Upregulation of cAMP prevents antibody-mediated thrombus formation in COVID-19

Thromboembolic events are frequently reported in patients infected with the SARS-CoV-2 virus. The exact mechanisms of COVID-19-associated hypercoagulopathy, however, remain elusive. Recently, we observed that platelets (PLTs) from patients with severe COVID-19 infection express high levels of procoagulant markers, which were found to be associated with increased risk for thrombosis. In the current study, we investigated the time course as well as the mechanisms leading to procoagulant PLTs in COVID-19. Our study demonstrates the presence of PLT-reactive IgG antibodies that induce marked changes in PLTs in terms of increased inner-mitochondrial transmembrane potential (Δψ) depolarization, phosphatidylserine (PS) externalization, and P-selectin expression. The IgG-induced procoagulant PLTs and increased thrombus formation were mediated by ligation of PLT Fc-γ RIIA (FcγRIIA). In addition, contents of calcium and cyclic-adenosine-monophosphate (cAMP) in PLTs were identified to play a central role in antibody-induced procoagulant PLT formation. Most importantly, antibody-induced procoagulant events, as well as increased thrombus formation in severe COVID-19, were inhibited by Iloprost, a clinically approved therapeutic agent that increases the intracellular cAMP levels in PLTs. Our data indicate that upregulation of cAMP could be a potential therapeutic target to prevent antibody-mediated coagulopathy in COVID-19 disease.

Vibrational Spectroscopic and Molecular Docking Studies of Amrinone, a Cardiotonic Inotropic Drug-=SUP=-*-=/SUP=-

Amrinone is a class I cardiotonic inotropic agent, which is known to increase the cyclic adenosine monophosphate (cAMP) level by inhibiting the phosphodiesterase 3 (PDE3) enzyme. In this study the theoretically possible stable conformations of the amrinone, was examined first by conformational analysis method and then the obtained most stable conformation was optimized by DFT/wb97xd/6-311++G(d,p) level of theory using Gaussian 03 program. The credibility of the theoretical model was confirmed by comparison of experimental and theoretical vibrational spectra of the title molecule. The fundamental vibrational wavenumbers, IR and Raman intensities of the optimized structure of amrinone were determined using DFT/wb97xd/6-311++G(d,p) level of theory and compared with the experimental vibrational spectra. To investigate the influence of amrinone on cAMP enhancement, the docking simulations towards PDE3B were carried out and the main binding interactions of amrinone with PDE3 were elucidated. Cytochrome P450s (CYPs) are very important phase I metabolizing enzymes. The interaction between amrinone and CYPs (CYP1A2, CYP2C9 and CYP2C19) was investigated by docking simulations. Moreover, molecular docking of the title molecule with different proteins and receptors were studied to reveal potential mechanisms for therapeutic applications. Molecular docking simulations revealed that amrinone showed strong binding affinity to integrins α5β1 (Delta G=-6.6 kcal/mol) and αIIbβ3 (-6.6 kcal/mol), and DNA (-6.5 kcal/mol). The results correlated with its anticancer activity. The drug likeness and ADMET properties of amrinone were analyzed for the prediction of pharmacokinetic profiles. Key words: amrinone, DFT calculations, FTIR, Molecular Docking, ADMET.

Members of the KCTD family are major regulators of cAMP signaling

Cyclic adenosine monophosphate (cAMP) is a pivotal second messenger with an essential role in neuronal function. cAMP synthesis by adenylyl cyclases (AC) is controlled by G protein–coupled receptor (GPCR) signaling systems. However, the network of molecular players involved in the process is incompletely defined. Here, we used CRISPR/Cas9–based screening to identify that members of the potassium channel tetradimerization domain (KCTD) family are major regulators of cAMP signaling. Focusing on striatal neurons, we show that the dominant isoform KCTD5 exerts its effects through an unusual mechanism that modulates the influx of Zn2+ via the Zip14 transporter to exert unique allosteric effects on AC. We further show that KCTD5 controls the amplitude and sensitivity of stimulatory GPCR inputs to cAMP production by Gβγ-mediated AC regulation. Finally, we report that KCTD5 haploinsufficiency in mice leads to motor deficits that can be reversed by chelating Zn2+. Together, our findings uncover KCTD proteins as major regulators of neuronal cAMP signaling via diverse mechanisms.

Pharmacological Actions of Myricetin in the Nervous System: A Comprehensive Review of Preclinical Studies in Animals and Cell Models

Myricetin is a natural flavonoid extracted from a variety of plants, such as medicinal herbs, vegetables, berries, and tea leaves. A growing body of evidence has reported that myricetin supplementation display therapeutic activities in a lot of nervous system disorders, such as cerebral ischemia, Alzheimer’s disease, Parkinson’s disease, epilepsy, and glioblastoma. Myricetin supplementation can also protect against pathological changes and behavioral impairment induced by multiple sclerosis and chronic stress. On the basis of these pharmacological actions, myricetin could be developed as a potential drug for the prevention and/or treatment of nervous system disorders. Mechanistic studies have shown that inhibition of oxidative stress, cellular apoptosis, and neuroinflammatory response are common mechanisms for the neuroprotective actions of myricetin. Other mechanisms, including the activation of the nuclear factor E2-related factor 2 (Nrf2), extracellular signal-regulated kinase 1/2 (ERK1/2), protein kinase B (Akt), cyclic adenosine monophosphate-response element binding protein (CREB), and brain-derived neurotrophic factor (BDNF) signaling, inhibition of intracellular Ca2+ increase, inhibition of c-Jun N-terminal kinase (JNK)-p38 activation, and suppression of mutant protein aggregation, may also mediate the neuroprotective effects of myricetin. Furthermore, myricetin treatment has been shown to promote the activation of the inhibitory neurons in the hypothalamic paraventricular nucleus, which subsequently produces anti-epilepsy effects. In this review, we make a comprehensive understanding about the pharmacological effects of myricetin in the nervous system, aiming to push the development of myricetin as a novel drug for the treatment of nervous system disorders.

High-Throughput Screening Campaign Identified a Potential Small Molecule RXFP3/4 Agonist

Relaxin/insulin-like family peptide receptor 3 (RXFP3) belongs to class A G protein-coupled receptor family. RXFP3 and its endogenous ligand relaxin-3 are mainly expressed in the brain with important roles in the regulation of appetite, energy metabolism, endocrine homeostasis and emotional processing. It is therefore implicated as a potential target for treatment of various central nervous system diseases. Since selective agonists of RXFP3 are restricted to relaxin-3 and its analogs, we conducted a high-throughput screening campaign against 32,021 synthetic and natural product-derived compounds using a cyclic adenosine monophosphate (cAMP) measurement-based method. Only one compound, WNN0109-C011, was identified following primary screening, secondary screening and dose-response studies. Although displayed agonistic effect in cells overexpressing the human RXFP3, it also showed cross-reactivity with the human RXFP4. This hit compound may provide not only a chemical probe to investigate the function of RXFP3/4, but also a novel scaffold for the development of RXFP3/4 agonists.

Synergistic Induction of Chicken Antimicrobial Host Defense Peptide Gene Expression by Butyrate and Sugars

Antimicrobial resistance is a major concern to public health demanding effective alternative strategies to disease control and prevention. Modulation of endogenous host defense peptide (HDP) synthesis has emerged as a promising antibiotic alternative approach. This study investigated a potential synergy between sugars and butyrate in inducing HDP gene expression in chickens. Our results revealed that sugars differentially regulated HDP expression in both gene- and sugar-specific manners in chicken HD11 macrophage cells. Among eight mono- and disaccharides tested, all were potent inducers of avian β-defensin 9 (AvBD9) gene (p<0.05), but only galactose, trehalose, and lactose obviously upregulated cathelicidin-B1 (CATHB1) gene expression. The expression of AvBD14 gene, on the other hand, was minimally influenced by sugars. Moreover, all sugars exhibited a strong synergy with butyrate in enhancing AvBD9 expression, while only galactose, trehalose, and lactose were synergistic with butyrate in CATHB1 induction. No synergy in AvBD14 induction was observed between sugars and butyrate. Although lactose augmented the expression of nearly all HDP genes, its synergy with butyrate was only seen with several, but not all, HDP genes. Mucin-2 gene was also synergistically induced by a combination of lactose and butyrate. Furthermore, lactose synergized with butyrate to induce AvBD9 expression in chicken jejunal explants (p<0.05). Mechanistically, hyper-acetylation of histones was observed in response to both butyrate and lactose, relative to individual compounds. Mitogen-activated protein kinase, NF-κB, and cyclic adenosine monophosphate signaling pathways were also found to be involved in butyrate- and lactose-mediated synergy in AvBD9 induction. Collectively, a combination of butyrate and a sugar with both HDP-inducing and barrier protective activities holds the promise to be developed as an alternative to antibiotics for disease control and prevention.

The 3-phosphoinositide–dependent protein kinase 1 is an essential upstream activator of protein kinase A in malaria parasites

Cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) signalling is essential for the proliferation of Plasmodium falciparum malaria blood stage parasites. The mechanisms regulating the activity of the catalytic subunit PfPKAc, however, are only partially understood, and PfPKAc function has not been investigated in gametocytes, the sexual blood stage forms that are essential for malaria transmission. By studying a conditional PfPKAc knockdown (cKD) mutant, we confirm the essential role for PfPKAc in erythrocyte invasion by merozoites and show that PfPKAc is involved in regulating gametocyte deformability. We furthermore demonstrate that overexpression of PfPKAc is lethal and kills parasites at the early phase of schizogony. Strikingly, whole genome sequencing (WGS) of parasite mutants selected to tolerate increased PfPKAc expression levels identified missense mutations exclusively in the gene encoding the parasite orthologue of 3-phosphoinositide–dependent protein kinase-1 (PfPDK1). Using targeted mutagenesis, we demonstrate that PfPDK1 is required to activate PfPKAc and that T189 in the PfPKAc activation loop is the crucial target residue in this process. In summary, our results corroborate the importance of tight regulation of PfPKA signalling for parasite survival and imply that PfPDK1 acts as a crucial upstream regulator in this pathway and potential new drug target.

Identification and characterization of an atypical Gαs-biased β2AR agonist that fails to evoke airway smooth muscle cell tachyphylaxis

G protein–coupled receptors display multifunctional signaling, offering the potential for agonist structures to promote conformational selectivity for biased outputs. For β2-adrenergic receptors (β2AR), unbiased agonists stabilize conformation(s) that evoke coupling to Gαs (cyclic adenosine monophosphate [cAMP] production/human airway smooth muscle [HASM] cell relaxation) and β-arrestin engagement, the latter acting to quench Gαs signaling, contributing to receptor desensitization/tachyphylaxis. We screened a 40-million-compound scaffold ranking library, revealing unanticipated agonists with dihydroimidazolyl-butyl-cyclic urea scaffolds. The S-stereoisomer of compound C1 shows no detectable β-arrestin engagement/signaling by four methods. However, C1-S retained Gαs signaling—a divergence of the outputs favorable for treating asthma. Functional studies with two models confirmed the biasing: β2AR-mediated cAMP signaling underwent desensitization to the unbiased agonist albuterol but not to C1-S, and desensitization of HASM cell relaxation was observed with albuterol but not with C1-S. These HASM results indicate biologically pertinent biasing of C1-S, in the context of the relevant physiologic response, in the human cell type of interest. Thus, C1-S was apparently strongly biased away from β-arrestin, in contrast to albuterol and C5-S. C1-S structural modeling and simulations revealed binding differences compared with unbiased epinephrine at transmembrane (TM) segments 3,5,6,7 and ECL2. C1-S (R2 = cyclohexane) was repositioned in the pocket such that it lost a TM6 interaction and gained a TM7 interaction compared with the analogous unbiased C5-S (R2 = benzene group), which appears to contribute to C1-S biasing away from β-arrestin. Thus, an agnostic large chemical-space library identified agonists with receptor interactions that resulted in relevant signal splitting of β2AR actions favorable for treating obstructive lung disease.