Levels Of Second Messengers Research Articles

Structures of the multi-domain oxygen sensor DosP: remote control of a c-di-GMP phosphodiesterase by a regulatory PAS domain

The heme-based direct oxygen sensor DosP degrades c-di-GMP, a second messenger nearly unique to bacteria. In stationary phase Escherichia coli, DosP is the most abundant c-di-GMP phosphodiesterase. Ligation of O2 to a heme-binding PAS domain (hPAS) of the protein enhances the phosphodiesterase through an allosteric mechanism that has remained elusive. We determine six structures of full-length DosP in its aerobic or anaerobic conformations, with or without c-di-GMP. DosP is an elongated dimer with the regulatory heme containing domain and phosphodiesterase separated by nearly 180 Å. In the absence of substrate, regardless of the heme status, DosP presents an equilibrium of two distinct conformations. Binding of substrate induces DosP to adopt a single, ON-state or OFF-state conformation depending on its heme status. Structural and biochemical studies of this multi-domain sensor and its mutants provide insights into signal regulation of second-messenger levels.

Structures of the multi-domain oxygen sensor DosP: remote control of a c-di-GMP phosphodiesterase by a regulatory PAS domain.

The heme-based direct oxygen sensor DosP degrades c-di-GMP, a second messenger nearly unique to bacteria. In stationary phase Escherichia coli, DosP is the most abundant c-di-GMP phosphodiesterase. Ligation of O2 to a heme-binding PAS domain (hPAS) of the protein enhances the phosphodiesterase through an allosteric mechanism that has remained elusive. We determined six structures of full-length DosP in its aerobic or anaerobic conformations, with or without c-di-GMP. DosP is an elongated dimer with the regulatory heme and phosphodiesterase separated by nearly 180 Å. In the absence of substrate, regardless of the heme status, DosP presents an equilibrium of two distinct conformations. Binding of substrate induces DosP to adopt a single, ON-state or OFF-state conformation depending on its heme status. Structural and biochemical studies of this multi-domain sensor and its mutants provide insights into signal regulation of second-messenger levels.

Regulation Mechanism of Dopamine Receptor 1 in Low Temperature Response of Marsupenaeus japonicus.

Dopamine receptors (DARs) are important transmembrane receptors responsible for receiving extracellular signals in the DAR-mediated signaling pathway, and are involved in a variety of physiological functions. Herein, the D1 DAR gene from Marsupenaeus japonicus (MjDAD1) was identified and characterized. The protein encoded by MjDAD1 has the typical structure and functional domains of the G-protein coupled receptor family. MjDAD1 expression was significantly upregulated in the gills and hepatopancreas after low temperature stress. Moreover, double-stranded RNA-mediated silencing of MjDAD1 significantly changed the levels of protein kinases (PKA and PKC), second messengers (cyclic AMP (cAMP), cyclic cGMP, calmodulin, and diacyl glycerol), and G-protein effectors (adenylate cyclase and phospholipase C). Furthermore, MjDAD1 silencing increased the apoptosis rate of gill and hepatopancreas cells. Thus, following binding to their specific receptors, G-protein effectors are activated by MjDAD1, leading to DAD1-cAMP/PKA pathway-mediated regulation of caspase-dependent mitochondrial apoptosis. We suggest that MjDAD1 is indispensable for the environmental adaptation of M. japonicus.

Presence of Small-Conductance Calcium-Activated Potassium (SK) Channels in the Central and Peripheral Nervous Systems and Their Role in Health and Disease.

Potassium (K+) channels establish and maintain the resting potential of most living cells. Their activity is predominantly regulated by the membrane voltage or the K+ gradient across the cell membrane. However, many cells also express small-conductance calcium-activated potassium (SK) channels, which have the unique ability to translate changes in the level of the intracellular second messenger, Ca2+ to changes in the membrane K+ conductance and, therefore, the resting membrane potential. This article reviews the structure, presence, distribution, and function of SK channels, their pharmacological modulation, and their role in health and disease, emphasizing nociception and pain.

The injection of one recombinant C-type lectin (LvLec) induced the immune response of hemocytes in Litopenaeus vannamei

To explore the immune function of C-type lectin in shrimp, one recombinant C-type lectin (LvLec) was injected into Litopenaeus vannamei. There were four treatments in the experiment: saline group (as control group), recombinant C-type lectin group (LvLec, 1 mg mL−1), Vibrio harveyi group (V. harveyi, 106 cfu mL−1) and recombinant C-type lectin combined with Vibrio harveyi group (LvLec + V. harveyi, 1 mg mL−1 + 106 cfu mL−1). The sampling time was set at 0, 3, 6, 9, 12, 24 h after the injection. The results showed that the total hemocyte count decreased significantly and the phagocytic activity improved notably after the injection of LvLec, V. harveyi or LvLec + V. harveyi. Prophenoloxidase (proPO) activity decreased, while phenoloxidase (PO) activity increased and the changing degree of each group exhibited a significant difference. The hemagglutinating activity and bacteriolytic activity improved significantly, while the antimicrobial activity did not show a remarkable change in all of the groups. There were also changes that occurred in the levels of second messengers (cAMP, cGMP) and protein kinase (PKA, PKG). After the injection of LvLec, V. harveyi or LvLec + V. harveyi, the concentration of cGMP and PKA increased significantly, while the concentration of cAMP and PKG did not change remarkably. The results above suggested that rLvLec could induce nonspecific immune response, including phagocytosis, release of PO, hemagglutination and bacteriolysis through cGMP-PKA pathway in vivo.

Hyperinsulinemia Induced Altered Insulin Signaling Pathway in Muscle of High Fat- and Carbohydrate-Fed Rats: Effect of Exercise.

Insulin resistance is a state of impaired responsiveness to insulin action. This condition not only results in deficient glucose uptake but increases the risk for cardiovascular diseases (CVD), stroke, and obesity. The present work investigates the molecular mechanisms of high carbohydrate and fat diet in inducing prediabetic hyperinsulinemia and effect of exercise on InsR signaling events, muscular AChE, and lactate dehydrogenase activity. Adult male Wistar rats were divided into the control (C) diet group, high-carbohydrate diet (HCD) group, high-fat diet (HFD) group, and HCD and HFD groups with exercise (HCD Ex and HFD Ex, respectively). Acetyl choline esterase activity, lactate dehydrogenase activity, total lactate levels, IRS1 phosphorylations, and Glut4 expression patterns were studied in the muscle tissue among these groups. High carbohydrate and fat feeding led to hyperinsulinemic status with reduced acetylcholine esterase (AChE) activity and impaired phosphorylation of IRS1 along with increased lactate concentrations in the muscle. Exercise significantly upregulated phosphoinositide 3 kinase (PI3K) docking site phosphorylation and downregulated the negative IRS1 phosphorylations thereby increasing the glucose transporter (GLUT) expressions and reducing the lactate accumulation. Also, the levels of second messengers like IP3 and cAMP were increased with exercise. Increased second messenger levels induce calcium release thereby activating the downstream pathway promoting the translocation of GLUT4 to the plasma membrane. Our results showed that the metabolic and signaling pathway dysregulations seen during diet-induced hyperinsulinemia, a metabolic condition seen during the early stages in the development of prediabetes, were improved with vigorous physical exercise. Thus, exercise can be considered as an excellent management approach over drug therapy for diabetes and its complications.

A Bacteriophytochrome Mediates Interplay between Light Sensing and the Second Messenger Cyclic Di-GMP to Control Social Behavior and Virulence

Bacteriophytochromes are the most abundant and ubiquitous light-sensing receptors in bacteria and are involved in time-of-day behavior or responses. However, their biological and regulatory role in non-photosynthetic bacteria is poorly understood, and even less is known about how they regulate diverse cellular processes. Here, we show that a bacteriophytochrome (XooBphP) from the plant pathogen Xanthomonas oryzae pv. oryzae perceives light signals and transduces a signal through its EAL-mediated phosphodiesterase activity, modulating the intracellular level of the ubiquitous bacterial second messenger c-di-GMP. We discover that light-mediated fine-tuning of intracellular c-di-GMP levels by XooBphP regulates production of virulence functions, iron metabolism, and transition from a sessile to a free-swimming motile lifestyle, contributing to its colonization of the host and virulence. XooBphP thus plays a crucial role in integrating photo-sensing with intracellular signaling to control the pathogenic lifestyle and social behavior.

Molecular Properties of New Enzyme Rhodopsins with Phosphodiesterase Activity.

The choanoflagellate Salpingoeca rosetta contains a chimeric rhodopsin protein composed of an N-terminal rhodopsin (Rh) domain and a C-terminal cyclic nucleotide phosphodiesterase (PDE) domain. The Rh-PDE enzyme (SrRh-PDE), which decreases the concentrations of cyclic nucleotides such as cGMP and cAMP in light, is a useful tool in optogenetics. Recently, eight additional Rh-PDE enzymes were found in choanoflagellate species, four from Choanoeca flexa and the other four from other species. In this paper, we studied the molecular properties of these new Rh-PDEs, which were compared with SrRh-PDE. Upon expression in HEK293 cells, four Rh-PDE proteins, including CfRh-PDE2 and CfRh-PDE3, exhibited no PDE activity when assessed by in-cell measurements and in vitro HPLC analysis. On the other hand, CfRh-PDE1 showed light-dependent PDE activity toward cGMP, which absorbed maximally at 491 nm. Therefore, CfRh-PDE1 is presumably responsible for colony inversion in C. flexa by absorbing blue-green light. The molecular properties of MrRh-PDE were similar to those of SrRh-PDE, although the λmax of MrRh-PDE (516 nm) was considerably red-shifted from that of SrRh-PDE (492 nm). One Rh-PDE, AsRh-PDE, did not contain the retinal-binding Lys at TM7 and showed cAMP-specific PDE activity both in the dark and light. These results provide mechanistic insight into rhodopsin-mediated, light-dependent regulation of second-messenger levels in eukaryotic microbes.

A Bacteriophytochrome Mediates Interplay between Light Sensing and the Second Messenger Cyclic Di-GMP to Control Bacterial Social Behaviors

Bacteriophytochromes are the most abundant and ubiquitous light sensing receptors in bacteria and are involved in time-of-day behavior or responses. However, their biological and regulatory role in non-photosynthetic bacteria is poorly understood, and even less is known about how it regulates diverse cellular processes. Here we show that a bacteriophytochrome (XooBphP), from the plant pathogen Xanthomonas oryzae pv. oryzae, perceives light signal and transduces through its EAL-mediated phosphodiesterase activity, modulating the intracellular level of the ubiquitous bacterial second messenger c-di-GMP. We discover that the light mediated fine-tuning of the intracellular c-di-GMP levels by XooBphP regulate the production of virulence associated functions, iron metabolism and transition of sessile to free-swimming motile lifestyle, contributing to the host colonization and virulence. Altogether, our results reveal that XooBphP plays a crucial role in integrating photo-sensing with intracellular signaling to control pathogenic lifestyle and bacterial social behaviors.

Methods to Investigate Signal Transduction Pathways in Trypanosoma cruzi: Cyclic Nucleotide Phosphodiesterases Assay Protocols.

Intracellular levels of cyclic nucleotide second messengers are regulated predominantly by a large superfamily of phosphodiesterases (PDEs). Most of the different PDE variants play specific physiological functions; in fact, PDEs can associate with other proteins allowing them to be strategically anchored throughout the cell. In this regard, precise cellular expression and compartmentalization of these enzymes produce the specific control of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) gradients in cells and enable their integration with other signaling pathways.In trypanosomatids, some PDEs are essential for their survival and play fundamental roles in the adaptation of these parasites to different environmental stresses, as well as in the differentiation between their different life cycle forms. Given that these enzymes not only are similar to human PDEs but also have differential biochemical properties, and due to the great knowledge of drugs that target human PDEs, trypanosomatid PDEs could be postulated as important therapeutic targets through the repositioning of drugs.In this chapter, we describe a simple and sensitive radioisotope-based method to measure cyclic 3',5'-nucleotide phosphodiesterase using [3H]cAMP.

Application of Free Energy Perturbation (FEP+) to Understanding Ligand Selectivity: A Case Study to Assess Selectivity Between Pairs of Phosphodiesterases (PDE's).

Cyclic nucleotide phosphodiesterases (PDE's) are metalloenzymes that play a key role in regulating the levels of the ubiquitous second messengers, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). In humans, 11 PDE protein families mediate numerous biochemical pathways throughout the body and are effective drug targets for the treatment of diseases ranging from central nervous system disorders to heart and pulmonary diseases. PDE's also share a highly conserved catalytic site (about 50%), thus making the design of selective drug candidates very challenging with classical structure-based design approaches given also the lack of publicly available co-crystal structures of pairs of PDE's with consistent biological data to be compared, as we show in our work. In this retrospective study, we apply free energy perturbation (FEP+) to predict the selectivity of inhibitors that bind two pairs of closely related PDE families: PDE9/1 and PDE5/6 where only 1 co-crystal structure per pair is publicly available. As another challenge, the p Ka of the PDE5/6 inhibitor is close to the experimental pH, making unclear the exact protonation state that should be used in the computational workflow. We demonstrate that running FEP+ on homology models constructed for these metalloenzymes accurately reproduces experimentally observed selectivity profiles also addressing the unclear protonation state to be used during computation with our recently developed p Ka-correction method. Based on these data, we conclude that FEP+ is a robust method for prediction of selectivity for this target class and may be helpful to address related lead optimization challenges in drug discovery.

Spectroscopic study of the transmembrane domain of a rhodopsin–phosphodiesterase fusion protein from a unicellular eukaryote

The choanoflagellate Salpingoeca rosetta contains a chimeric rhodopsin protein composed of an N-terminal rhodopsin (Rh) domain and a C-terminal cyclic nucleotide phosphodiesterase (PDE) domain. The Rh-PDE enzyme light-dependently decreases the concentrations of cyclic nucleotides such as cGMP and cAMP. Photoexcitation of purified full-length Rh-PDE yields an "M" intermediate with a deprotonated Schiff base, and its recovery is much faster than that of the enzyme domain. To gain structural and mechanistic insights into the Rh domain, here we expressed and purified the transmembrane domain of Rh-PDE, Rh-PDE(TMD), and analyzed it with transient absorption, light-induced difference UV-visible, and FTIR spectroscopy methods. These analyses revealed that the "K" intermediate forms within 0.005 ms and converts into the M intermediate with a time constant of 4 ms, with the latter returning to the original state within 4 s. FTIR spectroscopy revealed that all-trans to 13-cis photoisomerization occurs as the primary event during which chromophore distortion is located at the middle of the polyene chain, allowing the Schiff base to form a stronger hydrogen bond. We also noted that the peptide backbone of the α-helix becomes deformed upon M intermediate formation. Results from site-directed mutagenesis suggested that Glu-164 is protonated and that Asp-292 acts as the only Schiff base counterion in Rh-PDE. A strong reduction of enzymatic activity in a D292N variant, but not in an E164Q variant, indicated an important catalytic role of the negative charge at Asp-292. Our findings provide further mechanistic insights into rhodopsin-mediated, light-dependent regulation of second-messenger levels in eukaryotic microbes.

Expression and purification of human diacylglycerol kinase α from baculovirus-infected insect cells for structural studies.

Diacylglycerol kinases (DGKs) are lipid kinases that modulate the levels of lipid second messengers, diacylglycerol and phosphatidic acid. Recently, increasing attention has been paid to its α isozyme (DGKα) as a potential target for cancer immunotherapy. DGKα consists of the N-terminal regulatory domains including EF-hand motifs and C1 domains, and the C-terminal catalytic domain (DGKα-CD). To date, however, no structures of mammalian DGKs including their CDs have yet been reported, impeding our understanding on the catalytic mechanism of DGKs and the rational structure-based drug design. Here we attempted to produce DGKα-CD or a full-length DGKα using bacterial and baculovirus-insect cell expression system for structural studies. While several DGKα-CD constructs produced using both bacterial and insect cells formed insoluble or soluble aggregates, the full-length DGKα expressed in insect cells remained soluble and was purified to near homogeneity as a monomer with yields (1.3 mg/mL per one L cell culture) feasible for protein crystallization. Following enzymatic characterization showed that the purified DGKα is in fully functional state. We further demonstrated that the purified enzyme could be concentrated without any significant aggregation, and characterized its secondary structure by circular dichroism. Taken together, these results suggest that the presence of N-terminal regulatory domains suppress protein aggregation likely via their intramolecular interactions with DGKα-CD, and demonstrate that the baculovirus-insect cell expression of the full-length form of DGKα, not DGKα-CD alone, represents a promising approach to produce protein sample for structural studies of DGKα. Thus, our study will encourage future efforts to determine the crystal structure of DGK, which has not been determined since it was first identified in 1959.

AKR1B10 activates diacylglycerol (DAG) second messenger in breast cancer cells.

Aldo-keto reductase 1B10 (AKR1B10) is upregulated in breast cancer and promotes tumor growth and metastasis. However, little is known of the molecular mechanisms of action. Herein we report that AKR1B10 activates lipid second messengers to stimulate cell proliferation. Our data showed that ectopic expression of AKR1B10 in breast cancer cells MCF-7 promoted lipogenesis and enhanced levels of lipid second messengers, including phosphatidylinositol bisphosphate (PIP2), diacylglycerol (DAG), and inositol triphosphate (IP3). In contrast, silencing of AKR1B10 in breast cancer cells BT-20 and colon cancer cells HCT-8 led to decrease of these lipid messengers. Qualitative analyses by liquid chromatography-mass spectrum (LC-MS) revealed that AKR1B10 regulated the cellular levels of total DAG and majority of subspecies. This in turn modulated the phosphorylation of protein kinase C (PKC) isoforms PKCδ (Thr505), PKCµ (Ser744/748), and PKCα/βII (Thr638/641) and activity of the PKC-mediated c-Raf/MEK/ERK signaling cascade. A pan inhibitor of PKC (Go6983) blocked ERK1/2 activation by AKR1B10. In these cells, phospho-p90RSK, phospho-MSK, and Cyclin D1 expression was increased by AKR1B10, and pharmacological inhibition of the ERK signaling cascade with MEK1/2 inhibitors U0126 and PD98059 eradicated induction of phospho-p90RSK, phospho-MSK, and Cyclin D1. In breast cancer cells, AKR1B10 promoted the clonogenic growth and proliferation of breast cancer cells in two-dimension (2D) and three-dimension (3D) cultures and tumor growth in immunodeficient female nude mice through activation of the PKC/ERK pathway. These data suggest that AKR1B10 stimulates breast cancer cell growth and proliferation through activation of DAG-mediated PKC/ERK signaling pathway.

Nitrate Sensing and Metabolism Inhibit Biofilm Formation in the Opportunistic Pathogen Burkholderia pseudomallei by Reducing the Intracellular Concentration of c-di-GMP.

The opportunistic pathogen Burkholderia pseudomallei is a saprophytic bacterium and the causative agent of melioidosis, an emerging infectious disease associated with high morbidity and mortality. Although melioidosis is most prevalent during the rainy season in endemic areas, domestic gardens and farms can also serve as a reservoir for B. pseudomallei during the dry season, in part due to irrigation and fertilizer use. In the environment, B. pseudomallei forms biofilms and persists in soil near plant root zones. Biofilms are dynamic bacterial communities whose formation is regulated by extracellular cues and corresponding changes in the nearly universal secondary messenger cyclic dimeric GMP. Recent studies suggest B. pseudomallei loads are increased by irrigation and the addition of nitrate-rich fertilizers, whereby such nutrient imbalances may be linked to the transmission epidemiology of this important pathogen. We hypothesized that exogenous nitrate inhibits B. pseudomallei biofilms by reducing the intracellular concentration of c-di-GMP. Bioinformatics analyses revealed B. pseudomallei 1026b has the coding capacity for nitrate sensing, metabolism, and transport distributed on both chromosomes. Using a sequence-defined library of B. pseudomallei 1026b transposon insertion mutants, we characterized the role of denitrification genes in biofilm formation in response to nitrate. Our results indicate that the denitrification pathway is implicated in B. pseudomallei biofilm growth dynamics and biofilm formation is inhibited by exogenous addition of sodium nitrate. Genomics analysis identified transposon insertional mutants in a predicted two-component system (narX/narL), a nitrate reductase (narGH), and a nitrate transporter (narK-1) required to sense nitrate and alter biofilm formation. Additionally, the results presented here show that exogenous nitrate reduces intracellular levels of the bacterial second messenger c-di-GMP. These results implicate the role of nitrate sensing in the regulation of a c-di-GMP phosphodiesterase and the corresponding effects on c-di-GMP levels and biofilm formation in B. pseudomallei 1026b.

Beyond Intracellular Signaling: The Ins and Outs of Second Messengers Microdomains.

A typical characteristic of eukaryotic cells compared to prokaryotes is represented by the spatial heterogeneity of the different structural and functional components: for example, most of the genetic material is surrounded by a highly specific membrane structure (the nuclear membrane), continuous with, yet largely different from, the endoplasmic reticulum (ER); oxidative phosphorylation is carried out by organelles enclosed by a double membrane, the mitochondria; in addition, distinct domains, enriched in specific proteins, are present in the plasma membrane (PM) of most cells. Less obvious, but now generally accepted, is the notion that even the concentration of small molecules such as second messengers (Ca2+ and cAMP in particular) can be highly heterogeneous within cells. In the case of most organelles, the differences in the luminal levels of second messengers depend either on the existence on their membrane of proteins that allow the accumulation/release of the second messenger (e.g., in the case of Ca2+, pumps, exchangers or channels), or on the synthesis and degradation of the specific molecule within the lumen (the autonomous intramitochondrial cAMP system). It needs stressing that the existence of a surrounding membrane does not necessarily imply the existence of a gradient between the cytosol and the organelle lumen. For example, the nuclear membrane is highly permeable to both Ca2+ and cAMP (nuclear pores are permeable to solutes up to 50kDa) and differences in [Ca2+] or [cAMP] between cytoplasm and nucleoplasm are not seen in steady state and only very transiently during cell activation. A similar situation has been observed, as far as Ca2+ is concerned, in peroxisomes.

Investigation of PDE5/PDE6 and PDE5/PDE11 selective potent tadalafil-like PDE5 inhibitors using combination of molecular modeling approaches, molecular fingerprint-based virtual screening protocols and structure-based pharmacophore development

The essential biological function of phosphodiesterase (PDE) type enzymes is to regulate the cytoplasmic levels of intracellular second messengers, 3′,5′-cyclic guanosine monophosphate (cGMP) and/or 3′,5′-cyclic adenosine monophosphate (cAMP). PDE targets have 11 isoenzymes. Of these enzymes, PDE5 has attracted a special attention over the years after its recognition as being the target enzyme in treating erectile dysfunction. Due to the amino acid sequence and the secondary structural similarity of PDE6 and PDE11 with the catalytic domain of PDE5, first-generation PDE5 inhibitors (i.e. sildenafil and vardenafil) are also competitive inhibitors of PDE6 and PDE11. Since the major challenge of designing novel PDE5 inhibitors is to decrease their cross-reactivity with PDE6 and PDE11, in this study, we attempt to identify potent tadalafil-like PDE5 inhibitors that have PDE5/PDE6 and PDE5/PDE11 selectivity. For this aim, the similarity-based virtual screening protocol is applied for the “clean drug-like subset of ZINC database” that contains more than 20 million small compounds. Moreover, molecular dynamics (MD) simulations of selected hits complexed with PDE5 and off-targets were performed in order to get insights for structural and dynamical behaviors of the selected molecules as selective PDE5 inhibitors. Since tadalafil blocks hERG1 K channels in concentration dependent manner, the cardiotoxicity prediction of the hit molecules was also tested. Results of this study can be useful for designing of novel, safe and selective PDE5 inhibitors.

Effect of 24-epibrassinolide on Brassica napus alternative respiratory pathway, guard cells movements and phospholipid signaling under salt stress

Using Brassica napus roots we observed statistically significant increase in alternative respiratory pathway in response to exogenous 24-epibrassinolide (EBL) under optimal conditions and salinity. Also we observed activation of phospholipid signaling under the same conditions in response to EBL by measuring levels of lipid second messengers – diacylglycerol (DAG) and phosphatidic acid (PA). We found that brassinosteroids cause closure of stomata in isolated leaf disks while inhibitors of alternative oxidase cancelled these effects. This study demonstrates that BRs activate total respiration rate, alternative respiratory pathway, production of PA and DAG, stimulate stomata closure and growth under optimal conditions and salinity. Also, specific inhibitor of brassinosteroids biosynthesis decreased alternative respiratory pathway and production of lipid messengers in rape plants.

In silico design of novel hERG-neutral sildenafil-like PDE5 inhibitors

Cyclic nucleotide phosphodiesterase enzymes (PDEs) have functions in regulating the levels of intracellular second messengers, 3′, 5′-cyclic adenosine monophosphate (cAMP) and 3′, 5′-cyclic guanosine monophosphate (cGMP), via hydrolysis and decomposing mechanisms in cells. They take essential roles in modulating various cellular activities such as memory and smooth muscle functions. PDE type 5 (PDE5) inhibitors enhance the vasodilatory effects of cGMP in the corpus cavernosum and they are used to treat erectile dysfunction. Patch clamp experiments showed that the IC50 values of the human ether-à-go-go-related gene (hERG1) potassium (K) ion channel blocking affinity of PDE5 inhibitors sildenafil, vardenafil, and tadalafil as 33, 12, and 100 μM, respectively. hERG1 channel is responsible for the regulation of the action potential of human ventricular myocyte by contributing the rapid component of delayed rectifier K+ current (IKr) component of the cardiac action potential. In this work, interaction patterns and binding affinity predictions of selected PDE5 inhibitors against the hERG1 channel are studied. It is attempted to develop PDE5 inhibitor analogs with lower binding affinity to hERG1 ion channel while keeping their pharmacological activity against their principal target PDE5 using in silico methods. Based on detailed analyses of docking poses and predicted interaction energies, novel analogs of PDE5 inhibitors with lower predicted binding affinity to hERG1 channels without loosing their principal target activity were proposed. Moreover, molecular dynamics (MD) simulations and post-processing MD analyses (i.e. Molecular Mechanics/Generalized Born Surface Area calculations) were performed. Detailed analysis of molecular simulations helped us to better understand the PDE5 inhibitor–target binding interactions in the atomic level. Results of this study can be useful for designing of novel and safe PDE5 inhibitors with enhanced activity and other tailored properties.

Second Messengers.

Second messengers are small molecules and ions that relay signals received by cell-surface receptors to effector proteins. They include a wide variety of chemical species and have diverse properties that allow them to signal within membranes (e.g., hydrophobic molecules such as lipids and lipid derivatives), within the cytosol (e.g., polar molecules such as nucleotides and ions), or between the two (e.g., gases and free radicals). Second messengers are typically present at low concentrations in resting cells and can be rapidly produced or released when cells are stimulated. The levels of second messengers are exquisitely controlled temporally and spatially, and, during signaling, enzymatic reactions or opening of ion channels ensure that they are highly amplified. These messengers then diffuse rapidly from the source and bind to target proteins to alter their properties (activity, localization, stability, etc.) to propagate signaling.