Potent Enantiomer Research Articles

Chiral Fluorescent Antifungal Azole Probes Detect Resistance, Uptake Dynamics, and Subcellular Distribution in Candida Species.

Azoles are essential for fungal infection treatment, yet the increasing resistance highlights the need for innovative diagnostic tools and strategies to revitalize this class of antifungals. We developed two enantiomers of a fluorescent antifungal azole probe (1 S and 1 R ), analyzing 60 Candida strains via live-cell microscopy. A database of azole distribution images in strains of Candida albicans, Candida glabrata, and Candida parapsilosis, among the most important pathogenic Candida species, was established and analyzed. This analysis revealed distinct populations of yeast cells based on the correlation between fluorescent probe uptake and cell diameter. Varied uptake levels and subcellular distribution patterns were observed in C. albicans, C. glabrata, and C. parapsilosis, with the latter displaying increased localization to lipid droplets. Comparison of the more potent fluorescent antifungal azole probe enantiomer 1 S with the moderately potent enantiomer 1 R highlighted time-dependent differences in the uptake profiles. The former displayed a marked elevation in uptake after approximately 150 min, indicating the time required for significant cell permeabilization to occur and its association with the azole's antifungal activity potency. Divergent uptake levels between susceptible and high efflux-based azole-resistant strains were detected, offering a rapid diagnostic approach for identifying azole resistance. This study highlights unique insights achievable through fluorescent antifungal azole probes, unraveling the complexities of azole resistance, subcellular dynamics, and uptake within fungal pathogens.

Inhibition of RHOA activity preserves the survival and hemostasis function of long-term cold-stored platelets

AbstractPatients with thrombocytopenia require platelet transfusion to prevent and stop hemorrhage. Cold storage of platelets results in complex molecular lesions, including changes in membrane microdomains that are recognized by host macrophages and hepatocyte counter-receptors, resulting in phagocytosis and clearance upon transfusion. For this reason, platelets are stored at room temperature, a method that confers increased risk of bacterial contamination. By applying signaling analysis and genetic and pharmacological approaches, we identified that cold-induced activation of RAS homolog family, member A (RHOA) GTPase causes the major hallmarks of platelet cold storage lesions. RHOA deficiency renders murine platelets insensitive to cold storage–induced damage, and pharmacological inhibition by a RHOA activation inhibitor, R-G04, can prevent the cold storage–induced lesions. RHOA inhibition prevents myosin activation and clathrin-independent formation and internalization of lipid rafts enriched in active glycosyltransferases as well as abnormal distribution of GPIbα. RHOA inhibition further prevents the metabolic reprogramming of cold storage–induced lesions and allows the maintenance of glycolytic flux and mitochondria-dependent respiration. Importantly, human platelets transfused in mice after cold storage, in the presence of R-G04 or its more potent enantiomer S-G04, can circulate in vivo at similar levels as room temperature–stored platelets while retaining their hemostatic activity in vivo, as assessed by bleeding time correction in aspirin-treated mice. Our studies provide a mechanism-based translational approach to prevent cold storage–induced damage, which is useful for human platelet transfusion in patients with thrombocytopenia.

Identification of Novel Series of Potent and Selective Relaxin Family Peptide Receptor 1 (RXFP1) Agonists.

Relaxin H2 is a clinically relevant peptide agonist for relaxin family peptide receptor 1 (RXFP1), but a combination of this hormone's short plasma half-life and the need for injectable delivery limits its therapeutic potential. We sought to overcome these limitations through the development of a potent small molecule (SM) RXFP1 agonist. Although two large SM HTS campaigns failed in identifying suitable hit series, we uncovered novel chemical space starting from the only known SM RXFP1 agonist series, represented by ML290. Following a design-make-test-analyze strategy based on improving early dose to man ranking, we discovered compound 42 (AZ7976), a highly selective RXFP1 agonist with sub-nanomolar potency. We used AZ7976, its 10 000-fold less potent enantiomer 43 and recombinant relaxin H2 to evaluate in vivo pharmacology and demonstrate that AZ7976-mediated heart rate increase in rats was a result of RXFP1 agonism. As a result, AZ7976 was selected as lead for continued optimization.

A key role of miR-132-5p in the prefrontal cortex for persistent prophylactic actions of (R)-ketamine in mice

(R,S)-ketamine is known to elicit persistent prophylactic effects in rodent models of depression. However, the precise molecular mechanisms underlying its action remain elusive. Using RNA-sequencing analysis, we searched for novel molecular target(s) that contribute to the prophylactic effects of (R)-ketamine, a more potent enantiomer of (R,S)-ketamine in chronic restraint stress (CRS) model. Pretreatment with (R)-ketamine (10 mg/kg, 1 day before CRS) significantly ameliorated body weight loss, increased immobility time of forced swimming test, and decreased sucrose preference of sucrose preference test in CRS-exposed mice. RNA-sequencing analysis of prefrontal cortex (PFC) revealed that several miRNAs such as miR-132-5p might contribute to sustained prophylactic effects of (R)-ketamine. Methyl CpG binding protein 2 (MeCP2) is known to regulate brain-derived neurotrophic factor (BDNF) expression. Quantitative RT-PCR confirmed that (R)-ketamine significantly attenuated altered expression of miR-132-5p and its regulated genes (Bdnf, Mecp2, Tgfb1, Tgfbr2) in the PFC of CRS-exposed mice. Furthermore, (R)-ketamine significantly attenuated altered expression of BDNF, MeCP2, TGF-β1 (transforming growth factor β1), and synaptic proteins (PSD-95, and GluA1) in the PFC of CRS-exposed mice. Administration of agomiR-132-5p decreased the expression of Bdnf and Tgfb1 in the PFC, resulting in depression-like behaviors. In contrast, administration of antagomiR-132-5p blocked the increased expression of miR-132-5p and decreased expression of Bdnf in the PFC of CRS-exposed mice, resulting in antidepressant-like effects. In conclusion, our data show a novel role of miR-132-5p in the PFC underlying depression-like phenotypes in CRS model and the sustained prophylactic effects of (R)-ketamine.

Nuclear factor of activated T cells 4 in the prefrontal cortex is required for prophylactic actions of (R)-ketamine

(R, S)-ketamine has prophylactic antidepressant-like effects in rodents; however, the precise molecular mechanisms underlying its action remain unknown. Using RNA-sequencing analysis, we searched novel molecular target(s) that contribute to the prophylactic effects of (R)-ketamine, a more potent enantiomer of (R, S)-ketamine. Pretreatment with (R)-ketamine (10 mg/kg, 6 days before) significantly ameliorated body weight loss, splenomegaly, and increased immobility time of forced swimming test in lipopolysaccharide (LPS: 1.0 mg/kg)-treated mice. RNA-sequencing analysis of prefrontal cortex (PFC) and subsequent IPA (Ingenuity Pathway Analysis) revealed that the nuclear factor of activated T cells 4 (NFATc4) signaling might contribute to sustained prophylactic effects of (R)-ketamine. Quantitative RT-PCR confirmed that (R)-ketamine significantly attenuated the increased gene expression of NFATc4 signaling (Nfatc4, Cd4, Cd79b, H2-ab1, H2-aa) in the PFC of LPS-treated mice. Furthermore, pretreatment with NFAT inhibitors (i.e., NFAT inhibitor and cyclosporin A) showed prophylactic effects in the LPS-treated mice. Similar to (R)-ketamine, gene knockdown of Nfatc4 gene by bilateral injection of adeno-associated virus (AAV) into the mPFC could elicit prophylactic effects in the LPS-treated mice. In conclusion, our data implicate a novel NFATc4 signaling pathway in the PFC underlying the prophylactic effects of (R)-ketamine for inflammation-related depression.

Intranasal administration of transforming growth factor-β1 elicits rapid-acting antidepressant-like effects in a chronic social defeat stress model: A role of TrkB signaling

(R,S)-ketamine causes rapid-acting and sustained antidepressant effects in treatment-resistant patients with depression although the precise molecular mechanisms underlying its antidepressant action remain unclear. We recently reported that transforming growth factor (TGF)-β1 might contribute to the antidepressant-like effects of (R)-ketamine that is a more potent enantiomer in rodents. Although TrkB signaling plays a role in the antidepressant-like actions of (R,S)-ketamine and its enantiomers, the role of TrkB signaling in the antidepressant effects of TGF-β1 remains unclear. Using behavioral tests such as tail-suspension test (TST), forced swimming test (FST), and 1% sucrose preference test (SPT), we investigated whether a single intranasal administration of the recombinant TGF-β1 (1.5 and 3.0 μg/kg) causes rapid and sustained antidepressant-like effects in a chronic social defeat stress (CSDS) model. Both doses of TGF-β1 significantly attenuated the increased immobility time of TST and FST in the CSDS susceptible mice. High dose of TGF-β1, but not low dose, significantly ameliorated the decreased sucrose preference of SPT in the CSDS susceptible mice. Pretreatment with a TrkB antagonist ANA-12 (0.5 mg/kg) blocked the antidepressant-like effects of TGF-β1 in CSDS susceptible mice. The data suggest that intranasal administration of TGF-β1 could elicit rapid-acting antidepressant-like effects via TrkB stimulation in a CSDS model. Therefore, it is likely that intranasal administration of TGF-β1 would be a novel therapeutic approach for depression.

A Novel Steroidogenic Factor-1 Antagonist, OR-449, as a Targeted Therapy for Adrenocortical Cancer

Abstract Adrenocortical carcinoma (ACC) is a rare cancer with an annual incidence of 0.7-2.0 cases per million in the US and EU. Surgical resection combined with adjuvant chemotherapy remains the primary treatment for patients with advanced disease. Although surgery is an option for most patients, oftentimes cases presenting with locally advanced or metastatic disease are not candidates for surgical resection. Furthermore, the non-specific adrenolytic and highly toxic FDA-approved chemotherapy drug, mitotane, is only marginally effective in adult and pediatric ACC. Steroidogenic factor-1 (SF-1 or NR5A1), an orphan nuclear receptor that is essential for the growth and development of the adrenal gland, is highly expressed in adult and pediatric ACC. Moreover, SF- is the major, active transcription factor in ACC (1,2). To address the unmet need for a targeted therapy in ACC, we identified potent small molecule SF-1 antagonists that block SF-1 transcriptional activity through the SF-1 ligand-binding domain (IC50 = 15-20 nM in a CHO cell reporter assay). In short-term dissociated cell cultures established from SJ-ACC3 (3), a pediatric ACC patient-derived tumor xenograft (PDX), OR-449 and a related SF-1 antagonist, OR-907S, blocked DNA synthesis as measured by inhibition of EdU incorporation in SF-1+ cells (IC50 = 500-600 nM, >80% efficacy at 10 μM) whereas OR-907R, the 100-fold less potent enantiomer of OR-907S, is nearly inactive. OR-449, which has >20% oral bioavailability and a long plasma half-life (> 9 h) in mouse, rat and dog, inhibited growth of SJ-ACC3 tumors grown in immunocompromised mice following daily oral dosing (30 mg/kg) for 4 weeks. SF-1-responsive genes, both down- and up-regulated, identified by RNAseq (by comparison of OR-907S and OR-907R in SJ-ACC3 dissociated cell culture), also responded to OR-449 in SJ-ACC3 xenografts following 7 days of dosing, indicating transcriptional regulation of SF-1 by OR-449. Importantly, in an exploratory 2-week mouse safety study, OR-449 showed no adverse effects when dosed up to 100 mg/kg. Taken together, these findings suggest that SF-1 antagonists could provide a safe and effective targeted therapy for ACC.

Superhydrophobic SiO2 /poly(vinylidene fluoride) composite membranes for the gravity-driven separation of drug enantiomers from emulsions

Membrane technology has been widely used to separate oil and water emulsions. However, the applications on this field has been limited to the simple separation of the oil and water phases from synthetic emulsions. This study explores the application of porous superhydrophobic composite SiO2/PVDF membranes to separate the emulsion and recover the organic phase containing S(+) ibuprofen ester after the enzyme-catalyzed kinetic resolution of racemic (R,S) ibuprofen by Candida rugosa lipase. Herein, 0.5 wt% - 2.0 wt% superhydrophobic SiO2 aerogel powder derived from methyltrimethoxysilane (MTMS) precursors was blended into the casting solution to obtain superhydrophobic composite membranes. The evaluation of the separation kinetics of the pristine and modified membranes were carried out by gravity-driven filtration of water-in-toluene, water-in-hexane, water-in-isooctane emulsions. Fast separation of the 50 mL feed emulsions was achieved because of the high porosity (>70%) and large pore size (>0.5 μm) of the membranes. Among the different conditions prepared, the optimal SiO2 loading was determined to be at 1.0 wt%. Thus, this composite membrane was chosen to separate the drug emulsion. According to the enzyme activity test, the permeate exhibited 0.12 absorbance units which is highly comparable to the absorbance of the controlled sample (0.10 units). This indicates that the membrane was able to effectively separate the emulsion and recover a high purity organic phase containing the potent enantiomer useful for pharmaceutical applications. Hence, it is realized in this study that membrane technology can be applied in pharmaceutical industries and can provide a more efficient and economical approach to separate drug emulsions.

SUN-LB23 Translational Feasibility of Steroidogenic Factor-1 Antagonists as a Novel Targeted Therapy for Adrenocortical Cancer

Adrenocortical carcinoma (ACC) is an aggressive cancer with devastating outcomes. ACC is usually locally advanced or metastatic at diagnosis and, despite tumor resection plus chemotherapy, has a high rate of recurrence. The 5-year survival rate among metastatic ACC patients is less than 15%. ACC responds poorly to the single FDA-approved drug, mitotane, which is non-specifically adrenolytic and highly toxic. Other chemotherapy regimens tested have been unsuccessful in improving overall survival. Steroidogenic factor-1 (SF-1 or NR5A1) is an orphan nuclear receptor essential for growth and development of the adrenal gland and is the major, active transcription factor in ACC (1,2). To address the need for a targeted therapy in ACC, we have identified potent small molecule SF-1 antagonists that block SF-1 transcriptional activity through the ligand-binding domain (IC50 = 15-20 nM in a CHO cell reporter assay). In short-term dissociated cell cultures established from SJ-ACC3 (3), a pediatric ACC patient-derived tumor xenograft (PDX), the SF-1 antagonists OR-907S and OR-070 blocked DNA synthesis as measured by inhibition of EdU incorporation in SF-1+ cells (IC50 = 500-600 nM, >80% efficacy at 10 μM) whereas OR-907R, the 100-fold less potent enantiomer of OR-907S, is nearly inactive. Because the SF-1 antagonist sensitivity of the dissociated SJ-ACC3 cells declines markedly with repeated passage of the PDX in immunocompromised (C.B-17 SCID) mice, we have utilized an alternative model system for evaluating tumor target engagement and growth inhibition: the rat Leydig tumor cell line (R2C), which is growth-inhibited by the SF-1 antagonists OR-907S and OR-070 in vitro (IC50 = 60-100 nM) and as a xenograft in immunocompromised mice (CD-1 nude). The SF-1-responsive gene signature identified by RNAseq in R2C cell cultures by comparison of OR-907S and OR-907R was replicated by OR-070 and other orally-bioavailable lead antagonists in R2C xenografts following 3 days of dosing, indicating engagement of SF-1 by these compounds. Significantly, R2C tumors were growth-inhibited following daily oral dosing for 4 weeks with OR-070 (10-30 mg/kg). These findings suggest that SF-1 antagonists could be a targeted therapy for ACC. OR-070 has >30% oral bioavailability in rat and dog, indicating that this structural class of SF-1 antagonists has potential for clinical development.

Development of a three-dimensional in vitro co-culture model to increase drug selectivity for humans.

Insulin resistance is a metabolic state where insulin sensitivity is lower than normal condition and strongly related to type 2 diabetes. However, an in vitro model mimicking insulin resistance is rare and thus screening drugs for insulin resistance severely depends on an in vivo model. Here, to increase anti-diabetic drug selectivity for humans, 3D ADMSCs and macrophages were co-cultured with in-house fabricated co-culture plates. 3D co-culture plates were designed to load ADMSCs and RAW264.7 cells containing hydrogels in separate wells while allowing cell-cell interaction with co-culturing media. Hydrogels were constructed using a 3D cell-printing system containing 20 mg/ml alginate, 0.5 mg/ml gelatin and 0.5 mg/ml type I collagen. Cells containing hydrogels in 3D co-culture plates were incubated for 10 min to allow stabilization before the experiment. 3D co-culture plates were incubated with the CaCl2 solution for 5 min to complete the cross linking of alginate hydrogel. Cells in 3D co-culture plates were cultured for up to 12 days depending on the experiment and wells containing adipocytes and macrophages were separated and used for assays. KR-1, KR-2 and KR-3 compounds were applied during differentiation (12 days) in 3D co-cultured mouse 3T3-L1 adipocytes and 3D co-cultured human ADMSCs. Glucose uptake assay using 2-DG6P and 2-NBDG and western blot analysis were performed to investigate changes of insulin resistance in the 3D co-cultured model for interspecies selectivity of drug screening. KR-1 (mouse potent enantiomer) and KR-3 (racemic mixture) showed improvement of 2-DG and 2-NBDG uptake compared with KR-2 (human potent enantiomer) in 3D co-cultured 3T3-L1 adipocytes. In connection with insulin resistance in a 3D 3T3-L1 co-cultured model, KR-1 and KR-3 showed improvement of insulin sensitivity compared to KR-2 by markedly increasing GLUT4 expression. In contrast to the result of 3D co-cultured 3T3-L1 adipocytes, KR-1 failed to significantly improve 2-DG and 2-NBDG uptake in 3D co-cultured ADMSC adipocytes. Results of 2-NBDG accumulation and western blot analysis also showed that KR-2 and KR-3 improved insulin sensitivity relatively better than KR-1. Our 3D co-culture model with/without 3D co-culture plates can successfully mimic insulin resistance while allowing investigation of the effects of anti-obesity or anti-diabetic drugs on human or mouse co-culturing cell type. This 3D co-culture system may accelerate screening of drugs for insulin resistance depending on species.

Enantioselective resolution of Rac-terbutaline and evaluation of optically pure R-terbutaline hydrochloride as an efficient anti-asthmatic drug.

Terbutaline is a β2 -adrenoceptor agonist for the treatment of asthma and chronic obstructive pulmonary disease (COPD). Among the two isomers of terbutaline (TBT 2), R-isomer was found to be the potent enantiomer in generating therapeutic effect, while S-isomer has been reported to show side effects. In this study, R-terbutaline hydrochloride (R-TBH 6) was synthesized through chiral resolution from the racemic terbutaline sulfate (rac-TBS 1) with 99.9% enantiomeric excess (ee) in good overall yield (53.6%). Further, R-TBH 6 nebulized solution was prepared in half dosage of Bricanyl®, which is a marketed product of racemic terbutaline and evaluated in vitro aerosol performance and in vivo anti-asthmatic effect on guinea pigs via. pulmonary delivery. From the investigation, it is evident that R-TBH 6 nebulized solution of half dosage performed similar fine aerosol characteristics and anti-asthmatic effect with Bricanyl®.

Quantitative chiral and achiral determination of ketamine and its metabolites by LC–MS/MS in human serum, urine and fecal samples

Ketamine (KET) is a widely used anesthetic drug which is metabolized by CYP450 enzymes to norketamine (n-KET), dehydronorketamine (DHNK), hydroxynorketamine (HNK) and hydroxyketamine (HK). Ketamine is a chiral compound and S-ketamine is known to be the more potent enantiomer. Here, we present the development and validation of three LC–MS/MS assays; the first for the quantification of racemic KET, n-KET and DHNK in human serum, urine and feces; the second for the separation and quantification of the S- and R-enantiomers of KET, n-KET and DHNK, and the third for separation and quantification of 2S,6S-hydroxynorketamine (2S,6S-HNK) and 2R,6R-hydroxynorketamine (2R,6R-HNK) in serum and urine with the ability to separate and detect 10 additional hydroxylated norketamine metabolites of racemic ketamine. Sample preparation was done by liquid-liquid extraction using methyl tert-butyl ether. For achiral determination of KET and its metabolites, an isocratic elution with ammonium acetate (pH 3.8; 5mM) and acetonitrile on a C18 column was performed. For the separation of S- and R-enantiomers of KET, n-KET and DHNK, a gradient elution was applied using a mobile phase of ammonium acetate (pH 7.5; 10mM) and isopropanol on the CHIRAL-AGP® column. The enantioselective separation of the HNK metabolites was done on the chiral column Lux®-Amylose-2 with a gradient method using ammonium acetate (pH 9; 5mM) and a mixture of isopropanol and acetonitrile (4:1). The mass spectrometric detection monitored for each analyte 2–3 mass/charge transitions. D4-ketamine and D4-n-KET were used as internal standards. The assays were successfully validated according to current bioanalytical guidelines and applied to a pilot study in one healthy volunteer. Compared to previously published methods, our assays have superior analytical features such as a lower amount of required matrix, faster sample preparation, shorter analytical run time and higher sensitivity (LLOQ up to 0.1ng/ml). Moreover, our assay enables for the first time the enantioselective determination of 2R,6R- and 2S,6S-HNK which were shown to be responsible for the promising antidepressant effects of ketamine.

Amphetamine activates calcium channels through dopamine transporter-mediated depolarization

Amphetamine (AMPH) and its more potent enantiomer S(+)AMPH are psychostimulants used therapeutically to treat attention deficit hyperactivity disorder and have significant abuse liability. AMPH is a dopamine transporter (DAT) substrate that inhibits dopamine (DA) uptake and is implicated in DA release. Furthermore, AMPH activates ionic currents through DAT that modify cell excitability presumably by modulating voltage-gated channel activity. Indeed, several studies suggest that monoamine transporter-induced depolarization opens voltage-gated Ca2+ channels (CaV), which would constitute an additional AMPH mechanism of action. In this study we co-express human DAT (hDAT) with Ca2+ channels that have decreasing sensitivity to membrane depolarization (CaV1.3, CaV1.2 or CaV2.2). Although S(+)AMPH is more potent than DA in transport-competition assays and inward-current generation, at saturating concentrations both substrates indirectly activate voltage-gated L-type Ca2+ channels (CaV1.3 and CaV1.2) but not the N-type Ca2+ channel (CaV2.2). Furthermore, the potency to achieve hDAT-CaV electrical coupling is dominated by the substrate affinity on hDAT, with negligible influence of L-type channel voltage sensitivity. In contrast, the maximal coupling-strength (defined as Ca2+ signal change per unit hDAT current) is influenced by CaV voltage sensitivity, which is greater in CaV1.3- than in CaV1.2-expressing cells. Moreover, relative to DA, S(+)AMPH showed greater coupling-strength at concentrations that induced relatively small hDAT-mediated currents. Therefore S(+)AMPH is not only more potent than DA at inducing hDAT-mediated L-type Ca2+ channel currents but is a better depolarizing agent since it produces tighter electrical coupling between hDAT-mediated depolarization and L-type Ca2+ channel activation.

Lactam and oxazolidinone derived potent 5-hydroxytryptamine 6 receptor antagonists

Lactam and oxazolidinone derived potent 5-hydroxytryptamine 6 (5-HT6) receptor antagonists have been disclosed. One potent member from the lactam series, racemic compound 14 (Ki of 2.6nM in binding assay, IC50 of 15nM in functional cAMP antagonism assay) was separated into corresponding enantiomers that displayed the effect of chirality on binding potency (Ki of 1.6nM and 3000nM, respectively). The potent enantiomer displayed an IC50 of 8nM in cAMP antagonism assay, selectivity against a number of family members as well as brain permeability in rats after 6h post oral administration.

1‐Thia‐4,7‐diaza‐spiro[4.4]nonane‐3,6‐dione: A Structural Motif for 5‐hydroxytryptamine 6 Receptor Antagonism

A series of potent 5-hydroxytryptamine 6 (5-HT₆) receptor antagonists based on 1-thia-4,7-diaza-spiro[4.4]nonane-3,6-dione motif has been disclosed. Enantiomers of potent racemate compound 8a (K(i) = 26 nM) displayed difference in activity (K(i) of 15 nM versus 855 nM) signaling the influence of the stereochemistry of the chiral center on potency. In addition, the potent enantiomer displayed significant selectivity in biological activities over several related family members.

The Discovery of Novel Potent trans-3,4-Disubstituted Pyrrolidine Inhibitors of the Human Aspartic Protease Renin from in Silico Three-Dimensional (3D) Pharmacophore Searches

The small-molecule trans-3,4-disubstituted pyrrolidine 6 was identified from in silico three-dimensional (3D) pharmacophore searches based on known X-ray structures of renin-inhibitor complexes and demonstrated to be a weakly active inhibitor of the human enzyme. The unexpected binding mode of the more potent enantiomer (3S,4S)-6a in an extended conformation spanning the nonprime and S1' pockets of the recombinant human (rh)-renin active site was elucidated by X-ray crystallography. Initial structure-activity relationship work focused on modifications of the hydrophobic diphenylamine portion positioned in S1 and extending toward the S2 pocket. Replacement with an optimized P3-P1 pharmacophore interacting to the nonsubstrate S3(sp) cavity eventually resulted in significantly improved in vitro potency and selectivity. The prototype analogue (3S,4S)-12a of this new class of direct renin inhibitors exerted blood pressure lowering effects in a hypertensive double-transgenic rat model after oral administration.

Beneficial effect of (–)Schisandrin B against 3‐nitropropionic acid‐induced cell death in PC12 cells

Huntington's disease (HD) is characterized by the dysfunction of mitochondrial energy metabolism, which is associated with the functional impairment of succinate dehydrogenase (mitochondrial complex II), and pyruvate dehydrogenase (PDH). Treatment with 3-nitropropionic acid (3-NP), a potent irreversible inhibitor of succinate dehydrogenase, replicates most of the pathophysiological features of HD. In the present study, we investigated the effect of (-)schisandrin B [(-)Sch B, a potent enantiomer of schisandrin B] on 3-NP-induced cell injury in rat differentiated neuronal PC12 cells. The 3-NP caused cell necrosis, as assessed by lactate dehydrogenase (LDH) leakage, and mitochondrion-dependent cell apoptosis, as assessed by caspase-3 and caspase-9 activation, in differentiated PC12 cells. The cytotoxicity induced by 3-NP was associated with a depletion of cellular reduced glutathione (GSH) as well as the activation of redox-sensitive c-Jun N-terminal kinase (JNK) pathway and the inhibition of PDH. (-)Sch B pretreatment (5 and 15 μM) significantly reduced the extent of necrotic and apoptotic cell death in 3-NP-challenged cells. The cytoprotection afforded by (-)Sch B pretreatment was associated with the attenuation of 3-NP-induced GSH depletion as well as JNK activation and PDH inhibition. (-)Sch B pretreatment enhanced cellular glutathione redox status and ameliorated the 3-NP-induced cellular energy crisis, presumably by suppressing the activated JNK-mediated PDH inhibition, thereby protecting against necrotic and apoptotic cell death in differentiated PC12 cells.

Chiral proton catalysis of secondary nitroalkane additions to azomethine: synthesis of a potent GlyT1 inhibitor

The first enantioselective synthesis of a potent GlyT1 inhibitor is described. A 3-nitroazetidine donor is used in an enantioselective aza-Henry reaction catalyzed by a bis(amidine)-triflic acid salt organocatalyst, delivering the key intermediate with 92% ee. This adduct is reductively denitrated and converted to the target through a short sequence, thereby allowing assignment of the absolute configuration of the more potent enantiomer.

Prooxidant-Induced Glutathione Antioxidant Response <i>in Vitro</i> and <i>in Vivo</i>: A Comparative Study between Schisandrin B and Curcumin

We investigated whether two naturally-occurring prooxidants, namely, schisandrin B (Sch B) and curcumin, and a synthetic prooxidant, menadione, can invariably elicit cyto/hepatoprotective responses against oxidant-induced injury. Results showed that (-)Sch B (a potent enantiomer of Sch B, 15 μM), curcumin (7.5 μM) and menadione (2 μM) induced a similar extent of reactive oxygen species production in AML12 cells. The relative potencies of cytoprotection in vitro were in a descending order of curcumin>menadione>(-)Sch B, which were parallel to the extent of stimulation in cellular reduced glutathione level. We further examined their hepatoprotection in vivo. Pretreatment with Sch B (800 mg/kg) and curcumin (737 mg/kg), but not menadione (344 mg/kg), protected against CCl(4) toxicity, with the degree of protection afforded by Sch B being much larger than that of curcumin. The attenuated hepatoprotection afforded by curcumin may be attributed to its low bioavailability in vivo. This postulation is supported by the findings that intraperitoneal injections of Sch B (400 mg/kg) and curcumin (368 mg/kg) and the long term, low dose treatment with Sch B (20 mg/kg/d×15) and curcumin (18 mg/kg/d×15) induced glutathione antioxidant response and hepatoprotection to similar extents in vivo. The inability of menadione to induce hepatoprotection may be related to its extensive intestinal metabolism and/or hepatotoxicity. Taken together, prooxidants can invariably induce the glutathione antioxidant response and confer cytoprotection in vitro. Whether or not the prooxidant can produce a similar response in vivo would depend on its bioavailability and potential toxic effect.

Effect of hyperlipidemia on ketoconazole–midazolam drug–drug interaction in rat

Hyperlipidemia (HL) was previously shown to lower liver uptake of the more potent (-) enantiomer of ketoconazole (KTZ) in rat. The current study examined the possible modifying influence of experimental HL on a KTZ pharmacokinetic interaction with midazolam (MDZ). Normolipidemic and hyperlipidemic rats were administered a single intravenous dose of MDZ (5 mg/kg) with or without a single oral dose of racemic KTZ (40 mg/kg). Serial blood samples were collected over 8 h following MDZ injections via jugular vein cannulas. Plasma was jointly assayed for MDZ and KTZ concentrations using a validated assay. MDZ mean clearance (CL) was unchanged by KTZ coadministration. HL caused a significantly 61% lower MDZ-unbound fraction and decreases in volume of distribution (VD) but by itself had no effect on MDZ CL. This suggested that MDZ could bind to lipoproteins. With KTZ coadministered to hyperlipidemic rats, there were significant decreases in MDZ CL and VD. HL caused a decrease in unbound plasma fraction of oral KTZ but no significant difference in its pharmacokinetics. HL caused a more pronounced KTZ-associated inhibition of MDZ CL. This may be related to the decrease of MDZ's unbound fraction and perhaps to attenuation of CYP3A by HL in the rat.