Decarbamoylation of acetylcholinesterases is markedly slowed as carbamoyl groups increase in size.

Carbamates are esters of substituted carbamic acids that react with acetylcholinesterase (AChE) by initially transferring the carbamoyl group to a serine residue in the enzyme active site accompanied by loss of the carbamate leaving group followed by hydrolysis of the carbamoyl enzyme. This hydrolysis, or decarbamoylation, is relatively slow, and half-lives of carbamoylated AChEs range from 4 min to more than 30 days. Therefore, carbamates are effective AChE inhibitors that have been developed as insecticides and as therapeutic agents. We show here, in contrast to a previous report, that decarbamoylation rate constants are independent of the leaving group for a series of carbamates with the same carbamoyl group. When the alkyl substituents on the carbamoyl group increased in size from N-monomethyl- to N,N-dimethyl-, N-ethyl-N-methyl-, or N,N-diethyl-, the decarbamoylation rate constants decreased by 4-, 70-, and 800-fold, respectively. We suggest that this relationship arises as a result of active site distortion, particularly in the acyl pocket of the active site. Furthermore, solvent deuterium oxide isotope effects for decarbamoylation decreased from 2.8 for N-monomethylcarbamoyl AChE to 1.1 for N,N-diethylcarbamoyl AChE, indicating a shift in the rate-limiting step from general acid-base catalysis to a likely conformational change in the distorted active site.

γ-Secretase processing and effects of γ-secretase inhibitors and modulators on long Aß peptides in cells.

Understanding how different species of Aß are generated by γ-secretase cleavage has broad therapeutic implications, because shifts in γ-secretase processing that increase the relative production of Aßx-42/43 can initiate a pathological cascade, resulting in Alzheimer disease. We have explored the sequential stepwise γ-secretase cleavage model in cells. Eighteen BRI2-Aß fusion protein expression constructs designed to generate peptides from Aß1-38 to Aß1-55 and C99 (CTFß) were transfected into cells, and Aß production was assessed. Secreted and cell-associated Aß were detected using ELISA and immunoprecipitation MALDI-TOF mass spectrometry. Aß peptides from 1-38 to 1-55 were readily detected in the cells and as soluble full-length Aß proteins in the media. Aß peptides longer than Aß1-48 were efficiently cleaved by γ-secretase and produced varying ratios of Aß1-40:Aß1-42. γ-Secretase cleavage of Aß1-51 resulted in much higher levels of Aß1-42 than any other long Aß peptides, but the processing of Aß1-51 was heterogeneous with significant amounts of shorter Aßs, including Aß1-40, produced. Two PSEN1 variants altered Aß1-42 production from Aß1-51 but not Aß1-49. Unexpectedly, long Aß peptide substrates such as Aß1-49 showed reduced sensitivity to inhibition by γ-secretase inhibitors. In contrast, long Aß substrates showed little differential sensitivity to multiple γ-secretase modulators. Although these studies further support the sequential γ-secretase cleavage model, they confirm that in cells the initial γ-secretase cleavage does not precisely define subsequent product lines. These studies also raise interesting issues about the solubility and detection of long Aß, as well as the use of truncated substrates for assessing relative potency of γ-secretase inhibitors.

Gedunin inactivates the co-chaperone p23 protein causing cancer cell death by apoptosis.

Pharmacological inhibition of Hsp90 is an exciting option for cancer therapy. The clinical efficacy of Hsp90 inhibitors is, however, less than expected. Binding of the co-chaperone p23 to Hsp90 and induced overexpression of anti-apoptotic proteins Hsp70 and Hsp27 are thought to contribute to this outcome. Herein, we report that the natural product gedunin may provide a new alternative to inactivate the Hsp90 machine. We show that gedunin directly binds to p23 and inactivates it, without overexpression of Hsp27 and relatively modest induction of Hsp70. Using molecular docking and mutational analysis, we mapped the gedunin-binding site on p23. Functional analysis shows that gedunin inhibits the p23 chaperoning activity, blocks its cellular interaction with Hsp90, and interferes with p23-mediated gene regulation. Cell treatment with gedunin leads to cancer cell death by apoptosis through inactivation of p23 and activation of caspase 7, which cleaves p23 at the C terminus. These results provide important insight into the molecular mechanism of action of this promising lead compound.

Substrate-targeting gamma-secretase modulators.

Selective lowering of Abeta42 levels (the 42-residue isoform of the amyloid-beta peptide) with small-molecule gamma-secretase modulators (GSMs), such as some non-steroidal anti-inflammatory drugs, is a promising therapeutic approach for Alzheimer's disease. To identify the target of these agents we developed biotinylated photoactivatable GSMs. GSM photoprobes did not label the core proteins of the gamma-secretase complex, but instead labelled the beta-amyloid precursor protein (APP), APP carboxy-terminal fragments and amyloid-beta peptide in human neuroglioma H4 cells. Substrate labelling was competed by other GSMs, and labelling of an APP gamma-secretase substrate was more efficient than a Notch substrate. GSM interaction was localized to residues 28-36 of amyloid-beta, a region critical for aggregation. We also demonstrate that compounds known to interact with this region of amyloid-beta act as GSMs, and some GSMs alter the production of cell-derived amyloid-beta oligomers. Furthermore, mutation of the GSM binding site in the APP alters the sensitivity of the substrate to GSMs. These findings indicate that substrate targeting by GSMs mechanistically links two therapeutic actions: alteration in Abeta42 production and inhibition of amyloid-beta aggregation, which may synergistically reduce amyloid-beta deposition in Alzheimer's disease. These data also demonstrate the existence and feasibility of 'substrate targeting' by small-molecule effectors of proteolytic enzymes, which if generally applicable may significantly broaden the current notion of 'druggable' targets.

Abca1 deficiency affects Alzheimer's disease-like phenotype in human ApoE4 but not in ApoE3-targeted replacement mice.

ATP-binding cassette transporter A1 (ABCA1) transporter regulates cholesterol efflux and is an essential mediator of high-density lipoprotein (HDL) formation. In amyloid precursor protein (APP) transgenic mice, Abca1 deficiency increased amyloid deposition in the brain paralleled by decreased levels of Apolipoprotein E (ApoE). The APOEε4 allele is the major genetic risk factor of sporadic Alzheimer's disease (AD). Here, we reveal the effect of Abca1 deficiency on phenotype in mice expressing human ApoE3 or ApoE4. We used APP/E3 and APP/E4 mice generated by crossing APP/PS1ΔE9 transgenic mice to human APOE3- and APOE4-targeted replacement mice and examined Abca1 gene dose effect on amyloid deposition and cognition. The results from two behavior tests demonstrate that lack of one copy of Abca1 significantly exacerbates memory deficits in APP/E4/Abca1(-/+) but not in APP/E3/Abca1(-/+) mice. The data for amyloid plaques and insoluble amyloid-ß (Aß) also show that Abca1 hemizygosity increases Aß deposition only in APP/E4/Abca1(-/+) but not in APP/E3/Abca1(-/+) mice. Our in vivo microdialysis assays indicate that Abca1 deficiency significantly decreases Aß clearance in ApoE4-expressing mice, while the effect of Abca1 on Aß clearance in ApoE3-expressing mice was insignificant. In addition, we demonstrate that plasma HDL and Aß42 levels in APP/E4/Abca1(-/+) mice are significantly decreased, and there is a negative correlation between plasma HDL and amyloid plaques in brain, suggesting that plasma lipoproteins may be involved in Aß clearance. Overall, our results prove that the presence of functional Abca1 significantly influences the phenotype of APP mice expressing human ApoE4 and further substantiate therapeutic approaches in AD based on ABCA1-APOE regulatory axis.

Synthesis of a DOTA (Gd3+)-conjugate of proton-pump inhibitor pantoprazole for gastric wall imaging studies.

Magnetic resonance imaging (MRI) is used to evaluate gastrointestinal (GI) structure and functions in humans. Despite filling the viscus lumen with a contrast agent, visualization of the viscus wall is limited. To overcome this limitation, we de novo synthesized a conjugate that covalently combines a Gd-based MRI contrast agent, encaged with a chelating agent (DOTA), with pantoprazole, which is a widely used proton pump inhibitor that binds to proton pumps in the stomach and colon. The DOTA linkage was installed at a mechanism-based strategic location in the pantoprazole molecule to minimize a possible negative effect of the structural modification on the drug. It is anticipated that by defining the wall of the stomach and colon, this compound will facilitate functional MRI of the GI tract in humans.

Diverse compounds mimic Alzheimer disease-causing mutations by augmenting Abeta42 production.

Increased Abeta42 production has been linked to the development of Alzheimer disease. We now identify a number of compounds that raise Abeta42. Among the more potent Abeta42-raising agents identified are fenofibrate, an antilipidemic agent, and celecoxib, a COX-2-selective NSAID. Many COX-2-selective NSAIDs tested raised Abeta42, including multiple COX-2-selective derivatives of two Abeta42-lowering NSAIDs. Compounds devoid of COX activity and the endogenous isoprenoids FPP and GGPP also raised Abeta42. These compounds seem to target the gamma-secretase complex, increasing gamma-secretase-catalyzed production of Abeta42 in vitro. Short-term in vivo studies show that two Abeta42-raising compounds increase Abeta42 levels in the brains of mice. The elevations in Abeta42 by these compounds are comparable to the increases in Abeta42 induced by Alzheimer disease-causing mutations in the genes encoding amyloid beta protein precursor and presenilins, raising the possibility that exogenous compounds or naturally occurring isoprenoids might increase Abeta42 production in humans.

Liver X receptor agonist treatment ameliorates amyloid pathology and memory deficits caused by high-fat diet in APP23 mice.

High-fat diet and certain dietary patterns are associated with higher incidence of sporadic Alzheimer's disease (AD) and cognitive decline. However, no specific therapy has been suggested to ameliorate the negative effects of high fat/high cholesterol levels on cognition and amyloid pathology. Here we show that in 9-month-old APP23 mice, a high-fat/high-cholesterol (HF) diet provided for 4 months exacerbates the AD phenotype evaluated by behavioral, morphological, and biochemical assays. To examine the therapeutic potential of liver X receptor (LXR) ligands, APP23 mice were fed HF diet supplemented with synthetic LXR agonist T0901317 (T0). Our results demonstrate that LXR ligand treatment causes a significant reduction of memory deficits observed during both acquisition and retention phases of the Morris water maze. Moreover, the effects of T0 on cognition correlate with AD-like morphological and biochemical parameters. We found a significant decrease in amyloid plaque load, insoluble Abeta and soluble Abeta oligomers. In vitro experiments with primary glia demonstrate that Abca1 is essential for the proper lipidation of ApoE and mediates the effects of T0 on Abeta degradation by microglia. Microdialysis experiments performed on awake freely moving mice showed that T0 decreased Abeta levels in the interstitial fluid of the hippocampus, supporting the conclusion that this treatment increases Abeta clearance. The data presented conclusively shows that LXR activation in the context of a metabolic challenge has critical effects on AD phenotype progression by attenuating Abeta deposition and facilitating its clearance.

Celastrol inhibits Hsp90 chaperoning of steroid receptors by inducing fibrillization of the Co-chaperone p23.

Hsp90 is an ATP-dependent molecular chaperone. The best characterized inhibitors of Hsp90 target its ATP binding pocket, causing nonselective degradation of Hsp90 client proteins. Here, we show that the small molecule celastrol inhibits the Hsp90 chaperoning machinery by inactivating the co-chaperone p23, resulting in a more selective destabilization of steroid receptors compared with kinase clients. Our in vitro and in vivo results demonstrate that celastrol disrupts p23 function by altering its three-dimensional structure, leading to rapid formation of amyloid-like fibrils. This study reveals a unique inhibition mechanism of p23 by a small molecule that could be exploited in the dissection of protein fibrillization processes as well as in the therapeutics of steroid receptor-dependent diseases.

Notch signaling mediates G1/S cell-cycle progression in T cells via cyclin D3 and its dependent kinases.

Notch signaling plays a role in normal lymphocyte development and function. Activating Notch1-mutations, leading to aberrant downstream signaling, have been identified in human T-cell acute lymphoblastic leukemia (T-ALL). While this highlights the contribution of Notch signaling to T-ALL pathogenesis, the mechanisms by which Notch regulates proliferation and survival in normal and leukemic T cells are not fully understood. Our findings identify a role for Notch signaling in G(1)-S progression of cell cycle in T cells. Here we show that expression of the G(1) proteins, cyclin D3, CDK4, and CDK6, is Notch-dependent both in vitro and in vivo, and we outline a possible mechanism for the regulated expression of cyclin D3 in activated T cells via CSL (CBF-1, mammals; suppressor of hairless, Drosophila melanogaster; Lag-1, Caenorhabditis elegans), as well as a noncanonical Notch signaling pathway. While cyclin D3 expression contributes to cell-cycle progression in Notch-dependent human T-ALL cell lines, ectopic expression of CDK4 or CDK6 together with cyclin D3 shows partial rescue from gamma-secretase inhibitor (GSI)-induced G(1) arrest in these cell lines. Importantly, cyclin D3 and CDK4 are highly overexpressed in Notch-dependent T-cell lymphomas, justifying the combined use of cell-cycle inhibitors and GSI in treating human T-cell malignancies.

Notch regulates cytolytic effector function in CD8+ T cells.

The maturation of naive CD8(+) T cells into effector CTLs is a critical feature of a functional adaptive immune system. Development of CTLs depends, in part, upon the expression of the transcriptional regulator eomesodermin (EOMES), which is thought to regulate expression of two key effector molecules, perforin and granzyme B. Although EOMES is important for effector CTL development, the precise mechanisms regulating CD8(+) effector cell maturation remains poorly understood. In this study, we show that Notch1 regulates the expression of EOMES, perforin, and granzyme B through direct binding to the promoters of these crucial effector molecules. By abrogating Notch signaling, both biochemically as well as genetically, we conclude that Notch activity mediates CTL activity through direct regulation of EOMES, perforin, and granzyme B.

A gamma-secretase inhibitor and quinacrine reduce prions and prevent dendritic degeneration in murine brains.

In prion-infected mice, both the Notch-1 intracellular domain transcription factor (NICD) and the disease-causing prion protein (PrP(Sc)) increase in the brain preceding dendritic atrophy and loss. Because the drug LY411575 inhibits the gamma-secretase-catalyzed cleavage of Notch-1 that produces NICD, we asked whether this gamma-secretase inhibitor (GSI) might prevent dendritic degeneration in mice with scrapie. At 50 d postinoculation with Rocky Mountain Laboratory (RML) prions, mice were given GSI orally for 43-60 d. Because we did not expect GSI to produce a reduction of PrP(Sc) levels in brain, we added quinacrine (Qa) to the treatment regimen. Qa inhibits PrP(Sc) formation in cultured cells. The combination of GSI and Qa reduced PrP(Sc) by approximately 95% in the neocortex and hippocampus but only approximately 50% in the thalamus at the site of prion inoculation. The GSI plus Qa combination prevented dendritic atrophy and loss, but GSI alone did not. Even though GSI reduced NICD levels to a greater extent than GSI plus Qa, it was unable to prevent dendritic degeneration. Whether a balance between NICD and dendrite growth-stimulating factors was achieved with GSI plus Qa but not GSI alone remains to be determined. Although the combination of GSI and Qa diminished PrP(Sc) in the brains of RML-infected mice, GSI toxicity prevented us from being able to assess the effect the GSI plus Qa combination on incubation times. Whether less toxic GSIs can be used in place of LY411575 to prolong survival remains to be determined.

Pigment epithelium-derived factor maintains retinal pigment epithelium function by inhibiting vascular endothelial growth factor-R2 signaling through gamma-secretase.

Wet age-related macular degeneration (AMD) attacks the integrity of the retinal pigment epithelium (RPE) barrier system. The pathogenic process was hypothesized to be mediated by vascular endothelial growth factor (VEGF) and antagonized by pigment epithelium-derived factor (PEDF). To dissect these functional interactions, monolayer cultures of RPE cells were established, and changes in transepithelial resistance were evaluated after administration of PEDF, placenta growth factor (VEGF-R1 agonist), and VEGF-E (VEGF-R2 agonist). A recently described mechanism of VEGF inhibition in endothelia required the release of VEGF-R1 intracellular domain by gamma-secretase. To evaluate this pathway in the RPE, cells were pretreated with inhibitors DAPT or LY411575. Processing of VEGF receptors was assessed by Western blot analysis. Administration of VEGF-E rapidly increased RPE permeability, and PEDF inhibited the VEGF-E response dose-dependently. Both gamma-secretase antagonists prevented the inhibitory effects of PEDF. The co-administration of PEDF and VEGF-E depleted the amount of VEGF-R2 in the membrane and increased the amount of VEGF-R2 ectodomain in the media. Therefore, the inhibitory effect of PEDF appears to be mediated via the processing of VEGF-R2 by gamma-secretase. gamma-Secretase generates the amyloid-beta (Abeta) peptide of Alzheimer disease from its precursor (amyloid precursor protein). This peptide is also a component of drusen in dry AMD. The results support the hypothesis that misregulation of gamma-secretase may not only lead to Abeta deposits in dry AMD but can also be damaging to RPE function by blocking the protective effects of PEDF to prevent VEGF from driving the dry to wet AMD transition.

Iothalamate quantification by tandem mass spectrometry to measure glomerular filtration rate.

BACKGROUND: Glomerular filtration rate (GFR) can be determined by measuring renal clearance of the radiocontrast agent iothalamate. Current analytic methods for quantifying iothalamate concentrations in plasma and urine using liquid chromatography or capillary electrophoresis have limitations such as long analysis times and susceptibility to interferences. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to overcome these limitations. METHODS: Urine and plasma samples were deproteinized using acetonitrile and centrifugation. The supernatant was diluted in water and analyzed by LC-MS/MS using a water:methanol gradient. We monitored 4 multiple reaction monitoring transitions: m/z 614.8-487.0, 614.8-456.0, 614.8-361.1, and 614.8-177.1. We compared the results to those obtained via our standard capillary electrophoresis (CE-UV) on samples from 53 patients undergoing clinical GFR testing. RESULTS: Mean recovery was 90%-110% in both urine and plasma matrices. Imprecision was

Notch1 and TGFbeta1 cooperatively regulate Foxp3 expression and the maintenance of peripheral regulatory T cells.

Notch and its ligands have been implicated in the regulation and differentiation of various CD4(+) T-helper cells. Regulatory T cells (T(regs)), which express the transcription factor Foxp3, suppress aberrant immune responses that are typically associated with autoimmunity or excessive inflammation. Previous studies have shown that transforming growth factor beta (TGFbeta1) induces Foxp3 expression and a regulatory phenotype in peripheral T cells. Here, we show that pharmacologic inhibition of Notch signaling using gamma-secretase inhibitor (GSI) treatment blocks (1) TGFbeta1-induced Foxp3 expression, (2) the up-regulation of Foxp3-target genes, and (3) the ability to suppress naive T-cell proliferation. In addition, the binding of Notch1, CSL, and Smad to conserved binding sites in the foxp3 promoter can be inhibited by treatment with GSI. Finally, in vivo administration of GSI results in reduced Foxp3 expression and development of symptoms consistent with autoimmune hepatitis, a disease previously found to result from dysregulation of TGFbeta signaling and regulatory T cells. Together, these findings indicate that the Notch and TGFbeta signaling pathways cooperatively regulate Foxp3 expression and regulatory T-cell maintenance both in vitro and in vivo.

A convenient synthesis of (Z)-4-hydroxy-N-desmethyltamoxifen (endoxifen).

A mixture of the (Z)- and (E)-isomers of 4-hydroxy-N-desmethyltamoxifen was conveniently prepared in four steps. These geometrical isomers were then neatly separated by semi-preparative Reverse Phase High Performance Liquid Chromatography (RP-HPLC) using specified conditions. Additionally, the isolated E-isomer could be equilibrated in aqueous strong acid in acetonitrile or trifluoroacetic acid/dichloromethane to give a clean 1:1 mixture of Z/E isomers that was re-subjected to HPLC separation. In this way, most of the undesired (E)-isomer could be readily converted to the desired (Z)-isomer providing quick access to over 200mg quantities of pure endoxifen (Z-isomer), a potent antiestrogenic metabolite of tamoxifen traditionally used in breast cancer treatment.

Chemical synthesis of deuterium-labeled and unlabeled very long chain polyunsaturated fatty acids.

First syntheses of a deuterium-labeled very long C34-containing polyunsaturated fatty acid, C34:5n5.d(2), and three other unlabeled very long chain C30-32-containing polyunsaturated fatty acids are reported here. These syntheses were achieved by coupling chemically modified C22- and C20-containing polyunsaturated fatty acids with carbanions derived from arylalkyl sulfones, followed by sodium amalgam-mediated desulfonylation.

An NSAID-like compound, FT-9, preferentially inhibits gamma-secretase cleavage of the amyloid precursor protein compared to its effect on amyloid precursor-like protein 1.

Inhibition of gamma-secretase cleavage of the amyloid precursor protein (APP) is a prime target for the development of therapeutics for treating Alzheimer's disease; however, complete inhibition of this activity would also impair the processing of many other proteins, including the APP homologues, amyloid precursor-like protein (APLP) 1 and 2. To prevent unwanted side effects, therapeutically useful gamma-secretase inhibitors should specifically target APP processing while sparing cleavage of other gamma-substrates. Thus, since APLP1 and APLP2 are more similar to APP than any of the other known gamma-secretase substrates and have important physiological roles in their own right, we reasoned that comparison of the effect of gamma-secretase inhibitors on APLP processing should provide a sensitive indicator of the selectivity of putative inhibitors. To address this issue, we have optimized microsome and cell culture assays to monitor the gamma-secretase proteolysis of APP and APLPs. Production of the gamma-secretase-generated intracellular domain (ICD) occurs more rapidly from APLP1 than from either APLP2 or APP, suggesting that APLP1 is a better gamma-substrate and that substrate recognition is not restricted to the highly conserved amino acid sequences surrounding the epsilon-site. As expected, the well-characterized gamma-secretase modulator, fenofibrate, did not inhibit ICD release, whereas a related compound, FT-9, inhibited gamma-secretase both in microsomes and in whole cells. Importantly, FT-9 displayed a preferential effect, inhibiting cleavage of APP much more effectively than cleavage of APLP1. These findings suggest that selective inhibitors can be developed and that screening of compounds against APP and APLPs should assist in this process.

Cisplatin and dimethyl sulfoxide react to form an adducted compound with reduced cytotoxicity and neurotoxicity.

PURPOSE: We present work that demonstrates that cisplatin reacts rapidly with dimethyl sulfoxide (DMSO) in solution and identify the structure and reactivity of the resulting compound. METHODS: Electrospray ionization-mass spectrometry (ESI-MS) and NMR were used to identify the chemical structure of compounds formed when DMSO reacts with cisplatin. We studied the reactivity of the identified compound with DNA. In vitro toxicity studies in neurons and cancer cells and in vivo toxicity studies in rats were used to determine both the cancer chemotherapeutic and toxic effects of the identified compound. RESULTS: Cisplatin binds rapidly with DMSO to form a DMSO adduct. The resulting compound has reduced ability to bind to double-stranded DNA both in vitro and in cells. This compound has reduced toxicity for cancer cells and neurons in vitro. In vivo nephrotoxicity studies show that the adducted compound has different nephrotoxicity and elimination characteristics than cisplatin. CONCLUSIONS: From this work, we conclude that dissolving cisplatin in DMSO results in formation of an adducted compound with different therapeutic and biological characteristics. Furthermore, future studies which propose using DMSO in combination with cisplatin for chemotherapeutic treatment in patients must be reconsidered. Due to the rapidity and nature of the reaction, DMSO and cisplatin should not be combined for patient treatment.

Down-regulation of amyloid precursor protein by peptide nucleic acid in vivo.

Alzheimer's disease (AD) is a neurodegenerative disease associated with increased expression of amyloid precursor protein (APP) and the deposition of its proteolytic cleavage products, the amyloid-beta peptides, Abeta(1-40) and Abeta(1-42). Peptide nucleic acids (PNAs) have been shown to block the expression of proteins at transcriptional and translational levels. In this study we used a sense and an antisense PNA specifically targeted to APP to inhibit the transcription and translation of APP by complementary binding to DNA or mRNA, respectively. Using Western blotting, APP showed a drastic decrease (50% and 90% reduction, in two separate experiments, as compared with saline control) with the injection of sense APP. mRNA levels were higher at the same time point after injection of APP sense PNA, most probably because of a compensatory mechanism in response to the drop of APP that might have occurred at an earlier time point (0-1 h) and was reflected in a drop at the protein level at 1 h. The injection of antisense PNA showed about 70% decrease in APP as measured by Western blotting. Unmodified PNA can be used in vivo to reduce the levels of APP, which plays a critical role in the development of AD.