Search

Enhancing stress resilience in at-risk populations could significantly reduce the incidence of stress-related psychiatric disorders. We have previously reported that the administration of (R,S)-ketamine prevents stress-induced depressive-like behavior in male mice, perhaps by altering α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated transmission in hippocampal CA3. However, it is still unknown whether metabolites of (R,S)-ketamine can be prophylactic in both sexes. We administered (R,S)-ketamine or its metabolites (2R,6R)-hydroxynorketamine ((2R,6R)-HNK) and (2S,6S)-hydroxynorketamine ((2S,6S)-HNK) at various doses 1 week before one of a number of stressors in male and female 129S6/SvEv mice. Patch clamp electrophysiology was used to determine the effect of prophylactic drug administration on glutamatergic activity in CA3. To examine the interaction between ovarian hormones and stress resilience, female mice also underwent ovariectomy (OVX) surgery and a hormone replacement protocol prior to drug administration. (2S,6S)-HNK and (2R,6R)-HNK protected against distinct stress-induced behaviors in both sexes, with (2S,6S)-HNK attenuating learned fear in male mice, and (2R,6R)-HNK preventing stress-induced depressive-like behavior in both sexes. (R,S)-ketamine and (2R,6R)-HNK, but not (2S,6S)-HNK, attenuated large-amplitude AMPAR-mediated bursts in hippocampal CA3. All three compounds reduced N-methyl-D-aspartate receptor (NMDAR)-mediated currents 1 week after administration. Furthermore, ovarian-derived hormones were necessary for and sufficient to restore (R,S)-ketamine- and (2R,6R)-HNK-mediated prophylaxis in female mice. Our data provide further evidence that resilience-enhancing prophylactics may alter AMPAR-mediated glutamatergic transmission in CA3. Moreover, we show that prophylactics against stress-induced depressive-like behavior can be developed in a sex-specific manner and demonstrate that ovarian hormones are necessary for the prophylactic efficacy of (R,S)-ketamine and (2R,6R)-HNK in female mice.

Phosphodiesterase 5 (PDE5) hydrolyzes cyclic guanosine monophosphate (cGMP) leading to increased levels of the cAMP response element binding protein (CREB), a transcriptional factor involved with learning and memory processes. We previously reported potent quinoline-based PDE5 inhibitors (PDE5Is) for the treatment of Alzheimer’s disease (AD). However, the low aqueous solubility rendered them undesirable drug candidates. Here we report a series of novel PDE5Is with two new scaffolds, 1,2,3,4-tetrahydrobenzo[b][1,6]naphthyridine and 2,3-dihydro-1H-pyrrolo[3,4-b]quinolin-1-one. Among them, compound 6c, 2-acetyl-10-((3-chloro-4-methoxybenzyl)amino)-1,2,3,4-tetrahydrobenzo[b][1,6]naphthyridine-8-carbonitrile, the most potent compound, has an excellent in vitro IC(50) (0.056 nM) and improved aqueous solubility as well as good efficacy in a mouse model of AD. Furthermore, we are proposing two plausible binding modes obtained through in silico docking, which provide insights into the structural basis of the activity of the two series of compounds reported herein.

Nitric oxide (NO) is a gaseous molecule that plays a multifactorial role in several cellular processes. In the central nervous system, the NO dual nature in neuroprotection and neurotoxicity has been explored to unveil its involvement in Alzheimer's disease (AD). A growing body of research shows that the activation of the NO signaling pathway leading to the phosphorylation of the transcription factor cyclic adenine monophosphate responsive element binding protein (CREB) (so-called NO/cGMP/PKG/CREB signaling pathway) ameliorates altered neuroplasticity and memory deficits in AD animal models. In addition to NO donors, several other pharmacological agents, such as phosphodiesterase 5 (PDE5) inhibitors have been used to activate the pathway and rescue memory disorders. PDE5 inhibitors, including sildenafil, tadalafil and vardenafil, are marketed for the treatment of erectile dysfunction and arterial pulmonary hypertension due to their vasodilatory properties. The ability of PDE5 inhibitors to interfere with the NO/cGMP/PKG/CREB signaling pathway by increasing the levels of cGMP has prompted the hypothesis that PDE5 inhibition might be used as an effective therapeutic strategy for the treatment of AD. To this end, newly designed PDE5 inhibitors belonging to different chemical classes with improved pharmacologic profile (e.g. higher potency, improved selectivity, and blood-brain barrier penetration) have been synthesized and evaluated in several animal models of AD. In addition, recent medicinal chemistry effort has led to the development of agents concurrently acting on the PDE5 enzyme and a second target involved in AD. Both marketed and investigational PDE5 inhibitors have shown to reverse cognitive defects in young and aged wild type mice as well as transgenic mouse models of AD and tauopathy using a variety of behavioral tasks. These studies confirmed the therapeutic potential of PDE5 inhibitors as cognitive enhancers. However, clinical studies assessing cognitive functions using marketed PDE5 inhibitors have not been conclusive. Drug discovery efforts by our group and others are currently directed towards the development of novel PDE5 inhibitors tailored to AD with improved pharmacodynamic and pharmacokinetic properties. In summary, the present perspective reports an overview of the correlation between the NO signaling and AD, as well as an outline of the PDE5 inhibitors used as an alternative approach in altering the NO pathway leading to an improvement of learning and memory. The last two sections describe the preclinical and clinical evaluation of PDE5 inhibitors for the treatment of AD, providing a comprehensive analysis of the current status of the AD drug discovery efforts involving PDE5 as a new therapeutic target.

BACKGROUNDFemales are more likely than males to develop major depressive disorder (MDD) after exposure to stress. We previously reported that the administration of (R,S)-ketamine before stress can prevent stress-induced depressive-like behavior in male mice but have yet to assess efficacy in female mice or for other compounds, such as the metabolites of (R,S)-ketamine.METHODSWe administered (R,S)-ketamine or its metabolites (2R,6R)-hydroxynorketamine ((2R,6R)-HNK) and (2S,6S)-HNK at various doses 1 week before one of a number of stressors, including contextual fear conditioning (CFC), learned helplessness (LH), and chronic immobilization stress (CIS), in male and female 129S6/SvEv mice. To examine the interaction between ovarian hormones and stress resilience, female mice also underwent ovariectomy surgery (OVX) and a hormone replacement protocol prior to drug administration.RESULTS(R,S)-ketamine and (2S,6S)-HNK, but not (2R,6R)-HNK, attenuated learned fear in male mice. (R,S)-ketamine and (2R,6R)-HNK, but not (2S,6S)-HNK, significantly reduced stress-induced depressive-like behavior in male and female mice. (R,S)-ketamine and (2R,6R)-HNK) were prophylactically effective at a lower dose (10 mg/kg and 0.025 mg/kg, respectively) in female mice than in male mice (30 mg/kg and 0.075 mg/kg, respectively). Moreover, ovarian-derived hormones were necessary and sufficient for prophylaxis in female mice.CONCLUSIONSOur results suggest that prophylactics against stress-induced depressive-like behavior can be developed in a sex-specific manner and that ovarian hormones mediate prophylactic efficacy in females. To our knowledge, this is the first demonstration of the prophylactic efficacy of the metabolites of (R,S)-ketamine in male and female mice.

Supplemental Digital Content is Available in the Text. A novel and potent protein kinase G-1α (PKG-1α) inhibitor is used to demonstrate the important roles of PKG in capsaicin-induced acute pain and in persistent inflammatory pain., Activating PKG-1α induces a long-term hyperexcitability (LTH) in nociceptive neurons. Since the LTH correlates directly with chronic pain in many animal models, we tested the hypothesis that inhibiting PKG-1α would attenuate LTH-mediated pain. We first synthesized and characterized compound N46 (N-((3R,4R)-4-(4-(2-fluoro-3-methoxy-6-propoxybenzoyl)benzamido)pyrrolidin-3-yl)-1H-indazole-5-carboxamide). N46 inhibits PKG-1α with an IC50 of 7.5 nmol, was highly selective when tested against a panel of 274 kinases, and tissue distribution studies indicate that it does not enter the CNS. To evaluate its antinociceptive potential, we used 2 animal models in which the pain involves both activated PKG-1α and LTH. Injecting complete Freund's adjuvant (CFA) into the rat hind paw causes a thermal hyperalgesia that was significantly attenuated 24 hours after a single intravenous injection of N46. Next, we used a rat model of osteoarthritic knee joint pain and found that a single intra-articular injection of N46 alleviated the pain 14 days after the pain was established and the relief lasted for 7 days. Thermal hyperalgesia and osteoarthritic pain are also associated with the activation of the capsaicin-activated transient receptor protein vanilloid-1 (TRPV1) channel. We show that capsaicin activates PKG-1α in nerves and that a subcutaneous delivery of N46 attenuated the mechanical and thermal hypersensitivity elicited by exposure to capsaicin. Thus, PKG-1α appears to be downstream of the transient receptor protein vanilloid-1. Our studies provide proof of concept in animal models that a PKG-1α antagonist has a powerful antinociceptive effect on persistent, already existing inflammatory pain. They further suggest that N46 is a valid chemotype for the further development of such antagonists.

cGMP elevation does not influence motor, visible or exploratory behavior in mice treated with oTau. Evaluation of speed, latency, sensory threshold and exploratory behavior in mice treated with vehicle, oTau, 7a, 7a + oTau, 8pCPT-cGMP, 8pCPT-cGMP, +oTau. (PDF 171 kb)

The prevalence of obesity-induced type 2 diabetes (T2D) is increasing worldwide, and new treatment strategies are needed. We recently discovered that obesity activates a previously unknown pathway that promotes both excessive hepatic glucose production (HGP) and defective insulin signaling in hepatocytes, leading to exacerbation of hyperglycemia and insulin resistance in obesity. At the hub of this new pathway is a kinase cascade involving calcium/calmodulin-dependent protein kinase II (CaMKII), p38α mitogen-activated protein kinase (MAPK), and MAPKAPK2/3 (MK2/3). Genetic-based inhibition of these kinases improves metabolism in obese mice. Here, we report that treatment of obese insulin-resistant mice with an allosteric MK2/3 inhibitor, compound (cmpd) 28, ameliorates glucose homeostasis by suppressing excessive HGP and enhancing insulin signaling. The metabolic improvement seen with cmpd 28 is additive with the leading T2D drug, metformin, but it is not additive with dominant-negative MK2, suggesting an on-target mechanism of action. Allosteric MK2/3 inhibitors represent a potentially new approach to T2D that is highly mechanism based, has links to human T2D, and is predicted to avoid certain adverse effects seen with current T2D drugs.

Equilibrative transporters are potential drug targets; however, most functional assays involve radioactive substrate uptake that is unsuitable for high-throughput screens (HTS). We developed a robust yeast-based growth assay that is potentially applicable to many equilibrative transporters. As proof of principle, we applied our approach to Equilibrative Nucleoside Transporter 1 of the malarial parasite Plasmodium falciparum (PfENT1). PfENT1 inhibitors might serve as novel antimalarial drugs since PfENT1-mediated purine import is essential for parasite proliferation. To identify PfENT1 inhibitors, we screened 64 560 compounds and identified 171 by their ability to rescue the growth of PfENT1-expressing fui1Δ yeast in the presence of a cytotoxic PfENT1 substrate, 5-fluorouridine (5-FUrd). In secondary assays, nine of the highest activity compounds inhibited PfENT1-dependent growth of a purine auxotrophic yeast strain with adenosine as the sole purine source (IC50 0.2-2 μM). These nine compounds completely blocked [(3)H]adenosine uptake into PfENT1-expressing yeast and erythrocyte-free trophozoite-stage parasites (IC50 5-50 nM), and inhibited chloroquine-sensitive and -resistant parasite proliferation (IC50 5-50 μM). Wild-type (WT) parasite IC50 values were up to 4-fold lower compared to PfENT1-knockout (pfent1Δ) parasites. pfent1Δ parasite killing showed a delayed-death phenotype not observed with WT. We infer that, in parasites, the compounds inhibit both PfENT1 and a secondary target with similar efficacy. The secondary target identity is unknown, but its existence may reduce the likelihood of parasites developing resistance to PfENT1 inhibitors. Our data support the hypothesis that blocking purine transport through PfENT1 may be a novel and compelling approach for antimalarial drug development.

The identification of ligands that bind the protein Neutrophil Gelatinase-Associated Lipocalin (NGAL, Siderocalin, Lipocalin-2) have helped to elucidate its function. NGAL-Siderocalin binds and sequesters the iron loaded bacterial siderophore enterochelin (Ent), defining the protein as an innate immune effector. Simple metabolic catechols can also form tight complexes with NGAL-Siderocalin and ferric iron, suggesting that the protein may act as an iron scavenger even in the absence of Ent. While different catechols have been detected in human urine, they have not been directly purified from a biofluid and demonstrated to ligate iron with NGAL-Siderocalin. This paper describes a "natural products" approach to identify small molecules that mediate iron binding to NGAL-Siderocalin. A 10K filtrate of human urine was subjected to multiple steps of column chromatography and reverse-phase HPLC, guided by NGAL-Siderocalin-iron binding assays and LC-MS detection. The co-factor forming a ternary structure with iron and NGAL-Siderocalin was identified as authentic simple catechol (dihydroxybenze) by ESI-HR-Mass, UV, and NMR spectrometric analysis. Comparison of the binding strengths of different catechols demonstrated that the vicinal-dihydroxyl groups were the key functional groups and that steric compatibilities of the catechol ring have the strongest effect on binding. Although catechol was a known NGAL-Siderocalin co-factor, our purification directly confirmed its presence in urine as well as its capacity to serve as an iron trap with NGAL-Siderocalin.

We have previously identified and reported several potent piperidine-derived amide inhibitors of the human soluble epoxide hydrolase (sEH) enzyme. The inhibition of this enzyme leads to elevated levels of epoxyeicosatrienoic acids (EETs), which are known to possess anti-inflammatory, vasodilatory, and anti-fibrotic effects. Herein, we report the synthesis of 9 analogs of the lead sEH inhibitor and the follow-up structure-activity relationship and liver microsome stability studies. Our findings show that isosteric modifications that lead to significant alterations in the steric and electronic properties at a specific position in the molecule can reduce the efficacy by up to 75-fold. On the other hand, substituting hydrogen with deuterium produces a notable increase (∼30%) in the molecules' half-lives in both rat and human microsomes, while maintaining sEH inhibition potency. These data highlight the utility of isosteric replacement for improving bioavailability, and the newly-synthesized inhibitor structures may thus, serve as a starting point for preclinical development. Our docking study reveals that in the catalytic pocket of sEH, these analogs are in proximity of the key amino acids involved in hydrolysis of EETs.

Phosphoinositide 3-kinase (PI3K) and the proteasome pathway are both involved in activating the mechanistic target of rapamycin (mTOR). Because mTOR signaling is required for initiation of messenger RNA translation, we hypothesized that cotargeting the PI3K and proteasome pathways might synergistically inhibit translation of c-Myc. We found that a novel PI3K δ isoform inhibitor TGR-1202, but not the approved PI3Kδ inhibitor idelalisib, was highly synergistic with the proteasome inhibitor carfilzomib in lymphoma, leukemia, and myeloma cell lines and primary lymphoma and leukemia cells. TGR-1202 and carfilzomib (TC) synergistically inhibited phosphorylation of the eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1), leading to suppression of c-Myc translation and silencing of c-Myc-dependent transcription. The synergistic cytotoxicity of TC was rescued by overexpression of eIF4E or c-Myc. TGR-1202, but not other PI3Kδ inhibitors, inhibited casein kinase-1 ε (CK1ε). Targeting CK1ε using a selective chemical inhibitor or short hairpin RNA complements the effects of idelalisib, as a single agent or in combination with carfilzomib, in repressing phosphorylation of 4E-BP1 and the protein level of c-Myc. These results suggest that TGR-1202 is a dual PI3Kδ/CK1ε inhibitor, which may in part explain the clinical activity of TGR-1202 in aggressive lymphoma not found with idelalisib. Targeting CK1ε should become an integral part of therapeutic strategies targeting translation of oncogenes such as c-Myc.

Phosphodiesterase type 5 (PDE5) mediates the degradation of cGMP in a variety of tissues including brain. Recent studies have demonstrated the importance of the nitric oxide/cGMP/cAMP-responsive element-binding protein (CREB) pathway to the process of learning and memory. Thus, PDE5 inhibitors (PDE5Is) are thought to be promising new therapeutic agents for the treatment of Alzheimer's disease (AD), a neurodegenerative disorder characterized by memory loss. To explore this possibility, a series of quinoline derivatives were synthesized and evaluated. We found that compound 7a selectively inhibits PDE5 with an IC50 of 0.27 nM and readily crosses the blood brain barrier. In an in vivo mouse model of AD, compound 7a rescues synaptic and memory defects. Quinoline-based, CNS-permeant PDE5Is have potential for AD therapeutic development.

A series of potent amide non-urea inhibitors of soluble epoxide hydrolase (sEH) is disclosed. The inhibition of soluble epoxide hydrolase leads to elevated levels of epoxyeicosatrienoic acids (EETs), and thus inhibitors of sEH represent one of a novel approach to the development of vasodilatory and anti-inflammatory drugs. Structure-activities studies guided optimization of a lead compound, identified through high-throughput screening, gave rise to sub-nanomolar inhibitors of human sEH with stability in human liver microsomal assay suitable for preclinical development.

Many proteins have been proposed to act as surrogate markers of organ damage, yet for many candidates the essential biomarker characteristics that link the protein to the injured organ have not yet been described. We generated an Ngal reporter mouse by inserting a double-fusion reporter gene encoding luciferase-2 and mCherry (Luc2-mC) into the Ngal (Lcn2) locus. The Ngal-Luc2-mC reporter accurately recapitulated the endogenous message and illuminated injuries in vivo in real time. In the kidney, Ngal-Luc2-mC imaging showed a sensitive, rapid, dose-dependent, reversible, and organ- and cell-specific relationship with tubular stress, which correlated with the level of urinary Ngal (uNgal). Unexpectedly, specific cells of the distal nephron were the source of uNgal. Cells isolated from Ngal-Luc2-mC mice also revealed both the onset and the resolution of the injury, and the actions of NF-κB inhibitors and antibiotics during infection. Thus, imaging of Ngal-Luc2-mC mice and cells identified injurious and reparative agents that affect kidney damage.

Nuclear factor kappa B (NF-κB) is an important transcription factor. Aberrant regulation of the NF-κB pathway is frequently observed in a number of major ailments such as cancer and inflammatory diseases. Hence NF-κB modulators have been intensely pursued for their potential therapeutic applications. Numerous reviews have described recent progress in the development of these agents. More recently, a variety of structurally and functionally novel small molecules, identified through high-throughput screens conducted within the Molecular Libraries Screening Center Network (MLSCN) of the NIH Roadmap for Medical Research, have been added to the current list of NF-κB regulators. This review will discuss the inhibitors and activators newly discovered by Columbias Molecular Libraries Screening Center (MLSC) using a well-designed and stable cellular assay.

An IKKbeta inhibitor reported to block NF-kappaB transcriptional activities in Jurkat T cells, was found to enhance NF-kappaB translocation in HUVEC cells. These studies suggested a noncanonical NF-kappaB signaling pathway independent of IKKbeta in HUVEC cells.

Vesicular monoamine transporter type 2 (VMAT2) is a newly emerging target for both diagnostic and therapeutic applications in diabetes mellitus. In pursuit of novel VMAT2 antagonists, we identified a potent hypoglycemic agent with a novel dihydropyridone scaffold. Several analogs were designed and synthesized. A preliminary structure activity relationship (SARs) showed that the dihydropyridone scaffold is required for the activity.

An efficient synthesis of a series of N -8-quinolinyl benzenesultams as novel NF-κB inhibitors was described via diazotization-induced cyclization of easily accessible N -8-quinolinyl-2-aminobenzenesulfonamides.

During exercise, defects in calcium (Ca 2+ ) release have been proposed to impair muscle function. Here, we show that during exercise in mice and humans, the major Ca 2+ release channel required for excitation–contraction coupling (ECC) in skeletal muscle, the ryanodine receptor (RyR1), is progressively PKA-hyperphosphorylated, S -nitrosylated, and depleted of the phosphodiesterase PDE4D3 and the RyR1 stabilizing subunit calstabin1 (FKBP12), resulting in “leaky” channels that cause decreased exercise tolerance in mice. Mice with skeletal muscle-specific calstabin1 deletion or PDE4D deficiency exhibited significantly impaired exercise capacity. A small molecule (S107) that prevents depletion of calstabin1 from the RyR1 complex improved force generation and exercise capacity, reduced Ca 2+ -dependent neutral protease calpain activity and plasma creatine kinase levels. Taken together, these data suggest a possible mechanism by which Ca 2+ leak via calstabin1-depleted RyR1 channels leads to defective Ca 2+ signaling, muscle damage, and impaired exercise capacity.

Sitosterolemia is a disease characterized by very high levels of sitosterol and other plant sterols and premature atherothrombotic vascular disease. One theory holds that plant sterols can directly promote atherosclerosis, but the mechanism is not known. Unesterified, or "free," cholesterol (FC) is a potent inducer of macrophage death, which causes plaque necrosis, a precursor to atherothrombosis. FC-induced macrophage death, however, requires dysfunction of the sterol esterifying enzyme acyl-coenzyme A-cholesterol acyltransferase (ACAT), which likely occurs slowly during lesion progression. In contrast, plant sterols are relatively poorly esterified by ACAT, and so they may cause macrophage death and plaque necrosis in an accelerated manner. In support of this hypothesis, we show here that macrophages incubated with sitosterol-containing lipoproteins accumulate free sterols and undergo death in the absence of an ACAT inhibitor. As with FC loading, sitosterol-induced macrophage death requires sterol trafficking to the endoplasmic reticulum, and sitosterol-enriched endoplasmic reticulum membranes show evidence of membrane protein dysfunction. However, whereas FC induces caspase-dependent apoptosis through activation of the unfolded protein response and JNK, sitosterol-induced death is caspase-independent and involves neither the unfolded protein response nor JNK. Rather, cell death shows signs of necroptosis and autophagy and is suppressed by inhibitors of both processes. These data establish two new concepts. First, a relatively subtle change in sterol structure fundamentally alters the type of death program triggered in macrophages. Understanding the basis of this alteration should provide new insights into the molecular basis of death pathway signaling. Second, sitosterol-induced macrophage death does not require ACAT dysfunction and so may occur in an accelerated fashion. Pending future in vivo studies, this concept may provide at least one mechanism for accelerated plaque necrosis and atherothrombotic disease in patients with sitosterolemia.

Catalytic antibody 15A10 hydrolyzes the benzoyl ester of cocaine to form the nonpsychoactive metabolites benzoic acid and ecgonine methylester. Here, we report biochemical and structural studies that characterize the catalytic mechanism. The crystal structure of the cocaine-hydrolyzing monoclonal antibody (mAb) 15A10 has been determined at 2.35 A resolution. The binding pocket is fairly shallow and mainly hydrophobic but with a cluster of three hydrogen-bond donating residues (TrpL96, AsnH33, and TyrH35). Computational docking of the transition state analogue (TSA) indicates that these residues are appropriately positioned to coordinate the phosphonate moiety of the TSA and, hence, form an oxyanion hole. Tyrosine modification of the antibody with tetranitromethane reduced hydrolytic activity to background level. The contribution from these and other residues to catalysis and TSA binding was explored by site-directed mutagenesis of 15A10 expressed in a single chain fragment variable (scFv) format. The TyrH35Phe mutant had 4-fold reduced activity, and TrpL96Ala, TrpL96His, and AsnH33Ala mutants were all inactive. Comparison with an esterolytic antibody D2.3 revealed a similar arrangement of tryptophan, asparagine, and tyrosine residues in the oxyanion hole that stabilizes the transition state for ester hydrolysis. Furthermore, the crystal structure of the bacterial cocaine esterase (cocE) also showed that the cocE employs a tyrosine hydroxyl in the oxyanion hole. Thus, the biochemical and structural data are consistent with the catalytic antibody providing oxyanion stabilization as its major contribution to catalysis.

Ventricular arrhythmias can cause sudden cardiac death (SCD) in patients with normal hearts and in those with underlying disease such as heart failure. In animals with heart failure and in patients with inherited forms of exercise-induced SCD, depletion of the channel-stabilizing protein calstabin2 (FKBP12.6) from the ryanodine receptor–calcium release channel (RyR2) complex causes an intracellular Ca 2+ leak that can trigger fatal cardiac arrhythmias. A derivative of 1,4-benzothiazepine (JTV519) increased the affinity of calstabin2 for RyR2, which stabilized the closed state of RyR2 and prevented the Ca 2+ leak that triggers arrhythmias. Thus, enhancing the binding of calstabin2 to RyR2 may be a therapeutic strategy for common ventricular arrhythmias.

Cocaine mediates its reinforcing and toxic actions through a “loss of function” effect at multiple receptors. The difficulties inherent in blocking a pleiotropic blocker pose a great obstacle for the classical receptor–antagonist approach and have contributed to the failure (to date) to devise specific treatments for cocaine overdose and addiction. As an alternative, we have embarked on an investigation of catalytic antibodies, a programmable class of artificial enzyme, as “peripheral blockers”—agents designed to bind and degrade cocaine in the circulation before it partitions into the central nervous system to exert reinforcing or toxic effects. We synthesized transition-state analogs of cocaine's hydrolysis at its benzoyl ester, immunized mice, prepared hybridomas and developed the first anticocaine catalytic antibodies with the capacity to degrade cocaine to nonreinforcing, nontoxic products. We subsequently identified several families of anticocaine catalytic antibodies and found that the most potent antibody possessed sufficient activity to block cocaine-induced reinforcement, organ dysfunction and sudden death in rodent models of addiction, toxicity and overdose, respectively. With the potential to promote cessation of use, prolong abstinence and provide a treatment for acute overdose, the artificial enzyme approach comprehensively responds to the problem of cocaine.

Cocaine covalently modifies proteins through a reaction in which the methyl ester of cocaine acylates the ɛ-amino group of lysine residues. This reaction is highly specific in vitro , because no other amino acid reacts with cocaine, and only cocaine's methyl ester reacts with the lysine side chain. Covalently modified proteins were present in the plasma of rats and human subjects chronically exposed to cocaine. Modified endogenous proteins are immunogenic, and specific antibodies were elicited in mouse and detected in the plasma of human subjects. Covalent modification of proteins could explain cocaine's autoimmune effects and provide a new biochemical approach to cocaine's long-term actions.

A quinazoline that decreases polyglutamine aggregate burden in a cell-based assay was identified from a high-throughput screen of a chemical-compound library, provided by the NIH Molecular Libraries Small Molecule Repository (MLSMR). A structure and activity study yielded leads with submicromolar potency.

The spontaneous hydrolysis of the methyl-ester group of cocaine (1) in vivo contributes to the metabolic clearance of the drug in man. Neighboring-group participation by the tropane N-atom of cocaine in this hydrolysis was suggested by the normal stability of the methyl-ester groups of pseudococaine and N-acylnorcocaine. For cocaine, the relative rate of methyl-ester to benzoyl-ester hydrolysis was ca. 10:1 at pH≤7.4, and, although absolute rates increased with increasing pH, their ratio collapsed at pH>pKa (8.6). These data are consistent with intramolecular acid catalysis of alkaline hydrolysis of the cocaine methyl-ester group under physiologic conditions.

Accumulated evidence indicates that the interconversion of iron between ferric (Fe(3+)) and ferrous (Fe(2+)) can be realized through interaction with reactive oxygen species in the Fenton and Haber-Weiss reactions and thereby physiologically effects redox cycling. The imbalance of iron and ROS may eventually cause tissue damage such as renal proximal tubule injury and necrosis. Many approaches were exploited to ameliorate the oxidative stress caused by the imbalance. (-)-Epigallocatechin-3-gallate, the most active and most abundant catechin in tea, was found to be involved in the protection of a spectrum of renal injuries caused by oxidative stress. Most of studies suggested that EGCG works as an antioxidant. In this paper, Multivariate analysis of the LC-MS data of tea extracts and binding assays showed that the tea polyphenol EGCG can form stable complex with iron through the protein Ngal, a biomarker of acute kidney injury. UV-Vis and Luminescence spectrum methods showed that Ngal can inhibit the chemical reactivity of iron and EGCG through forming an Ngal-EGCG-iron complex. In thinking of the interaction of iron and ROS, we proposed that EGCG may work as both antioxidant and Ngal binding siderphore in protection of kidney from injuries.

Inhibition of soluble epoxide hydrolase (sEH) has been proposed as a new pharmaceutical approach for treating hypertension and vascular inflammation. The most potent sEH inhibitors reported in literature to date are urea derivatives. However, these compounds have limited pharmacokinetic profiles. We investigated non-urea amide derivatives as sEH inhibitors and identified a potent human sEH inhibitor 14-34 having potency comparable to urea-based inhibitors.

The first general stereocontrolled approach to C-3 alkyl analogs of cocaine is described. The route utilized versatile intermediates 2β-hydroxymethyl-3α-cyanotropane and 2β-hydroxymethyl-3β-cyanotropane.

The lymphoid tyrosine phosphatase (Lyp, PTPN22) is a critical negative regulator of T cell antigen receptor (TCR) signaling. A single-nucleotide polymorphism (SNP) in the ptpn22 gene correlates with the incidence of various autoimmune diseases, including type 1 diabetes, rheumatoid arthritis, and systemic lupus erythematosus. Since the disease-associated allele is a more potent inhibitor of TCR signaling, specific Lyp inhibitors may become valuable in treating autoimmunity. Using a structure-based approach, we synthesized a library of 34 compounds that inhibited Lyp with IC(50) values between 0.27 and 6.2 μM. A reporter assay was employed to screen for compounds that enhanced TCR signaling in cells, and several inhibitors displayed a dose-dependent, activating effect. Subsequent probing for Lyp's direct physiological targets by immunoblot analysis confirmed the ability of the compounds to inhibit Lyp in T cells. Selectivity profiling against closely related tyrosine phosphatases and in silico docking studies with the crystal structure of Lyp yielded valuable information for the design of Lyp-specific compounds.

I102 does C5, and with slightly greater efficiency. Kinetic properties for these reactions have not been determined yet, but the rate enhancements above background are not as large as for the original substrate 7. The presence of the amino group opens additional mechanisms for the reaction of 8 and 9 in which the amino group acts as a nucleophile or as a general base. The latter mechanism would parallel that demonstrated for C3 with substrate 7, with a neutral general base acting in place of a presumed anionic base.

The lipocalins are secreted proteins that bind small organic molecules. Scn-Ngal (also known as neutrophil gelatinase associated lipocalin, siderocalin, lipocalin 2) sequesters bacterial iron chelators, called siderophores, and consequently blocks bacterial growth. However, Scn-Ngal is also prominently expressed in aseptic diseases, implying that it binds additional ligands and serves additional functions. Using chemical screens, crystallography and fluorescence methods, we report that Scn-Ngal binds iron together with a small metabolic product called catechol. The formation of the complex blocked the reactivity of iron and permitted its transport once introduced into circulation in vivo. Scn-Ngal then recycled its iron in endosomes by a pH-sensitive mechanism. As catechols derive from bacterial and mammalian metabolism of dietary compounds, the Scn-Ngal-catechol-Fe(III) complex represents an unforeseen microbial-host interaction, which mimics Scn-Ngal-siderophore interactions but instead traffics iron in aseptic tissues. These results identify an endogenous siderophore, which may link the disparate roles of Scn-Ngal in different diseases.

Large-conductance, voltage- and Ca(2+)-gated potassium (BK) channels control excitability in a number of cell types. BK channels are composed of alpha subunits, which contain the voltage-sensor domains and the Ca(2+)- sensor domains and form the pore, and often one of four types of beta subunits, which modulate the channel in a cell-specific manner. beta 4 is expressed in neurons throughout the brain. Deletion of beta 4 in mice causes temporal lobe epilepsy. Compared with channels composed of alpha alone, channels composed of alpha and beta 4 activate and deactivate more slowly. We inferred the locations of the two beta 4 transmembrane (TM) helices TM1 and TM2 relative to the seven alpha TM helices, S0-S6, from the extent of disulfide bond formation between cysteines substituted in the extracellular flanks of these TM helices. We found that beta 4 TM2 is close to alpha S0 and that beta 4 TM1 is close to both alpha S1 and S2. At least at their extracellular ends, TM1 and TM2 are not close to S3-S6. In six of eight of the most highly crosslinked cysteine pairs, four crosslinks from TM2 to S0 and one each from TM1 to S1 and S2 had small effects on the V(50) and on the rates of activation and deactivation. That disulfide crosslinking caused only small functional perturbations is consistent with the proximity of the extracellular ends of TM2 to S0 and of TM1 to S1 and to S2, in both the open and closed states.

Memory loss, synaptic dysfunction, and accumulation of amyloid beta-peptides (A beta) are major hallmarks of Alzheimer's disease (AD). Downregulation of the nitric oxide/cGMP/cGMP-dependent protein kinase/c-AMP responsive element-binding protein (CREB) cascade has been linked to the synaptic deficits after A beta elevation. Here, we report that the phosphodiesterase 5 inhibitor (PDE5) sildenafil (Viagra), a molecule that enhances phosphorylation of CREB, a molecule involved in memory, through elevation of cGMP levels, is beneficial against the AD phenotype in a mouse model of amyloid deposition. We demonstrate that the inhibitor produces an immediate and long-lasting amelioration of synaptic function, CREB phosphorylation, and memory. This effect is also associated with a long-lasting reduction of A beta levels. Given that side effects of PDE5 inhibitors are widely known and do not preclude their administration to a senile population, these drugs have potential for the treatment of AD and other diseases associated with elevated A beta levels.

Vesicular monoamine transporter type 2 (VMAT2) is a newly emerging target for both diagnostic and therapeutic applications in diabetes mellitus. In pursuit of novel VMAT2 antagonists, we identified a potent hypoglycemic agent with a novel dihydropyridone scaffold. Several analogs were designed and synthesized. A preliminary structure activity relationship (SAR) showed that the dihydropyridone scaffold is required for the activity.

Nuclear factor kappa B (NF-kappaB) is an important transcription factor. Aberrant regulation of the NF-kappaB pathway is frequently observed in a number of major ailments such as cancer and inflammatory diseases. Hence NF-kappaB modulators have been intensely pursued for their potential therapeutic applications. Numerous reviews have described recent progress in the development of these agents. More recently, a variety of structurally and functionally novel small molecules, identified through high-throughput screens conducted within the Molecular Libraries Screening Center Network (MLSCN) of the NIH Roadmap for Medical Research, have been added to the current list of NF-kappaB regulators. This review will discuss the inhibitors and activators newly discovered by Columbia's Molecular Libraries Screening Center (MLSC) using a well-designed and stable cellular assay.