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The SARS-CoV-2 virus responsible for the COVID-19 pandemic has generated an explosion of interest both in the mechanisms of infection leading to dissemination and expression of this disease, and in potential risk factors that may have a mechanistic basis for disease propagation or control. Vitamin D has emerged as a factor that may be involved in these two areas. The focus of this article is to apply our current understanding of vitamin D as a facilitator of immunocompetence both with regard to innate and adaptive immunity and to consider how this may relate to COVID-19 disease. There are also intriguing potential links to vitamin D as a factor in the cytokine storm that portends some of the most serious consequences of SARS-CoV-2 infection, such as the acute respiratory distress syndrome. Moreover, cardiac and coagulopathic features of COVID-19 disease deserve attention as they may also be related to vitamin D. Finally, we review the current clinical data associating vitamin D with SARS-CoV-2 infection, a putative clinical link that at this time must still be considered hypothetical.

Although COVID-19 is most well known for causing substantial respiratory pathology, it can also result in several extrapulmonary manifestations. These conditions include thrombotic complications, myocardial dysfunction and arrhythmia, acute coronary syndromes, acute kidney injury, gastrointestinal symptoms, hepatocellular injury, hyperglycemia and ketosis, neurologic illnesses, ocular symptoms, and dermatologic complications. Given that ACE2, the entry receptor for the causative coronavirus SARS-CoV-2, is expressed in multiple extrapulmonary tissues, direct viral tissue damage is a plausible mechanism of injury. In addition, endothelial damage and thromboinflammation, dysregulation of immune responses, and maladaptation of ACE2-related pathways might all contribute to these extrapulmonary manifestations of COVID-19. Here we review the extrapulmonary organ-specific pathophysiology, presentations and management considerations for patients with COVID-19 to aid clinicians and scientists in recognizing and monitoring the spectrum of manifestations, and in developing research priorities and therapeutic strategies for all organ systems involved.

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.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the pathogen responsible for the coronavirus disease 2019 (COVID-19) pandemic, which has resulted in global healthcare crises and strained health resources. As the population of patients recovering from COVID-19 grows, it is paramount to establish an understanding of the healthcare issues surrounding them. COVID-19 is now recognized as a multi-organ disease with a broad spectrum of manifestations. Similarly to post-acute viral syndromes described in survivors of other virulent coronavirus epidemics, there are increasing reports of persistent and prolonged effects after acute COVID-19. Patient advocacy groups, many members of which identify themselves as long haulers, have helped contribute to the recognition of post-acute COVID-19, a syndrome characterized by persistent symptoms and/or delayed or long-term complications beyond 4 weeks from the onset of symptoms. Here, we provide a comprehensive review of the current literature on post-acute COVID-19, its pathophysiology and its organ-specific sequelae. Finally, we discuss relevant considerations for the multidisciplinary care of COVID-19 survivors and propose a framework for the identification of those at high risk for post-acute COVID-19 and their coordinated management through dedicated COVID-19 clinics.

Background and AimsThe COVID-19 pandemic has caused widespread mortality and mortality. Famotidine is commonly used for gastric acid suppression but has recently gained attention as an antiviral that may inhibit SARS-CoV-2 replication. This study tested whether famotidine use is associated with improved clinical outcomes in patients with COVID-19 initially hospitalized to a non-intensive care setting.MethodsThis was a retrospective cohort study conducted among consecutive hospitalized patients with COVID-19 infection from February 25 to April 13, 2020 at a single medical center. The primary exposure was famotidine, received within 24 hours of hospital admission. The primary outcome was intubation or death. Propensity score matching was used to balance the baseline characteristics of patients who did and did not use famotidine.Results1,620 hospitalized patients with COVID-19 were identified including 84 (5.1%) who received famotidine within 24 hours of hospital admission. 340 (21%) patients met the study composite outcome of death or intubation. Use of famotidine was associated with reduced risk for death or intubation (adjusted hazard ratio (aHR) 0.42, 95% CI 0.21-0.85) and also with reduced risk for death alone (aHR 0.30, 95% CI 0.11-0.80). After balancing baseline patient characteristics using propensity score matching, these relationships were unchanged (HR for famotidine and death or intubation: 0.43, 95% CI 0.21-0.88). Proton pump inhibitors, which also suppress gastric acid, were not associated with reduced risk for death or intubation.ConclusionFamotidine use is associated with reduced risk of intubation or death in hospitalized COVID-19 patients. Randomized controlled trials are warranted to determine whether famotidine therapy improves outcomes in hospitalized COVID-19 patients.

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.

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.

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.

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.

OBJECTIVEDelayed cerebral ischemia (DCI) is a significant contributor to poor outcomes after aneurysmal subarachnoid hemorrhage (aSAH). The neurotoxin 3-aminopropanal (3-AP) is upregulated in cerebral ischemia. This phase II clinical trial evaluated the efficacy of tiopronin in reducing CSF 3-AP levels in patients with aSAH.METHODSIn this prospective, randomized, double-blind, placebo-controlled, multicenter clinical trial, 60 patients were assigned to receive tiopronin or placebo in a 1:1 ratio. Treatment was commenced within 96 hours after aSAH onset, administered at a dose of 3 g daily, and continued until 14 days after aSAH or hospital discharge, whichever occurred earlier. The primary efficacy outcome was the CSF 3-AP level at 7 ± 1 days after aSAH.RESULTSOf the 60 enrolled patients, 29 (97%) and 27 (93%) in the tiopronin and placebo arms, respectively, received more than one dose of the study drug or placebo. At post-aSAH day 7 ± 1, CSF samples were available in 41% (n = 12/29) and 48% (n = 13/27) of patients in the tiopronin and placebo arms, respectively. No difference in CSF 3-AP levels at post-aSAH day 7 ± 1 was observed between the study arms (11 ± 12 nmol/mL vs 13 ± 18 nmol/mL; p = 0.766). Prespecified adverse events led to early treatment cessation for 4 patients in the tiopronin arm and 2 in the placebo arm.CONCLUSIONSThe power of this study was affected by missing data. Therefore, the authors could not establish or refute an effect of tiopronin on CSF 3-AP levels. Additional observational studies investigating the role of 3-AP as a biomarker for DCI may be warranted prior to its use as a molecular target in future clinical trials.Clinical trial registration no.: NCT01095731 (ClinicalTrials.gov)

Introduction: Subacute symptomatic neurological decline is a significant and preventable contributor to patient morbidity and mortality after aneurysmal subarachnoid hemorrhage (aSAH). 3-Aminopropanal (3-AP) is a potent neurotoxin that is upregulated in the setting of cerebral ischemia. Targeted neutralization has achieved a neuroprotective effect in preclinical models. This phase II trial evaluated the efficacy of Tiopronin administration in reducing 3-AP levels in patients with aSAH. Methods: We designed a prospective, randomized, double-blind, placebo-controlled clinical trial that was conducted in three centers in the United States. 60 patients were enrolled and assigned to receive Tiopronin or placebo in a 1:1 ratio from enrolment until the earlier to occur of post-bleed day 14 or hospital discharge. Patients received a total of 3 grams daily, in 3 divided doses. The primary efficacy endpoint was the change in serum and/or cerebrospinal fluid (CSF) 3-AP from enrolment to study drug discontinuation. Results: Of the 60 enrolled patients, 29 (97%) in the treatment arm and 27 (93%) in the control arm received more than one dose of the study drug. A greater relative reduction in serum 3-AP level was observed in the treatment arm (adj Beta=-0.689, 95% C.I.=-2.730, -0.008; p=0.049). This was most apparent, but did not reach significance, in patients with grade IV-V aSAH (adj Beta=-0.808; 95% C.I.=-3.872, 0.167; p=0.067). No difference in the change in CSF level was observed (adj Beta=0.048, 95% C.I.=-2.334, 2.589; p=0.903). Pre-specified adverse events led to early cessation of treatment for four patients in the treatment arm, and two in the control arm. Conclusion: Tiopronin attenuated the accumulation of serum 3-AP, providing the first translational evidence for the use of 3-AP antagonists in aSAH. Further investigation to evaluate for a dose-response relationship in Tiopronin’s ability to cross the blood-brain barrier is recommended.

We present a microfluidic aptamer-based biosensor for detection of low-molecular-weight biomarkers in patient samples. Using a microfluidic device that integrates aptamer-based specific analyte extraction, isocratic elution, and detection by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, we demonstrate rapid, sensitive and label-free detection of arginine vasopressin (AVP) in human plasma ultrafiltrate. AVP molecules in complex matrices are specifically captured by an aptamer that is immobilized on microbeads via affinity binding in a microchamber. After the removal of unbound, contaminating molecules through washing, aptamer-AVP complexes are thermally disrupted via on-chip temperature control. Released AVP molecules are eluted with purified water and transferred to a separate microchamber, and deposited onto a single spot on a MALDI plate via repeated, piezoelectrically actuated ejection, which enriches AVP molecules over the spot area. This integrated on-chip sample processing enables the quantitative detection of low-abundance AVP by MALDI-TOF mass spectrometry in a rapid and label-free manner. Our experimental results show the detection of AVP in human plasma ultrafiltrate as low as physiologically relevant picomolar concentrations via aptamer-based selective preconcentration, demonstrating the potential of our approach as a rapid (~ 1hr), sensitive clinical AVP assay.

Doctors are called to a life of compassionate service to human beings invested with intrinsic dignity. This essay is adapted from the commencement address that Dr. Landry delivered at the St. Louis University School of Medicine on July 18, 2011.

Organophosphate compounds (OPCs) are commonly used as pesticides and were developed as nerve agents for chemical warfare. Exposure to OPCs results in toxicity due to their covalent binding and inhibition of acetylcholinesterase (AChE). Treatment for toxicity due to OPC exposure has been largely focused on the reactivation of AChE by oxime-based compounds via direct nucleophilic attack on the phosphorous center. However, due to the disadvantages to existing oxime-based reactivators for treatment of OPC poisoning, we considered non-oxime mechanisms of reactivation. A high throughput screen of compound libraries was performed to discover previously unidentified reactivation compounds, followed by studies on their analogs. In the process, we discovered multiple non-oxime classes of compounds, the most robust of which we have already reported [1]. Herein, we report other classes of compounds we identified in our screen that are efficient at reactivation. During biochemical characterization, we also found some compounds with other activities that may inspire novel therapeutic approaches to OPC toxicity. Specifically, we found compounds that [1] increase the rate of substrate hydrolysis by AChE and, [2] protect the enzyme from inhibition by OPC. Further, we discovered that a subset of reactivator compounds recover activity from both AChE and the related enzyme butyrylcholinesterase (BuChE). We now report these compounds, their activities and discuss how each relates to therapeutic approaches that would provide alternatives to traditional oxime-based reactivation.

Acetylcholinesterase (AChE) that has been covalently inhibited by organophosphate compounds (OPCs), such as nerve agents and pesticides, has traditionally been reactivated by using nucleophilic oximes. There is, however, a clearly recognized need for new classes of compounds with the ability to reactivate inhibited AChE with improved in vivo efficacy. Here we describe our discovery of new functional groups--Mannich phenols and general bases--that are capable of reactivating OPC--inhibited AChE more efficiently than standard oximes and we describe the cooperative mechanism by which these functionalities are delivered to the active site. These discoveries, supported by preliminary in vivo results and crystallographic data, significantly broaden the available approaches for reactivation of AChE.

The catalytic bioscavenger phosphotriesterase (PTE) is experimentally an effective antidote for organophosphate poisoning. We are interested in the molecular engineering of this enzyme to confer additional functionality, such as improved in vivo longevity. To this aim, we developed PTE cysteine mutants with free sulfhydryls to allow macromolecular attachments to the protein. A library of PTE cysteine mutants were assessed for efficiency in hydrolysing the toxic pesticide metabolite paraoxon, and screened for attachment with a sulfhydryl-reactive small molecule, fluorescein 5-maleimide (F5M), to examine cysteine availability. We established that the newly incorporated cysteines were readily available for labelling, with R90C, E116C and S291C displaying the highest affinity for binding with F5M. Next, we screened for efficiency in attaching a large macromolecule, a 30 000 Da polyethylene glycol (PEG) molecule. Using a solid-phase PEGylation strategy, we found the E116C mutant to be the best single-mutant candidate for attachment with PEG30. Kinetic activity of PEGylated E116C, with paraoxon as substrate, displayed activity approaching that of the unPEGylated wild-type. Our findings demonstrate, for the first time, an efficient cysteine mutation and subsequent method for sulfhydryl-specific macromolecule attachment to PTE.

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.

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.

Severe bronchospasm refractory to β-agonists continues to cause significant morbidity and mortality in asthmatic patients. We questioned whether chloride channels/transporters are novel targets for the relaxation of airway smooth muscle (ASM). We have screened a library of compounds, derivatives of anthranilic and indanyloxyacetic acid, that were originally developed to antagonize chloride channels in the kidney. We hypothesized that members of this library would be novel calcium-activated chloride channel blockers for the airway. The initial screen of this compound library identified 4 of 20 compounds that relaxed a tetraethylammonium chloride-induced contraction in guinea pig tracheal rings. The two most effective compounds, compounds 1 and 13, were further studied for their potential to either prevent the initiation of or relax the maintenance phase of an acetylcholine (ACh)-induced contraction or to potentiate β-agonist-mediated relaxation. Both relaxed an established ACh-induced contraction in human and guinea pig ex vivo ASM. In contrast, the prevention of an ACh-induced contraction required copretreatment with the sodium-potassium-chloride cotransporter blocker bumetanide. The combination of compound 13 and bumetanide also potentiated relaxation by the β-agonist isoproterenol in guinea pig tracheal rings. Compounds 1 and 13 hyperpolarized the plasma cell membrane of human ASM cells and blocked spontaneous transient inward currents, a measure of chloride currents in these cells. These functional and electrophysiological data suggest that modulating ASM chloride flux is a novel therapeutic target in asthma and other bronchoconstrictive diseases.

To determine if the selective vasopressin type 1a receptor agonist selepressin (FE 202158) is as effective as the mixed vasopressin type 1a receptor/vasopressin V2 receptor agonist vasopressor hormone arginine vasopressin when used as a titrated first-line vasopressor therapy in an ovine model of Pseudomonas aeruginosa pneumonia-induced severe sepsis.Prospective, randomized, controlled laboratory experiment.University animal research facility.Forty-five chronically instrumented sheep.Sheep were anesthetized, insufflated with cooled cotton smoke via tracheostomy, and P. aeruginosa were instilled into their airways. They were then placed on assisted ventilation, awakened, and resuscitated with lactated Ringer's solution titrated to maintain hematocrit ± 3% from baseline levels. If, despite fluid management, mean arterial pressure fell by more than 10 mm Hg from baseline level, an additional continuous IV infusion of arginine vasopressin or selepressin was titrated to raise and maintain mean arterial pressure within no less than 10 mm Hg from baseline level. Effects of combination treatment of selepressin with the selective vasopressin V2 receptor agonist desmopressin were similarly investigated.In septic sheep, MAP fell by ~30 mm Hg, systemic vascular resistance index decreased by ~50%, and ~7 L of fluid were retained over 24 hours; this fluid accumulation was partially reduced by arginine vasopressin and almost completely blocked by selepressin; and combined infusion of selepressin and desmopressin increased fluid accumulation to levels similar to arginine vasopressin treatment.Resuscitation with the selective vasopressin type 1a receptor agonist selepressin blocked vascular leak more effectively than the mixed vasopressin type 1a receptor/vasopressin V2 receptor agonist arginine vasopressin because of its lack of agonist activity at the vasopressin V2 receptor.

Epigenetic dysregulation is a hallmark of germinal center derived B cell lymphomas. In particular, the monoallelic mutations in histone acetyltransferases (HATs), p300 and CBP are present in approximately 40% of DLBCL and FL patients. The haploinsufficiency of CBP leads to loss of MHC which contributes to both immune escape and lymphomagenesis. The intact wild-type allele offers an opportunity to leverage the normal enzyme to overcome the pathogenic impact of the mutated allele. We reported our lead compound YF2 out of a library (n=70) of HAT activators led to significant cytotoxic effect in lymphoma cell lines compared to its parent compound CTPB, which is not cell membrane permeable. YF2 induced p300-mediated histone acetylation as well as p53 acetylation in cell-free acetylation assay. YF2 also induced the acetylation of H3K14 and H3K27 at the cellular level. Since CREBBP mutations are more frequent than EP300 and are mutually exclusive in patients, it is of high insterest to determine if YF2 affects CBP-mediated acetylation. We measured CBP mediated histone acetylation in a cell free assay by combining the catalytic core of CBP, histone H3 and acetyl-CoA. YF2 increased the level of CBP-mediated Ac-H3K27 (EC50=175.4nM) and Ac-H3K18 (EC50=1330 nM). Studies have shown that in mice lacking Crebbp there is reduced MHC II surface expression on GC cells impairing antigen presentation. To determine if YF2 affects the expression of MHC class I, II and related genes in B cell lymphoma cells, qPCR based gene expression profiling was performed pre and post YF2 exposure. YF2 significantly induces mRNA expression level of HLA-DMA (2.9 fold), PSMB10 (2 fold), and reduced the expression of HLA-G (10 fold). Encode ChIP-seq data confirms enrichement of CBP and p300 in the promotor regions of HLA-DMA and PSMB10 in germinal center B cells, indicating a regulatory effect of YF2 on these genes via HAT modulation. HLA‐G is known to inhibit NK‐cell- and CTL‐mediated cytotoxicity, therefore a reduction in HLA-G may release the inhibition of NK-cell and CTL recognition. Taken together, YF2 may modulate p300 and CBP mediated HLA and related gene expression to induce improved immune surveillance. YF2 is selectively cytotoxic in EP300 mutated B cell lymphoma lines. Since HDAC inhibitors have been approved in the clinic for patients with T cell lymphoma (TCL), we further tested the cytotoxic effect of YF2 in 6 TCL cell lines. Interestingly, YF2 is equivalently cytotoxic in TCL cell lines with IC50 ranging from 4.4 to 14 uM compared to the IC50 values in EP300 mutated BCL lines (5 to 11 uM). Additionally, YF2 induces apoptosis and G1 arrest in a dose dependent manner demonstrated by flow cytometry. We collected three primary lymphoma patient samples including one FL sample carrying CREBBP and p53 mutations and observed that YF2 induced cell death in patient samples, but not in PBMCs from 3 healthy donors, indicating a good therapeutic window. Robust synergy with combination of YF2 and romidepsin was observed in 100% (6/6) EP300 mutated BCL, 40% (2/5) EP300 wild-type BCL and 60% (3/5) TCL cells (EOB = 28). In vivo, SCID‐beige mice were xenografted with SUDHL6 and treated with either vehicle, YF2 40mg/kg, romidepsin 2mg/kg or the combination. Single agent YF2 led to equivalent tumor growth delay as FDA-approved drug romidepsin. The YF2 plus romidepsin combination led to significant tumor growth delay compared to the single agents and control. The efficacy of YF2 and romidepsin in PDX model will be determined in an ongoing study. Given the fact that the RING domain of p300 functions as an inhibitory domain to regulate p300 activity, we hypothesized that YF2 may bind to the RING domain, change the confirmation to facilitate access to the HAT substrate-binding pocket. As predicted by LBias, small molecule binding sites on p300 are on the RING domain and bromodomain. We further checked the potential binding site by docking YF2-like small molecules on the RING domain. We discovered the potential binding site of YF2 in the pocket of RING domain. Kinome screening showed YF2 has no off-target effects on 97 kinases. Ongoing experiments will determine the binding of YF2 to the p300 and CBP catalytic cores. In summary, our lead compound YF2 induces HAT enzymes by activating not only p300 but also CBP mediated acetylation of histone. Modulating HLA genes by YF2 may potentially offer an opportunity to reverse immune evasion adopted by lymphoma cells as a mechanism for survival. Disclosures O'Connor: Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; ADCT Therapeutics, Affimed, Agensys, Merck, Seattle Genetics, Spectrum, Trillium, and Verastem Oncology.: Research Funding; TG Therapeutics: Other: Travel Support, Research Funding. Arancio:Appia Pharmaceuticals: Research Funding; Neurokine Therapeutics: Other: Founder. Amengual:Epizyme: Speakers Bureau; Appia Pharmaceuticals: Research Funding.

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.

The lymphoid tyrosine phosphatase (LYP), encoded by the PTPN22 gene, has recently been identified as a promising drug target for human autoimmunity diseases. Like the majority of protein-tyrosine phosphatases LYP can adopt two functionally distinct forms determined by the conformation of the WPD-loop. The WPD-loop plays an important role in the catalytic dephosphorylation by protein-tyrosine phosphatases. Here we investigate the binding modes of two chemotypes of small molecule LYP inhibitors with respect to both protein conformations using computational modeling. To evaluate binding in the active form, we built a LYP protein structure model of high quality. Our results suggest that the two different compound classes investigated, bind to different conformations of the LYP phosphatase domain. Binding to the closed form is facilitated by an interaction with Asp195 in the WPD-loop, presumably stabilizing the active conformation. The analysis presented here is relevant for the design of inhibitors that specifically target either the closed or the open conformation of LYP in order to achieve better selectivity over phosphatases with similar binding sites.

We present a microfluidic aptameric biosensor, or aptasensor, for selective detection of clinically relevant analytes with integrated analyte enrichment, isocratic elution and label-free detection by mass spectrometry. Using a microfluidic platform that is coupled to matrix assisted laser desorption/ionization mass spectrometry (MALDI–MS), we demonstrate specific purification, enrichment, and label-free detection of trace amounts of arginine vasopressin (AVP), a peptide hormone that is responsible for arterial vasoconstriction. During extreme physical trauma, in particular immunological shock or congestive heart failure, AVP is excreted abnormally and is hence a biomarker for such conditions. The device uses an aptamer, i.e., an oligonucleotide that binds specifically to an analyte via affinity interactions, to achieve highly selective analyte capture and enrichment. In addition, via thermally induced reversible disruption of the aptamer-analyte binding, the device can be easily regenerated for reuse and allows isocratic analyte elution, i.e., release and collection of analytes using a single aqueous solution. Furthermore, the device is coupled to MALDI–MS using a microfluidic flow gate, which directs the eluted analyte onto a MALDI sample plate for mass spectrometry. We first perform systematic characterization of kinetic and thermal release properties, as well as the overall timescale of the assay, using fluorescently labeled AVP. We then demonstrate MALDI–MS detection of unlabeled AVP at clinically relevant concentrations approaching 1 pM.

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.

Monoalleleic inactivating mutations in histone acetyltransferase (HAT) enzymes promote lymphomagenesis in germinal center derived B-cell lymphomas, follicular lymphoma (FL) and diffuse large B cell lymphoma (DLBCL), occurring in about 40% of patients. The intact wild-type allele offers an opportunity to leverage the normal enzyme to overcome the pathogenic impact of the mutated allele. We hypothesize that if inactivating mutations in HATs are critical to FL and DLBCL lymphomagenesis, then drugs capable of inducing enhanced function of the wild-type HAT allele product should be cytotoxic in cells harboring HAT mutations. We designed and synthesized a library of new chemical entities with HAT activating properties (N=70). The cytotoxic effects of the compounds (N=29) were evaluated via medium-throughput screening in 4 DLBCL cell lines. IC50 ranged from 3.6 to 43.2 µM. Focusing on 6 analogue compounds, which share the same Nphenylbenzamide scaffold, we evaluated cytotoxicity across an expanded panel of 11 DLBCL cell lines. The median IC50 of 6 analogues tested was lower in the EP300 mutated cell lines (median 9.6 µM, range 5 - 11 µM) compared to the wildtype lines (median, 17 µM, range 15 - 24 µM). YF2 was chosen as the lead compound because it was the most selective of the analogues in inducing cytotoxicity in cell lines harboring EP300 mutations compared to wildtype (IC50 5 µM and 19 µM respectively, p To determine YF2's functional effect on activating p300 in a cell free assay, p300-mediated histone and p53 acetylation was measured by combining recombinant p300, substrate and acetyl-CoA. YF2 increased p300-mediated histone H3 lysine27 acetylation (EC50=38.64 nM) and H3 lysine18 acetylation (EC50=1.656 nM). YF2 also induced acetylation of p53 by 5-fold in a dose dependent manner. In cellular assays, YF2 induces acetylation of histone (H3K27 2-fold and H3K14 1.6-fold) after exposure in the SUDHL-6 cell line (EP300 mutated) as measured by mass spectrometry and confirmed by immunoblot of histone extracts. To assess the pharmacokinetics (PK) and preliminary in vivo efficacy of YF2, SUDHL-6 (EP300-mutated) xenograft bearing mice were treated once daily i.p. for 6 days with YF2 doses of 40mg/kg or 60mg/kg. Serum and tumor samples were collected at sequential time points. The Cmax of YF2 60mg/kg was 2424 ng/mL (5.1 µM) whereas Cmax of 40mg/kg was 2091.96 ng/mL (4.5 µM) which is consistent with the cellular IC50. Both concentrations of YF2, 40mg/kg and 60mg/kg, accumulated in tumor with Cmax 9536.71 and 9858.15 ng/g, respectively. YF2 is rapidly absorbed in the serum (Tmax 0.25 h) and sustained in the tumor (Tmax 4h). Significant effects on tumor size were observed in 13 of 19 mice demonstrating decreased tumor volume following only 6 days of YF2 treatment. Mice treated with YF2 40mg/kg induced H3K27 acetylation in tumor specimens as determined by mass spectrometry.YF2 40mg/kg was well tolerated in SCID/beige mice for 30 days without significant weight loss, while 60mg/kg YF2 led to 20% weight loss during 6-days of treatment. Additionally, YF2 demonstrated cytotoxic effects in 2 primary patient lymphoma samples but were non-cytotoxic to peripheral blood mononuclear cells from healthy donors. Furthermore, we hypothesized that if DLBCL is sensitive to an enhanced acetylation state, then combined targeting of epigenetic machinery with HAT activators and HDAC inhibitors may induce profound epigenetic modification leading to synergistic induction of programmed cell death. The concentration : effect relationship of YF2 and the pan-HDAC inhibitor, romidepsin, was evaluated over time across a panel of lymphoma cell lines (N=7). Synergy was calculated by Excess over Bliss (EOB>10 connotes synergy). Combination of YF2 and romidepsin demonstrated strong synergism in DLBCL lines (EOB = 48). The combination led to enhanced histone acetylation compared to either single agents. The combination is safe in mice and murine xenograft studies of the combination are underway. In summary, YF2 induces HAT-mediated acetylation of histone and p53. It demonstrates selective cytotoxic effects in EP300-mutated DLBCL cell lines, and is both well tolerated and effective in xenograft mouse models of lymphoma suggesting potential clinical application and precision medicine opportunities for patients harboring this mutation. Disclosures O'Connor: Seattle Genetics: Research Funding; ADC Therapeutics: Research Funding; Celgene: Research Funding.

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.

Enhancing cocaine metabolism by administration of cocaine esterase (CocE) has been recognized as a promising treatment strategy for cocaine overdose and addiction, because CocE is the most efficient native enzyme for metabolizing the naturally occurring cocaine yet identified. A major obstacle to the clinical application of CocE is the thermoinstability of native CocE with a half-life of only a few minutes at physiological temperature (37 degrees C). Here we report thermostable variants of CocE developed through rational design using a novel computational approach followed by in vitro and in vivo studies. This integrated computational-experimental effort has yielded a CocE variant with a approximately 30-fold increase in plasma half-life both in vitro and in vivo. The novel design strategy can be used to develop thermostable mutants of any protein.

Vasodilation due to impaired vascular tone is common in liver failure. Vasoconstrictor drugs are almost always required during the anhepatic phase of a liver transplant to maintain blood pressure unless venovenous bypass is employed. Arginine-vasopressin can be used as a vasoconstrictor instead of or in addition to norepinephrine for this purpose, but the effect of vasopressin on the portal vein pressure and flow in this setting is unknown. Portal vein pressure, portal vein blood flow, hemodynamic variables, and plasma vasopressin levels were measured in 16 patients during liver transplantation after ligation of the hepatic artery before and after a vasopressin infusion of 3.8 +/- 1.1 units/hour. Measurements were performed on the native liver prior to caval clamping. After vasopressin infusion, the portal vein pressure decreased significantly from 24.0 +/- 6.5 to 21.5 +/- 7.4 mm Hg [mean +/- standard deviation (SD), P = 0.006]. The portal vein blood flow also decreased (from 1.01 +/- 0.53 to 0.76 +/- 0.53 L/minute, mean +/- SD, P < 0.0001), as did the portal vein blood flow to cardiac output ratio (from 0.14 +/- 0.06 to 0.10 +/- 0.07, mean +/- SD, P < 0.0001). In conclusion, vasopressin significantly decreased portal vein pressure and flow of the native liver without decreasing cardiac output or intestinal perfusion in patients undergoing liver transplantations.

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.

Eine wichtige Komponente der Therapie des vasodilatatorischen Schockgeschehens im Rahmen der Intensivmedizin ist der Einsatz von Katecholaminen. Bei prolongierter Anwendung kommt es jedoch zur Wirkungsverminderung durch Abnahme der Rezeptorzahl („Down-Regulation“) und Verminderung der Rezeptorsensibilitat (Desensibilisierung). Die Moglichkeit, diesem Effekt durch zusatzliche Gabe von Vasopressin und dessen Analogon Terlipressin entgegenzuwirken, haben Serpa Neto und Kollegen im Rahmen einer Metaanalyse untersucht.