Optimizing metabolic stability of phosphodiesterase 5 inhibitors: Discovery of a potent N-(pyridin-3-ylmethyl)quinoline derivative targeting synaptic plasticity.

Phosphodiesterase 5 (PDE5) is a cyclic guanosine monophosphate-degrading enzyme involved in numerous biological pathways. Inhibitors of PDE5 are important therapeutics for the treatment of neurodegenerative diseases, including Alzheimer's disease (AD). We previously reported the first generation of quinoline-based PDE5 inhibitors for the treatment of AD. However, the short in vitro microsomal stability rendered them unsuitable drug candidates. Here we report a series of new quinoline-based PDE5 inhibitors. Among them, compound 4b, 8-cyclopropyl-3-(hydroxymethyl)-4-(((6-methoxypyridin-3-yl)methyl)amino)quinoline-6-carbonitrile, shows a PDE5 IC50 of 20 nM and improved in vitro microsomal stability (t1/2 = 44.6 min) as well as excellent efficacy in restoring long-term potentiation, a type of synaptic plasticity to underlie memory formation, in electrophysiology experiments with a mouse model of AD. These results provide an insight into the development of a new class of PDE5 inhibitors for the treatment of AD.

Silencing c-Myc translation as a therapeutic strategy through targeting PI3Kδ and CK1ε in hematological malignancies.

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.

Discovery of New Classes of Compounds that Reactivate Acetylcholinesterase Inhibited by Organophosphates.

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.

Chloride channel blockade relaxes airway smooth muscle and potentiates relaxation by ß-agonists.

Severe bronchospasm refractory to ß-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 ß-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 ß-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.

The selective vasopressin type 1a receptor agonist selepressin (FE 202158) blocks vascular leak in ovine severe sepsis*.

OBJECTIVE: 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. DESIGN: Prospective, randomized, controlled laboratory experiment. SETTING: University animal research facility. SUBJECTS: Forty-five chronically instrumented sheep. INTERVENTIONS: 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. MEASUREMENTS AND MAIN RESULTS: 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. CONCLUSIONS: 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.

An Internally Attached Aptameric Graphene Nanosensor for Sensitive Vasopressin Measurement in Critical Patient Monitoring.

This paper presents an aptameric graphene nanosensor for rapid and sensitive measurement of arginine vasopressin (AVP) toward continuous monitoring of critical care patients. The nanosensor is a field-effect transistor (FET) with monolayer graphene as the conducting channel and is functionalized with a new custom-designed aptamer for specific AVP recognition. Binding between the aptamer and AVP induces a change in the carrier density in the graphene and resulting in measurable changes in FET characteristics for determination of the AVP concentration. The aptamer, based on the natural enantiomer D-deoxyribose, possess optimized kinetic binding properties and is attached at an internal position to the graphene for enhanced sensitivity to low concentrations of AVP. Experimental results show that this aptameric graphene nanosensor is highly sensitive (with a limit of detection of 0.3 pM and a resolution of 0.1 pM) to AVP, and rapidly responsive (within 90 s) to both increasing and decreasing AVP concentration changes. The device is also reversable (within 4%), repeatable (within 4%) and reproducible (within 5%) in AVP measurements.

Prophylactic (R,S)-ketamine and (2S,6S)-HNK decrease fear expression by differentially modulating fear neural ensembles.

BACKGROUND: We previously reported that a single injection of (R,S)-ketamine or its metabolite (2S,6S)-hydroxynorketamine (HNK) prior to stress attenuates learned fear. However, whether these drugs attenuate learned fear through divergent or convergent effects on neural activity remains to be determined. METHODS: 129S6/SvEv male mice were injected with saline, (R,S)-ketamine, or (2S,6S)-HNK one week before a 3-shock contextual fear conditioning (CFC) paradigm. Five days later, mice were re-exposed to the aversive context, and euthanized one hour later to quantify active cells. Brains were processed for c-fos immunoreactivity, and neural networks were built with a novel, wide-scale imaging pipeline. RESULTS: We found that (R,S)-ketamine and (2S,6S)-HNK attenuate learned fear. Fear-related neural activity was altered in: dorsal CA3 following (2S,6S)-HNK; ventral CA3 and CA1, infralimbic (IL) and prelimbic (PL) regions, insular cortex (IC), retrosplenial cortex (RSP), piriform cortex (PIR), nucleus reuniens (RE), and periaqueductal grey (PAG) following both (R,S)-ketamine and (2S,6S)-HNK; and in the paraventricular nucleus of thalamus (PVT) following (R,S)-ketamine. Dorsal CA3 and ventral hippocampus activation correlated with freezing in the (R,S)-ketamine group, and RSP activation correlated with freezing in both (R,S)-ketamine and (2S,6S)-HNK groups. (R,S)-ketamine increased connectivity between cortical and subcortical regions while (2S,6S)-HNK increased connectivity within these regions. CONCLUSIONS: This work identifies novel nodes in fear networks, involving the RE, PIR, IC, PAG and RSP, that can be targeted with neuromodulatory strategies or pharmaceutical compounds to treat fear-induced disorders. This approach could be used to optimize target engagement and dosing strategies of existing medications.

Exploring the Landscape of Aptamers: From Cross-Reactive to Selective to Specific, High-Affinity Receptors for Cocaine.

We reported over 20 years ago MNS-4.1, the first DNA aptamer with a micromolar affinity for cocaine. MNS-4.1 is based on a structural motif that is very common in any random pool of oligonucleotides, and it is actually a nonspecific hydrophobic receptor with wide cross-reactivity with alkaloids and steroids. Despite such weaknesses preventing broad applications, this aptamer became widely used in proof-of-concept demonstrations of new formats of biosensors. We now report a series of progressively improved DNA aptamers recognizing cocaine, with the final optimized receptors having low nanomolar affinity and over a thousand-fold selectivity over the initial cross-reactants. In the process of optimization, we tested different methods to eliminate cross-reactivities and improve affinity, eventually achieving properties that are comparable to those of the reported monoclonal antibody candidates for the therapy of overdose. Multiple aptamers that we now report share structural motifs with the previously reported receptor for serotonin. Further mutagenesis studies revealed a palindromic, highly adaptable, broadly cross-reactive hydrophobic motif that could be rebuilt through mutagenesis, expansion of linker regions, and selections into receptors with exceptional affinities and varying specificities.

Platelet integrin αIIbß3 plays a key role in venous thrombogenesis in a mouse model.

Venous thrombosis (VT) is a common vascular disease associated with reduced survival and a high recurrence rate. Previous studies have shown that the accumulation of platelets and neutrophils at sites of endothelial cell activation is a primary event in VT, but a role for platelet αIIbß3 in the initiation of venous thrombosis has not been established. This task has been complicated by the increased bleeding linked to partial agonism of current αIIbß3 inhibitory drugs such as tirofiban (Aggrastat ® ). Here, we show that m-tirofiban, an engineered version of tirofiban, is not a partial agonist of αIIbß3. This is based on its cryo-EM structure in complex with human full-length αIIbß3 and its inability to increase expression of an activation-sensitive epitope on platelet αIIbß3. m-tirofiban abolished agonist-induced platelet aggregation ex vivo at concentrations that preserved clot retraction and markedly suppressed the accumulation of platelets, neutrophils, and fibrin on thrombin-activated endothelium in real-time using intravital microscopy in a mouse model of venous thrombogenesis. Unlike tirofiban, however, m-tirofiban did not increase bleeding at the thrombosis-inhibitory dose. These findings establish a key role for αIIbß3 in the initiation of VT, provide a guiding principle for designing potentially safer inhibitors for other integrins, and suggest that pure antagonists of αIIbß3 like m-tirofiban merit further consideration as potential thromboprophylaxis agents in patients at high-risk for VT and hemorrhage.

Platelet integrin αIIbß3 plays a key role in a venous thrombogenesis mouse model.

Venous thrombosis (VT) is a common vascular disease associated with reduced survival and a high recurrence rate. VT is initiated by the accumulation of platelets and neutrophils at sites of endothelial cell activation. A role for platelet αIIbß3 in VT is not established, a task complicated by the increased bleeding risk caused by partial agonists such as tirofiban. Here, we show that m-tirofiban, a modified version of tirofiban, does not agonize αIIbß3 based on lack of neoepitope expression and the cryo-EM structure of m-tirofiban/full-length αIIbß3 complex. m-tirofiban abolishes agonist-induced platelet aggregation while preserving clot retraction ex vivo and, unlike tirofiban, it suppresses venous thrombogenesis in a mouse model without increasing bleeding. These findings establish a key role for αIIbß3 in VT initiation and suggest that m-tirofiban and compounds with a similar structurally-defined mechanism of action merit consideration as potential thromboprophylaxis agents in patients at high risk for VT and hemorrhage.

A pharmacological toolkit for human microglia identifies Topoisomerase I inhibitors as immunomodulators for Alzheimer's disease.

While efforts to identify microglial subtypes have recently accelerated, the relation of transcriptomically defined states to function has been largely limited to in silico annotations. Here, we characterize a set of pharmacological compounds that have been proposed to polarize human microglia towards two distinct states - one enriched for AD and MS genes and another characterized by increased expression of antigen presentation genes. Using different model systems including HMC3 cells, iPSC-derived microglia and cerebral organoids, we characterize the effect of these compounds in mimicking human microglial subtypes in vitro. We show that the Topoisomerase I inhibitor Camptothecin induces a CD74high/MHChigh microglial subtype which is specialized in amyloid beta phagocytosis. Camptothecin suppressed amyloid toxicity and restored microglia back to their homeostatic state in a zebrafish amyloid model. Our work provides avenues to recapitulate human microglial subtypes in vitro, enabling functional characterization and providing a foundation for modulating human microglia in vivo.

A functional group-guided approach to aptamers for small molecules.

Aptameric receptors are important biosensor components, yet our ability to identify them depends on the target structures. We analyzed the contributions of individual functional groups on small molecules to binding within 27 target-aptamer pairs, identifying potential hindrances to receptor isolation-for example, negative cooperativity between sterically hindered functional groups. To increase the probability of aptamer isolation for important targets, such as leucine and voriconazole, for which multiple previous selection attempts failed, we designed tailored strategies focused on overcoming individual structural barriers to successful selections. This approach enables us to move beyond standardized protocols into functional group-guided searches, relying on sequences common to receptors for targets and their analogs to serve as anchors in regions of vast oligonucleotide spaces wherein useful reagents are likely to be found.

Iron traffics in circulation bound to a siderocalin (Ngal)-catechol complex.

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.

Design, synthesis and evaluation of non-urea inhibitors of soluble epoxide hydrolase.

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 Vocation of a Doctor.

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.

Distinct functional and conformational states of the human lymphoid tyrosine phosphatase catalytic domain can be targeted by choice of the inhibitor chemotype.

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.

Vasopressin deficiency and vasodilatory state in end-stage liver disease.

OBJECTIVES: Relative vasopressin deficiency, a contributor to vasodilatory septic shock, also may be a cause of the vasodilatory state in liver disease. This study assessed endogenous vasopressin levels in patients with liver disease and their hemodynamic response to exogenous vasopressin. DESIGN: A prospective, observational study. SETTING: A single-center, tertiary hospital. PARTICIPANTS: Human subjects undergoing liver transplantation or major surgery. INTERVENTIONS: Vasopressin levels were measured in 28 patients with liver disease undergoing liver transplantation and 7 control patients with normal liver function. Additionally, intravenous vasopressin was administered to 20 liver transplant recipients, and the hemodynamic response was observed. MEASUREMENTS AND MAIN RESULTS: Patients with liver disease had significantly lower baseline vasopressin levels than controls (19.3 ± 27.1 pg/mL v 50.9 ± 36.7 pg/mL, p = 0.015). Patients with low vasopressin levels (≤20 pg/mL) were more likely to have lower baseline mean blood pressure (≤80 mmHg) than patients with high vasopressin levels (11/16 v 0/4, p = 0.013). Systemic vascular resistance increased by 33% 3 minutes after intravenous vasopressin. Thirteen of 16 patients with low vasopressin levels compared with 1 of 4 patients with high vasopressin levels responded to exogenous vasopressin, with an increase of mean blood pressure by more than 20% (p = 0.028). CONCLUSIONS: Patients with liver disease have lower vasopressin levels than controls and respond with a brisk vasoconstrictor response to exogenous vasopressin. Therefore, relative endogenous vasopressin deficiency may contribute to vasodilatory shock in liver disease similar to what has been observed in septic shock.

Locations of the beta1 transmembrane helices in the BK potassium channel.

BK channels are composed of alpha-subunits, which form a voltage- and Ca(2+)-gated potassium channel, and of modulatory beta-subunits. The beta1-subunit is expressed in smooth muscle, where it renders the BK channel sensitive to [Ca(2+)](i) in a voltage range near the smooth-muscle resting potential and slows activation and deactivation. BK channel acts thereby as a damped feedback regulator of voltage-dependent Ca(2+) channels and of smooth muscle tone. We explored the contacts between alpha and beta1 by determining the extent of endogenous disulfide bond formation between cysteines substituted just extracellular to the two beta1 transmembrane (TM) helices, TM1 and TM2, and to the seven alpha TM helices, consisting of S1-S6, conserved in all voltage-dependent potassium channels, and the unique S0 helix, which we previously concluded was partly surrounded by S1-S4. We now find that the extracellular ends of beta1 TM2 and alpha S0 are in contact and that beta1 TM1 is close to both S1 and S2. The extracellular ends of TM1 and TM2 are not close to S3-S6. In almost all cases, cross-linking of TM2 to S0 or of TM1 to S1 or S2 shifted the conductance-voltage curves toward more positive potentials, slowed activation, and speeded deactivation, and in general favored the closed state. TM1 and TM2 are in position to contribute, in concert with the extracellular loop and the intracellular N- and C-terminal tails of beta1, to the modulation of BK channel function.

Remodeling of ryanodine receptor complex causes "leaky" channels: a molecular mechanism for decreased exercise capacity.

During exercise, defects in calcium (Ca2+) release have been proposed to impair muscle function. Here, we show that during exercise in mice and humans, the major Ca2+ 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 Ca2+-dependent neutral protease calpain activity and plasma creatine kinase levels. Taken together, these data suggest a possible mechanism by which Ca2+ leak via calstabin1-depleted RyR1 channels leads to defective Ca2+ signaling, muscle damage, and impaired exercise capacity.

Post-acute COVID-19 syndrome.

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.