A retrospective observational study of the treatment with polymyxin B for liver transplantation recipients infected by multidrug-resistant gram-negative bacteria.

WHAT IS KNOWN AND OBJECTIVE: Only a few studies about polymyxin B (PMB) against multidrug-resistant gram-negative bacteria (MDR GNB) infection were conducted in liver transplantation recipients (LTRs). The purpose of this study was to investigate the efficacy and safety of PMB in the treatment of MDR-GNB in liver transplant recipients and to determine the risk factors affecting clinical cure and 30-day all-cause mortality. METHODS: Data of LTRs receiving PMB from January 2016 to February 2020 were collected. Clinical cure and 30-day all-cause mortality were the main efficacy outcomes, while the incidence of nephrotoxicity, neurotoxicity, and hyperpigmentation of PMB was the main safety outcome. RESULTS AND DISCUSSION: Data of 42 LTRs were included. Clinical cure with PMB was observed in 27 recipients (64.3%), and the 30-day all-cause mortality rate was 31.0% (13/42). The incidence of acute kidney injury (AKI), neurotoxicity, and hyperpigmentation was 57.1% (16/28), 4.8% (2/42), and 16.7% (7/42), respectively. Logistic regression analysis showed that Acute Physiology and Chronic Health Evaluation (APACHE) II score (OR, 1.203; 95% CI, 1.016-1.423, p = 0.032) was an independent risk factor for 30-day all-cause mortality, whereas renal replacement therapy (OR, 0.128; 95% CI, 0.019-0.860, p = 0.034) was an independent risk factor for clinical cure with PMB. WHAT IS NEW AND CONCLUSIONS: This is the first study to evaluate the application of PMB in LTRs. If there were no better therapeutic options left for LTRs other than PMB, it can be used against MDR GNB infection in LTRs. We should closely observe adverse events or reactions, and adjust the dose based on the balance of efficacy and safety.

Comparative pharmacokinetics of baicalin and geniposide in juvenile and adult rats after oral administration of Qingkailing Granules.

Objective: To explore the effect of age on Qingkailing Granules disposition by comparing the pharmacokinetics of geniposide and baicalin in juvenile and adult rats. Methods: A simple and rapid LC-MS/MS method was developed and validated to simultaneously determine geniposide and baicalin in rat plasma after a simple protein precipitation. The analytes were separated on an Agilent ZORBAX Extend-C18 column. The mobile phase consisted of acetonitrile and water with 0.1% (volume percent) formic acid at a flow rate of 0.6 mL/min. The ionization was conducted using an ESI source in negative ion mode. Multiple reaction monitoring was used for quantification at transitions of m/z 445.0 â†’ m/z 268.9 for baicalin, m/z 433.2 â†’ m/z 225.0 for geniposide, m/z 431.0 â†’ m/z 341.0 for vitexin (IS). Juvenile and adult rats were administrated Qingkailing Granules (3 g/kg) orally. Plasma concentrations of baicalin and geniposide were determined by LC-MS/MS. Results: The linear ranges of the analytes were 1-1000 ng/mL for baicalin and 2-2000 ng/mL for geniposide. The method was successfully applied to compare the pharmacokinetics of the analytes between juvenile and adult rats after oral administration of Qingkailing Granules. AUC was bigger in adult rats, while t 1/2 was longer in juvenile rats. Conclusion: These results suggested that the absorption and elimination of baicalin and geniposide in juvenile rats was lower than that in adult rats. Additional attention should be paid to the pharmacokinetic difference when Qingkailing Granules were used in children.

Megastigmane glycosides from Urena lobata.

Five new megastigmane glycosides, urenalobasides A-E (1-5), together with 11 known ones (6-16) were isolated from Urena lobata. Their structures were determined by extensive spectroscopic and spectrometric data (1D and 2D NMR, IR, and HRESIMS) and calculated electronic circular dichroism method. Compounds 1 and 2 are two unusual megastigmanes structurally containing a 6/5 fused ring system. Compound 3 exhibits inhibition of nitric oxide production in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells with IC50 value of 53.7 ±â€¯1.0 µM (positive control, dexamethasone, IC50 = 16.6 ±â€¯0.8 µM).

Nitric oxide inhibitory polyketides from Penicillium chrysogenum MT-12, an endophytic fungus isolated from Huperzia serrata.

Twelve new polyketides, penicichrysogenins A-L (1-10, 11a, and 11b) along with five known compounds (12a, 12b, and 13-15) were isolated from the solid substrate fermentation cultures of a Huperzia serrata endophytic fungus Penicillium chrysogenum MT-12. The structures of the new compounds were established using extensive spectroscopic (1D and 2D NMR, IR, and HRESIMS) and calculated electronic circular dichroism (ECD) methods. Compounds 11a/11b and 12a/12b were two pairs of enantiomers successfully separated by chiral HPLC resolution. Compounds 4, 5, 8, 9, 11a/11b, and 12a/12b exhibited inhibition of nitric oxide production in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells with IC50 values in the range of 17.5-98.4µM.

Synthesis of Unnatural 2-Substituted Quinolones and 1,3-Diketones by a Member of Type III Polyketide Synthases from Huperzia serrata.

A curcuminoids, benzalacetone-, and quinolone-producing type III polyketide synthase (HsPKS3) from Huperzia serrata uniquely catalyzes the formation of unnatural 2-substituted quinolones and 1,3-diketones via head-to-head condensation of two completely different substrates. The broad range of substrate tolerance of HsPKS3 facilitates accessing structurally diverse 2-substituted quinolones and 1,3-diketones.

Rapid P1 SAR of brain penetrant tertiary carbinamine derived BACE inhibitors.

This Letter describes the one pot synthesis of tertiary carbinamine 3 and related analogs of brain penetrant BACE-1 inhibitors via the alkylation of the Schiff base intermediate 2. The methodology developed for this study provided a convenient and rapid means to explore the P1 region of these types of inhibitors, where the P1 group is installed in the final step using a one-pot two-step protocol. Further SAR studies led to the identification of 10 which is twofold more potent in vitro as compared to the lead compound. This inhibitor was characterized in a cisterna magna ported rhesus monkey model, where significant lowering of CSF Abeta40 was observed.

SAR of tertiary carbinamine derived BACE1 inhibitors: role of aspartate ligand amine pKa in enzyme inhibition.

The optimization of tertiary carbinamine derived inhibitors of BACE1 from its discovery as an unstable lead to low nanomolar cell active compounds is described. Five-membered heterocycles are reported as stable and potency enhancing linkers. In the course of this work, we have discovered a clear trend where the activity of inhibitors at a given assay pH is dependent on pK(a) of the amino group that interacts directly with the catalytic aspartates. The potency of compounds as inhibitors of Alphabeta production in a cell culture assay correlated much better with BACE1 enzyme potency measured at pH 7.5 than at pH 4.5.

First demonstration of cerebrospinal fluid and plasma A beta lowering with oral administration of a beta-site amyloid precursor protein-cleaving enzyme 1 inhibitor in nonhuman primates.

beta-Site amyloid precursor protein (APP)-cleaving enzyme (BACE) 1 cleavage of amyloid precursor protein is an essential step in the generation of the potentially neurotoxic and amyloidogenic A beta 42 peptides in Alzheimer's disease. Although previous mouse studies have shown brain A beta lowering after BACE1 inhibition, extension of such studies to nonhuman primates or man was precluded by poor potency, brain penetration, and pharmacokinetics of available inhibitors. In this study, a novel tertiary carbinamine BACE1 inhibitor, tertiary carbinamine (TC)-1, was assessed in a unique cisterna magna ported rhesus monkey model, where the temporal dynamics of A beta in cerebrospinal fluid (CSF) and plasma could be evaluated. TC-1, a potent inhibitor (IC(50) approximately 0.4 nM), has excellent passive membrane permeability, low susceptibility to P-glycoprotein transport, and lowered brain A beta levels in a mouse model. Intravenous infusion of TC-1 led to a significant but transient lowering of CSF and plasma A beta levels in conscious rhesus monkeys because it underwent CYP3A4-mediated metabolism. Oral codosing of TC-1 with ritonavir, a potent CYP3A4 inhibitor, twice daily over 3.5 days in rhesus monkeys led to sustained plasma TC-1 exposure and a significant and sustained reduction in CSF sAPP beta, A beta 40, A beta 42, and plasma A beta 40 levels. CSF A beta 42 lowering showed an EC(50) of approximately 20 nM with respect to the CSF [TC-1] levels, demonstrating excellent concordance with its potency in a cell-based assay. These results demonstrate the first in vivo proof of concept of CSF A beta lowering after oral administration of a BACE1 inhibitor in a nonhuman primate.

Decrease in age-adjusted cerebrospinal fluid beta-secretase activity in Alzheimer's subjects.

OBJECTIVES: To develop a novel cerebrospinal fluid (CSF) beta-secretase-1 activity assay and evaluate beta-secretase-1 (BACE-1) activity as a potential biomarker in human Alzheimer's disease. METHODS: The assay consisted of an enzymatic reaction of CSF samples with an optimized beta-secretase peptide substrate and the cleavage products were detected using a neo-epitope specific antibody. RESULTS: The CSF BACE-1 activity assay described exhibits time, temperature, dose, and pH dependence, with sensitivity down to <1 pM of recombinant BACE-1 enzyme, and is completely blocked by BACE-1 inhibitors. The endogenous BACE-1 enzyme in CSF appears to exist as a c-terminally truncated protein, based on both western blotting and capture-based activity assays. In a small cohort of human subjects, an age-dependent increase in CSF BACE activity was observed (~1.0 pM/year, p<0.05). In Alzheimer's disease subjects, a significant decline in age-adjusted CSF BACE activity was observed compared to controls (56% in the log-transformed scale, p=0.02). CONCLUSION: We have developed a robust assay to measure CSF BACE-1 activity which could serve as a potential biomarker in human Alzheimer's disease subjects.

In vivo beta-secretase 1 inhibition leads to brain Abeta lowering and increased alpha-secretase processing of amyloid precursor protein without effect on neuregulin-1.

beta-Secretase (BACE) cleavage of amyloid precursor protein (APP) is one of the first steps in the production of amyloid beta peptide Abeta42, the putative neurotoxic species in Alzheimer's disease. Recent studies have shown that BACE1 knockdown leads to hypomyelination, putatively caused by a decline in neuregulin (NRG)-1 processing. In this study, we have tested a potent cell-permeable BACE1 inhibitor (IC(50) approximately 30 nM) by administering it directly into the lateral ventricles of mice, expressing human wild-type (WT)-APP, to determine the consequences of BACE1 inhibition on brain APP and NRG-1 processing. BACE1 inhibition, in vivo, led to a significant dose- and time-dependent lowering of brain Abeta40 and Abeta42. BACE1 inhibition also led to a robust brain secreted (s)APPbeta lowering that was accompanied by an increase in brain sAPPalpha levels. Although an increase in full-length NRG-1 levels was evident in 15-day-old BACE1 homozygous knockout (KO) (-/-) mice, in agreement with previous studies, this effect was also observed in 15-day-old heterozygous (+/-) mice, but it was not evident in 30-day-old and 2-year-old BACE1 KO (-/-) mice. Thus, BACE1 knockdown led to a transient decrease in NRG-1 processing in mice. Pharmacological inhibition of BACE1 in adult mice, which led to significant Abeta lowering, was without any significant effect on brain NRG-1 processing. Taken together, these results suggest that BACE1 is the major beta-site cleavage enzyme for APP and that its inhibition can lower brain Abeta and redirect APP processing via the potentially nonamyloidogenic alpha-secretase pathway, without significantly altering NRG-1 processing.

Effects of NaCl stress on changes in the ultraviolet-B radiation-induced cyclobutyl pyrimidine dimer and ultraviolet absorbing compound contents in mung bean.

In two mung bean cultivars (Phaseolus raditus L. cv. 'Qindou-20' and 'Zhonglv-1') with different sensitivities to UV-B grown in growth chamber under supplemental or no supplemental UV-B radiation (0.4 W/m(2)) with or without 0.4% NaCl, the effects of NaCl stress on UV-B-induced DNA damage and repair were studied. The results showed that, under NaCl stress, (i) CPD accumulation was lower in the tolerant cultivar 'Zhonglv-1' but was the same in the sensitive cultivar 'Qindou-20', (ii) CPD formation in both cultivars was weakened, (iii) the photorepair and dark repair capacity were higher in the tolerant cultivar and (iv) the photorepair was weakened and dark repair capacity did not change in the sensitive cultivar. There was a negative correlation between susceptibility of CPD formation and levels of UV-absorbing compounds. These results demonstrate that NaCl stress can affect not only the susceptibility to CPD formation, but also the capacities for photorepair and dark repair of DNA, which together result in the change in UV-B-induced CPD accumulation and thereby that in sensitivity of plant to UV-B. The results also suggest that the differences in susceptibilities to CPD formation are due to the differences in levels of UV-absorbing compounds.

Discovery of isonicotinamide derived beta-secretase inhibitors: in vivo reduction of beta-amyloid.

beta-Secretase inhibition offers an exciting opportunity for therapeutic intervention in the progression of Alzheimer's disease. A series of isonicotinamides derived from traditional aspartyl protease transition state isostere inhibitors has been optimized to yield low nanomolar inhibitors with sufficient penetration across the blood-brain barrier to demonstrate beta-amyloid lowering in a murine model.

Discovery of oxadiazoyl tertiary carbinamine inhibitors of beta-secretase (BACE-1).

We describe the discovery and optimization of tertiary carbinamine derived inhibitors of the enzyme beta-secretase (BACE-1). These novel non-transition-state-derived ligands incorporate a single primary amine to interact with the catalytic aspartates of the target enzyme. Optimization of this series provided inhibitors with intrinsic and functional potency comparable to evolved transition state isostere derived inhibitors of BACE-1.

LRRTM3 promotes processing of amyloid-precursor protein by BACE1 and is a positional candidate gene for late-onset Alzheimer's disease.

Rare familial forms of Alzheimer's disease (AD) are thought to be caused by elevated proteolytic production of the Abeta42 peptide from the beta-amyloid-precursor protein (APP). Although the pathogenesis of the more common late-onset AD (LOAD) is not understood, BACE1, the protease that cleaves APP to generate the N terminus of Abeta42, is more active in patients with LOAD, suggesting that increased amyloid production processing might also contribute to the sporadic disease. Using high-throughput siRNA screening technology, we assessed 15,200 genes for their role in Abeta42 secretion and identified leucine-rich repeat transmembrane 3 (LRRTM3) as a neuronal gene that promotes APP processing by BACE1. siRNAs targeting LRRTM3 inhibit the secretion of Abeta40, Abeta42, and sAPPbeta, the N-terminal APP fragment produced by BACE1 cleavage, from cultured cells and primary neurons by up to 60%, whereas overexpression increases Abeta secretion. LRRTM3 is expressed nearly exclusively in the nervous system, including regions affected during AD, such as the dentate gyrus. Furthermore, LRRTM3 maps to a region of chromosome 10 linked to both LOAD and elevated plasma Abeta42, and is structurally similar to a family of neuronal receptors that includes the NOGO receptor, an inhibitor of neuronal regeneration and APP processing. Thus, LRRTM3 is a functional and positional candidate gene for AD, and, given its receptor-like structure and restricted expression, a potential therapeutic target.

A genome wide analysis of ubiquitin ligases in APP processing identifies a novel regulator of BACE1 mRNA levels.

Proteolysis of beta-amyloid precursor protein (APP) into amyloid beta peptide (Abeta) by beta- and gamma-secretases is a critical step in the pathogenesis of Alzheimer's Disease (AD), but the pathways regulating secretases are not fully characterized. Ubiquitinylation, which is dysregulated in AD, may affect APP processing. Here, we describe a screen for APP processing modulators using an siRNA library targeting 532 predicted ubiquitin ligases. Seven siRNA pools diminished Abeta production. Of these, siRNAs targeting PPIL2 (hCyp-60) suppressed beta-site cleavage. Knockdown of PPIL2 mRNA decreased BACE1 mRNA, while overexpression of PPIL2 cDNA enhanced BACE1 mRNA levels. Microarray analysis of PPIL2 or BACE1 knockdown indicated that genes affected by BACE1 knockdown are a subset of those dependent upon PPIL2; suggesting that BACE1 expression is downstream of PPIL2. The association of PPIL2 with BACE expression and its requirement for Abeta production suggests new approaches to discover disease modifying agents for AD.

Aminoethylenes: a tetrahedral intermediate isostere yielding potent inhibitors of the aspartyl protease BACE-1.

A series of novel beta-site amyloid precursor protein cleaving enzyme (BACE-1) inhibitors containing an aminoethylene (AE) tetrahedral intermediate isostere were synthesized and evaluated in comparison to corresponding hydroxyethylene (HE) compounds. Enzymatic inhibitory values were similar for both isosteres, as were structure-activity relationships with respect to stereochemical preference and substituent variation (P2/P3, P1, and P2'); however, the AE compounds were markedly more potent in a cell-based assay for reduction of beta-secretase activity. The incorporation of preferred P2/P3, P1, and P2' substituents into the AE pharmacophore yielded compound 7, which possessed enzymatic and cell assay IC(50)s of 26 nM and 180 nM, respectively. A three-dimensional crystal structure of 7 in complex with BACE-1 revealed that the amino group of the inhibitor core engages the catalytic aspartates in a manner analogous to hydroxyl groups in HE inhibitors. The AE isostere class represents a promising advance in the development of BACE-1 inhibitors.

Biochemical and cell-based assays for characterization of BACE-1 inhibitors.

The deposition of beta-amyloid peptides (A beta42 and A beta40) in neuritic plaques is one of the hallmarks of Alzheimer's disease (AD). A beta peptides are derived from sequential cleavage of amyloid precursor protein (APP) by beta- and gamma-secretases. BACE-1 has been shown to be the major beta-secretase and is a primary therapeutic target for AD. In this article, two novel assays for the characterization of BACE-1 inhibitors are reported. The first is a sensitive 96-well HPLC biochemical assay that uses a unique substrate containing an optimized peptide cleavage sequence, NFEV, spanning from the P2-P2' positions This substrate was processed by BACE-1 approximately 10 times more efficiently than was the widely used substrate containing the Swedish (NLDA) sequence. As a result, the concentration of the enzyme required for the assay can be as low as 100 pM, permitting the evaluation of inhibitors with subnanomolar potency. The assay has also been applied to related aspartyl proteases such as cathepsin D (Cat D) and BACE-2. The second assay is a homogeneous electrochemiluminescence assay for the evaluation of BACE-1 inhibition in cultured cells that assesses the level of secreted amyloid EV40_NF from HEK293T cells stably transfected with APP containing the novel NFEV sequence. To illustrate the use of these assays, the properties of a potent, cell-active BACE-1 inhibitor are described.

Novel mutations introduced at the beta-site of amyloid beta protein precursor enhance the production of amyloid beta peptide by BACE1 in vitro and in cells.

Abnormal production and accumulation of amyloid-beta peptide (Abeta) plays a major role in the pathogenesis of Alzheimer's disease (AD). beta-secretase (BACE1) is responsible for the cleavage at thebeta-site in amyloid beta protein precursor (AbetaPP/APP) to generate the N-terminus of Abeta. Here we report the stepwise identification and characterization of a novel APP-beta-site mutant, "NFEV" (APP_NFEV) in vitro and in cells. In vitro, the APP_NFEV exhibits 100-fold enhanced cleavage rate relative to the "wild-type" substrate (APPwt) and 10-fold increase relative to the Swedish-type mutation variant (APPsw). In cells, it was preferably cleaved among 24 APP beta-site mutations tested. More importantly, the APP_NFEV mutant failed to generate any detectable Abeta peptides in BACE1-KO mouse fibroblast cells. The production of Abeta peptides was restored by co-transfecting human BACE1, demonstrating that BACE1 is the only enzyme responsible for the processing of APP_NFEV in these cells. Analysis of APP_NFEV cleavage products secreted in the media revealed that in cells BACE1 cleaves APP_NFEV at the position between NF and EV, identical to that observed in vitro. A BACE inhibitor blocked the processing of the APP_NFEV beta-site in vitro and in cells. Our data indicates that the "NFEV" mutant is not only an enhanced substrate for BACE1 in vitro, but also a specific substrate for BACE1 in cells.

Compounds that bind APP and inhibit Abeta processing in vitro suggest a novel approach to Alzheimer disease therapeutics.

Extracellular deposits of aggregated amyloid-beta (Abeta) peptides are a hallmark of Alzheimer disease; thus, inhibition of Abeta production and/or aggregation is an appealing strategy to thwart the onset and progression of this disease. The release of Abeta requires processing of the amyloid precursor protein (APP) by both beta- and gamma-secretase. Using an assay that incorporates full-length recombinant APP as a substrate for beta-secretase (BACE), we have identified a series of compounds that inhibit APP processing, but do not affect the cleavage of peptide substrates by BACE1. These molecules also inhibit the processing of APP and Abeta by BACE2 and selectively inhibit the production of Abeta(42) species by gamma-secretase in assays using CTF99. The compounds bind directly to APP, likely within the Abeta domain, and therefore, unlike previously described inhibitors of the secretase enzymes, their mechanism of action is mediated through APP. These studies demonstrate that APP binding agents can affect its processing through multiple pathways, providing proof of concept for novel strategies aimed at selectively modulating Abeta production.