition of the phosphorylation at Thr177 by CaMKK and inactivation of the enzymatic activity. Dithiothreitol (DTT), a small molecule reducing reagent, rescued these inhibitions. Conversely, mutated CaMKI (C179V) was resistant to the Na2S3-induced inactivation. In transfected cells expressing CaMKI, ionomycin-induced CaMKI activity was decreased upon treatment with Na2S4, whereas cells expressing mutant CaMKI (C179V) proved resistant to this treatment. A biotin-polyethylene glycol-conjugated maleimide capture assay revealed that CaMKI was a target for polysulfidation in cells. Furthermore, the polysulfidation of CaMKI protected Cys179 from its irreversible modification, known as protein succination. Thus, we propose that CaMKI was reversibly inhibited via polysulfidation of Cys179 by RSS, thereby protecting it from irreversible modification.

Investigation of original multivalent iminosugars as pharmacological chaperones for the treatment of Gaucher disease.

Multivalent iminosugars conjugated with a morpholine moiety and/or designed as prodrugs have been prepared and evaluated as new classes of pharmacological chaperones for the treatment of Gaucher disease. This study further confirms the interest of the prodrug concept and shows that the addition of a lysosome-targeting morpholine unit into iminosugar cluster structures has no significant impact on the chaperone activity on Gaucher cells.

Design, synthesis, and evaluation of guanylhydrazones as potential inhibitors or reactivators of acetylcholinesterase.

Analogs of pralidoxime, which is a commercial antidote for intoxication from neurotoxic organophosphorus compounds, were designed, synthesized, characterized, and tested as potential inhibitors or reactivators of acetylcholinesterase (AChE) using the Ellman's test, nuclear magnetic resonance, and molecular modeling. These analogs include 1-methylpyridine-2-carboxaldehyde hydrazone, 1-methylpyridine-2-carboxaldehyde guanylhydrazone, and six other guanylhydrazones obtained from different benzaldehydes. The results indicate that all compounds are weak AChE reactivators but relatively good AChE inhibitors. The most effective AChE inhibitor discovered was the guanylhydrazone derived from 2,4-dinitrobenzaldehyde and was compared with tacrine, displaying similar activity to this reference material. These results indicate that guanylhydrazones as well as future similar derivatives may function as drugs for the treatment of Alzheimer's disease.

In silico studies on the role of mutant Y337A to reactivate tabun inhibited mAChE with K048.

Organophosphorus compound (OP) tabun is resistant to reactivate by many oxime drugs after the formation of OP-conjugate with AChE. The reactivation of tabun-inhibited mAChE and site-directed mutants by bispyridinium oxime, K048 (N-[4-(4-hydroxyiminomethylpyridinio)butyl]-4-carbamoylpyridinium dibromide) showed that the mutations significantly poor the overall reactivation efficacy of K048. We have unravelled the lowered efficacy of K048 with the tabun-mutant mAChE(Y337A) using docking and steered molecular dynamics (SMD) simulations. The computed results showed some interesting features for the interaction of drug molecule K048 with tabun-mAChE(wild-type) and tabun-mutant mAChE(Y337A). The SMD simulations showed that the active pyridinium ring of K048 is directed towards the phosphorus atom conjugated to the active serine (SUN203) of tabun-mAChE(wild-type). The cradle shaped residues Tyr337-Phe338 present in the choline binding site stabilize the active pyridinium ring of K048 with π-π interaction and the residue Trp86 involved in T-shaped cation-π interaction. However, in the case of tabun-mutant mAChE(Y337A).K048 conjugate, the replacement of aromatic Tyr337 with the aliphatic alanine unit in the choline binding site, however, loses one of the π-π interaction between the active pyridinium ring of K048 and the Tyr337. The placement of aliphatic alanine unit resulted in the displacement of the side chain of Phe338 towards the His447. Such displacement is causing the inaccessibility of the drug towards the phosphorus atom conjugated to the active serine (SUN203) of tabun-mutant mAChE(Y337A). Furthermore, the unbinding of the K048 with SMD studies showed that the active pyridinium ring of the drug undergoes a complete turn along the gorge axis and is directed away from the phosphorus atom conjugated to the active serine of the tabun-mutant mAChE(Y337A). Such effects inside the gorge of tabun-mutant mAChE(Y337A) would lower the efficacy of the drug molecule (K048) for the reactivation process. The binding free energy computed for the tabun-mAChE(wild-type) and tabun-mutant mAChE(Y337A) with K048 showed that the drug molecule prefers to bind strongly with the former enzyme (∼30 kJ/mol) than the later one.

A novel mechanism of hepatocellular carcinoma cell apoptosis induced by lupeol via Brain-Derived Neurotrophic Factor Inhibition and Glycogen Synthase Kinase 3 beta reactivation.

Lupeol is a naturally available triterpenoid with selective anticancerous potential on various human cancer cells. The present study shows that lupeol can inhibit cell proliferation of hepatocellular carcinoma (HCC) HCCLM3 cells in a time- and dose-dependent manner, through caspase-3 dependent activation and Poly ADP-Ribose Polymerase (PARP) cleavage. Lupeol-induced cell death is associated with a marked decrease in the protein expression of Brain-Derived Neurotrophic Factor (BDNF) and ser-9-phosphoryltion of Glycogen Synthase Kinase 3 Beta (GSK-3ß), with concomitant suppression of Akt1, phosphatidyl inositol 3-kinase (PI3K), ß-catenin, c-Myc and Cyclin D1 mRNA expression. Suppressing overexpression of BDNF by lupeol results in decreased protein expression of p-Akt and PI3K (p110α), as well as reactivation of GSK-3ß function in HepG2 cells. Lupeol treatment also inhibits LiCl-induced activation of Wnt signaling pathway and exerts the in vitro anti-invasive activity in Huh-7 cells. LiCl-triggered high expression of ß-catenin, c-Myc and Cyclin D1 protein is reduced followed by lupeol exposure. The findings suggest a mechanistic link between caspase dependent pathway, BDNF secretion and Akt/PI3K/GSK-3ß in HCC cells. These results indicate that lupeol can suppress HCC cell proliferation by inhibiting BDNF secretion and phosphorylation of GSK-3ß(Ser-9), cooperated with blockade of Akt/PI3K and Wnt signaling pathway.

Aberrant CaMKII activity in the medial prefrontal cortex is associated with cognitive dysfunction in ADHD model rats.

Attention-deficit/hyperactivity disorder (ADHD) is a heterogeneous neurobehavioral disorder accompanied by cognitive and learning deficits, which is prevalent among boys. Juvenile male stroke-prone spontaneously hypertensive rats (SHRSP) exhibit ADHD-like behaviors including cognitive deficits and represent one animal model of ADHD. Here, we define a mechanism underlying cognitive dysfunction observed in SHRSP. Acute methylphenidate (MPH: 1mg/kg, p.o.) administration to SHRSP significantly improved not only inattention in a Y-maze task but also cognitive dysfunction in a novel object recognition test. Interestingly, Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activity, which is essential for memory and learning acquisition, was excessively elevated in the medial prefrontal cortex (mPFC) but not in the hippocampal CA1 region of SHRSP compared with Wistar-Kyoto (WKY) rats. We also confirmed that elevated CaMKII autophosphorylation in the mPFC causes increased phosphorylation of the CaMKII substrate α-amino-3-hydroxy-5-methyl-4-isoxazolpropionic acid-type glutamate receptor subunit 1 (GluR1) (Ser-831). Ca(2+)-dependent phosphorylation levels of factors such as extracellular signal-regulated kinase (ERK) and protein kinase C (PKC) were unchanged in the SHRSP mPFC. Also, protein levels of the dopamine D2 receptor (D2R) but not the dopamine D1 receptor (D1R) were increased in the SHRSP mPFC. Acute MPH (1mg/kg, p.o.) administration attenuated aberrant CaMKII activity and increased GluR1 phosphorylation observed in SHRSP. Taken together, we propose that cognitive impairment in SHRSP is associated with aberrant CaMKII activity in the mPFC.

Thiol-dependent recovery of catalytic activity from oxidized protein tyrosine phosphatases.

Protein tyrosine phosphatases (PTPs) play an important role in the regulation of mammalian signal transduction. During some cell signaling processes, the generation of endogenous hydrogen peroxide inactivates selected PTPs via oxidation of the enzyme's catalytic cysteine thiolate group. Importantly, low-molecular weight and protein thiols in the cell have the potential to regenerate the catalytically active PTPs. Here we examined the recovery of catalytic activity from two oxidatively inactivated PTPs (PTP1B and SHP-2) by various low-molecular weight thiols and the enzyme thioredoxin. All monothiols examined regenerated the catalytic activity of oxidized PTP1B, with apparent rate constants that varied by a factor of approximately 8. In general, molecules bearing low-pKa thiol groups were particularly effective. The biological thiol glutathione repaired oxidized PTP1B with an apparent second-order rate constant of 0.023 ± 0.004 M(-1) s(-1), while the dithiol dithiothreitol (DTT) displayed an apparent second-order rate constant of 0.325 ± 0.007 M(-1) s(-1). The enzyme thioredoxin regenerated the catalytic activity of oxidized PTP1B at a substantially faster rate than DTT. Thioredoxin (2 µM) converted oxidized PTP1B to the active form with an observed rate constant of 1.4 × 10(-3) s(-1). The rates at which these agents regenerated oxidized PTP1B followed the order Trx > DTT > GSHand comparable values observed at 2 µM Trx, 4 mM DTT, and 60 mM GSH. Various disulfides that are byproducts of the reactivation process did not inactivate native PTP1B at concentrations of 1-20 mM. The common biochemical reducing agent tris(2-carboxyethyl)phosphine regenerates enzymatic activity from oxidized PTP1B somewhat faster than the thiol-based reagents, with a rate constant of 1.5 ± 0.5 M(-1) s(-1). We observed profound kinetic differences between the thiol-dependent regeneration of activity from oxidized PTP1B and SHP-2, highlighting the potential for structural differences in various oxidized PTPs to play a significant role in the rates at which low-molecular weight thiols and thiol-containing enzymes such as thioredoxin and glutaredoxin return catalytic activity to these enzymes during cell signaling events.

Progress in the development of enzyme-based nerve agent bioscavengers.

Acetylcholinesterase is the physiological target for acute toxicity of nerve agents. Attempts to protect acetylcholinesterase from phosphylation by nerve agents, is currently achieved by reversible inhibitors that transiently mask the enzyme active site. This approach either protects only peripheral acetylcholinesterase or may cause side effects. Thus, an alternative strategy consists in scavenging nerve agents in the bloodstream before they can reach acetylcholinesterase. Pre- or post-exposure administration of bioscavengers, enzymes that neutralize and detoxify organophosphorus molecules, is one of the major developments of new medical counter-measures. These enzymes act either as stoichiometric or catalytic bioscavengers. Human butyrylcholinesterase is the leading stoichiometric bioscavenger. Current efforts are devoted to its mass production with care to pharmacokinetic properties of the final product for extended lifetime. Development of specific reactivators of phosphylated butyrylcholinesterase, or variants with spontaneous reactivation activity is also envisioned for rapid in situ regeneration of the scavenger. Human paraoxonase 1 is the leading catalytic bioscavenger under development. Research efforts focus on improving its catalytic efficiency toward the most toxic isomers of nerve agents, by means of directed evolution-based strategies. Human prolidase appears to be another promising human enzyme. Other non-human efficient enzymes like bacterial phosphotriesterases or squid diisopropylfluorophosphatase are also considered though their intrinsic immunogenic properties remain challenging for use in humans. Encapsulation, PEGylation and other modifications are possible solutions to address this problem as well as that of their limited lifetime. Finally, gene therapy for in situ generation and delivery of bioscavengers is for the far future, but its proof of concept has been established.

Phenyltetrahydroisoquinoline-pyridinaldoxime conjugates as efficient uncharged reactivators for the dephosphylation of inhibited human acetylcholinesterase.

Pyridinium and bis-pyridinium aldoximes are used as antidotes to reactivate acetylcholinesterase (AChE) inhibited by organophosphorus nerve agents. Herein, we described a series of nine nonquaternary phenyltetrahydroisoquinoline-pyridinaldoxime conjugates more efficient than or as efficient as pyridinium oximes to reactivate VX-, tabun- and ethyl paraoxon-inhibited human AChE. This study explores the structure-activity relationships of this new family of reactivators and shows that 1b-d are uncharged hAChE reactivators with a broad spectrum.

Nonquaternary reactivators for organophosphate-inhibited cholinesterases.

A new class of amidine-oxime reactivators of organophosphate (OP)-inhibited cholinesterases (ChE) was synthesized and tested in vitro and in vivo. Compared with 2-PAM, the most promising cyclic amidine-oxime (i.e., 12e) showed comparable or greater reactivation of OP-inactivated AChE and OP-inactivated BChE. To the best of our knowledge, this is the first report of a nonquaternary oxime that has, comparable to 2-PAM, in vitro potency for reactivation of Sarin (GB)-inhibited AChE and BChE. Amidine-oximes were tested in vitro, and reactivation rates for OP-inactivated butyrylcholinesterase (BChE) were greater than those for 2-PAM or MINA. Amidine-oxime reactivation rates for OP-inactivated acetylcholinesterase (AChE) were lower compared to 2-PAM but greater compared with MINA. Amidine-oximes were tested in vivo for protection against the toxicity of nerve agent model compounds. (i.e., a model of Sarin). Post-treatment (i.e., 5 min after OP exposure, i.p,) with amidine oximes 7a-c and 12a, 12c, 12e, 12f, and 15b (145 µmol/kg, i.p.) protected 100% of the mice challenged with the sarin model compound. Even at 25% of the initial dose of amidine-oxime (i.e., a dose of 36 µmol/kg, i.p.), 7b and 12e protected 100% of the animals challenged with the sarin nerve agent model compound that caused lethality in 6/11 animals without amidine-oxime.

Influence of 2,5-dichloro-1,4-benzoquinone on jack bean urease activity. Inhibitory effect, total reducing capacity and DPPH radical scavenging activity.

Inhibition of jack bean activity by 2,5-dichloro-1,4-benzoquinone (DCBQ) was studied in phosphate buffer, pH 7.0. It was found that DCBQ acted as a strong, time and concentration dependent inactivator of urease. Under the experimental conditions obeyed the terms of pseudo-first-order reaction, urease was totally inactivated. Application of Wilson-Kitz method proved that the urease-DCBQ interaction followed a simple bimolecular process and the presence of intermediate complex was undetectable. The determined second order rate constant of the inactivation was 0.053 (µM min)(-1). Thiols such as l-cysteine, glutathione and dithiothreitol (DTT) protected urease from inhibition by DCBQ but DCBQ-modified urease did not regain its activity after DTT application. The thiol protective studies indicated an essential role of urease thiol(s) in the inhibition. The irreversibility of the inactivation showed that the process was a result of a direct modification of urease thiol(s) by DCBQ (DCBQ chlorine(s) substitution). The decomposition of DCBQ in aqueous solution at natural light exposure was monitored by visible spectrophotometry, determination of the total reducing capacity (Folin-Ciocalteu method) and DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging ability. The DCBQ conversion resulted in a decrease of the inhibition power and was well correlated with the increase of the total reducing capacity and DPPH scavenging ability. These findings were attributed to DCBQ transformation by photolysis and the hydrolysis effect was found to be negligible.

Amidine-oximes: reactivators for organophosphate exposure.

A new class of amidine-oxime reactivators of organophosphate (OP)-inhibited cholinesterases (ChE) were designed, synthesized, and tested. These compounds represent a novel group of oximes with enhanced capabilities of crossing the blood-brain barrier. Lack of brain penetration is a major limitation for currently used oximes as antidotes of OP poisoning. The concept described herein relies on a combination of an amidine residue and oxime functionality whereby the amidine increases the binding affinity to the ChE and the oxime is responsible for reactivation. Amidine-oximes were tested in vitro and reactivation rates for OP-BuChE were greater than pralidoxime (2-PAM) or monoisonitrosoacetone (MINA). Amidine-oxime reactivation rates for OP-AChE were lower compared to 2-PAM but greater compared with MINA. After pretreatment for 30 min with oximes 15c and 15d (145 µmol/kg, ip) mice were challenged with a soman model compound. In addition, 15d was tested in a post-treatment experiment (145 µmol/kg, ip, administration 5 min after sarin model compound exposure). In both cases, amidine-oximes afforded 100% 24 h survival in an animal model of OP exposure.

Inactivation of porcine kidney betaine aldehyde dehydrogenase by hydrogen peroxide.

Concentrated urine formation in the kidney is accompanied by conditions that favor the accumulation of reactive oxygen species (ROS). Under hyperosmotic conditions, medulla cells accumulate glycine betaine, which is an osmolyte synthesized by betaine aldehyde dehydrogenase (BADH, EC 1.2.1.8). All BADHs identified to date have a highly reactive cysteine residue at the active site, and this cysteine is susceptible to oxidation by hydrogen peroxide. Porcine kidney BADH incubated with H(2)O(2) (0-500 µM) lost 25% of its activity. However, pkBADH inactivation by hydrogen peroxide was limited, even after 120 min of incubation. The presence of coenzyme NAD(+) (10-50 µM) increased the extent of inactivation (60%) at 120 min of reaction, but the ligands betaine aldehyde (50 and 500 µM) and glycine betaine (100 mM) did not change the rate or extent of inactivation as compared to the reaction without ligand. 2-Mercaptoethanol and dithiothreitol, but not reduced glutathione, were able to restore enzyme activity. Mass spectrometry analysis of hydrogen peroxide inactivated BADH revealed oxidation of M278, M243, M241 and H335 in the absence and oxidation of M94, M327 and M278 in the presence of NAD(+). Molecular modeling of BADH revealed that the oxidized methionine and histidine residues are near the NAD(+) binding site. In the presence of the coenzyme, these oxidized residues are proximal to the betaine aldehyde binding site. None of the oxidized amino acid residues participates directly in catalysis. We suggest that pkBADH inactivation by hydrogen peroxide occurs via disulfide bond formation between vicinal catalytic cysteines (C288 and C289).

Telomerase reactivation reverses tissue degeneration in aged telomerase-deficient mice.

An ageing world population has fuelled interest in regenerative remedies that may stem declining organ function and maintain fitness. Unanswered is whether elimination of intrinsic instigators driving age-associated degeneration can reverse, as opposed to simply arrest, various afflictions of the aged. Such instigators include progressively damaged genomes. Telomerase-deficient mice have served as a model system to study the adverse cellular and organismal consequences of wide-spread endogenous DNA damage signalling activation in vivo. Telomere loss and uncapping provokes progressive tissue atrophy, stem cell depletion, organ system failure and impaired tissue injury responses. Here, we sought to determine whether entrenched multi-system degeneration in adult mice with severe telomere dysfunction can be halted or possibly reversed by reactivation of endogenous telomerase activity. To this end, we engineered a knock-in allele encoding a 4-hydroxytamoxifen (4-OHT)-inducible telomerase reverse transcriptase-oestrogen receptor (TERT-ER) under transcriptional control of the endogenous TERT promoter. Homozygous TERT-ER mice have short dysfunctional telomeres and sustain increased DNA damage signalling and classical degenerative phenotypes upon successive generational matings and advancing age. Telomerase reactivation in such late generation TERT-ER mice extends telomeres, reduces DNA damage signalling and associated cellular checkpoint responses, allows resumption of proliferation in quiescent cultures, and eliminates degenerative phenotypes across multiple organs including testes, spleens and intestines. Notably, somatic telomerase reactivation reversed neurodegeneration with restoration of proliferating Sox2(+) neural progenitors, Dcx(+) newborn neurons, and Olig2(+) oligodendrocyte populations. Consistent with the integral role of subventricular zone neural progenitors in generation and maintenance of olfactory bulb interneurons, this wave of telomerase-dependent neurogenesis resulted in alleviation of hyposmia and recovery of innate olfactory avoidance responses. Accumulating evidence implicating telomere damage as a driver of age-associated organ decline and disease risk and the marked reversal of systemic degenerative phenotypes in adult mice observed here support the development of regenerative strategies designed to restore telomere integrity.

Kinetic analysis of interactions of paraoxon and oximes with human, Rhesus monkey, swine, rabbit, rat and guinea pig acetylcholinesterase.

Previous in vitro studies showed marked species differences in the reactivating efficiency of oximes between human and animal acetylcholinesterase (AChE) inhibited by organophosphorus (OP) nerve agents. These findings provoked the present in vitro study which was designed to determine the inhibition, aging, spontaneous and oxime-induced reactivation kinetics of the pesticide paraoxon, serving as a model compound for diethyl-OP, and the oximes obidoxime, pralidoxime, HI 6 and MMB-4 with human, Rhesus monkey, swine, rabbit, rat and guinea pig erythrocyte AChE. Comparable results were obtained with human and monkey AChE. Differences between human, swine, rabbit, rat and guinea pig AChE were determined for the inhibition and reactivation kinetics. A six-fold difference of the inhibitory potency of paraoxon with human and guinea pig AChE was recorded while only moderate differences of the reactivation constants between human and animal AChE were determined. Obidoxime was by far the most effective reactivator with all tested species. Only minor species differences were found for the aging and spontaneous reactivation kinetics. The results of the present study underline the necessity to determine the inhibition, aging and reactivation kinetics in vitro as a basis for the development of meaningful therapeutic animal models, for the proper assessment of in vivo animal data and for the extrapolation of animal data to humans.

Thiadiazole carbamates: potent inhibitors of lysosomal acid lipase and potential Niemann-Pick type C disease therapeutics.

Niemann-Pick type C (NPC) disease is a lysosomal storage disorder characterized at the cellular level by abnormal accumulation of cholesterol and other lipids in lysosomal storage organelles. Lysosomal acid lipase (LAL) has been recently identified as a potential therapeutic target for NPC. LAL can be specifically inhibited by a variety of 3,4-disubstituted thiadiazole carbamates. An efficient synthesis of the C(3) oxygenated/C(4) aminated analogues has been developed that furnishes the products in high yields and high degrees of purity. Common intermediates can also be used for the synthesis of the C(3) carbon substituted derivatives. Herein we tested various thiadiazole carbamates, amides, esters, and ketones for inhibition of LAL. In addition, we tested a diverse selection of commercially available non-thiadiazole carbamates. Our studies show that, among the compounds examined herein, only thiadiazole carbamates are effective inhibitors of LAL. We present a mechanism for LAL inhibition by these compounds whereby LAL transiently carbamoylates the enzyme similarly to previously described inhibition of acetylcholinesterase by rivastigmine and other carbamates as well as acylation of various lipases by orlistat.

Novel acetylcholinesterase reactivator--oxime K048--reactivation activity in vitro.

A novel acetylcholinesterase (AChE) reactivator, a bispyridinium aldoxime named K048, was first synthesized in 2003. It is a promising antidote against tabun poisoning. Afterwards, other studies on several cholinesterases (ChE) of different species (humans, rats, etc.) and models (in vitro or in vivo) were conducted. We tested this oxime against nine different AChE inhibitors using in vitro tests on rat brain homogenate as source of enzyme. Our results showed that oxime K048 reached promising reactivation activity in case of all tested AChE inhibitors, except cyclosarin, at oxime concentration 10(-3) M. At a concentration of 10(-5) M, which is more common for human use, only methylchlorpyrifos-inhibited AChE was reactivated.

Novel bisquaternary oximes--reactivation of acetylcholinesterase and butyrylcholinesterase inhibited by paraoxon.

Four novel bisquaternary aldoxime cholinesterase reactivators differing in their chemical structure were prepared. Afterwards, their biological activity was evaluated for their ability to reactivate acetylcholinesterase (AChE; EC 3.1.1.7) and butyrylcholinesterase (BuChE; EC 3.1.1.8) inhibited by paraoxon. Their reactivation activity was compared with standard reactivators--pralidoxime, obidoxime and HI-6--which are clinically used at present. As it resulted, none of the prepared compounds surpassed obidoxime, which is considered to be the most potent compound if used for reactivation of AChE inhibited by paraoxon. In case of BuChE reactivation, two compounds (K053 and K068) achieved similar results as obidoxime.