Innate immune pathways are the first line of cellular defense against pathogen infections ranging from bacteria to Metazoa. These pathways are activated following the recognition of pathogen associated molecular patterns (PAMPs) by membrane and cytosolic pattern recognition receptors. In addition, some of these cellular sensors can also recognize endogenous danger-associated molecular patterns (DAMPs) arising from damaged or dying cells and triggering innate immune responses. Among the cytosolic nucleic acid sensors, the cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS) plays an essential role in the activation of the type I interferon (IFNs) response and the production of pro-inflammatory cytokines. Indeed, upon nucleic acid binding, cGAS synthesizes cGAMP, a second messenger mediating the activation of the STING signaling pathway. The functional conservation of the cGAS-STING pathway during evolution highlights its importance in host cellular surveillance against pathogen infections. Apart from their functions in immunity, cGAS and STING also play major roles in nuclear functions and tumor development. Therefore, cGAS-STING is now considered as an attractive target to identify novel biomarkers and design therapeutics for auto-inflammatory and autoimmune disorders as well as infectious diseases and cancer. Here, we review the current knowledge about the structure of cGAS and the evolution from bacteria to Metazoa and present its main functions in defense against pathogens and cancer, in connection with STING. The advantages and limitations of in vivo models relevant for studying the cGAS-STING pathway will be discussed for the notion of species specificity and in the context of their integration into therapeutic screening assays targeting cGAG and/or STING.

Guanosine monophosphate, among the nucleotides, has the unique property to self-associate and form nanoscale cylinders consisting of hydrogen-bonded G-quartet disks, which are stacked on top of one another. Such self-assemblies describe not only the basic structural motif of G-quadruplexes formed by, e.g., telomeric DNA sequences, but are also interesting targets for supramolecular chemistry and nanotechnology. The G-quartet stacks serve as an excellent model to understand the fundamentals of their molecular self-association and to unveil their application spectrum. However, the thermodynamic stability of such self-assemblies over an extended temperature and pressure range is largely unexplored. Here, we report a combined FTIR and NMR study on the temperature and pressure stability of G-quartet stacks formed by disodium guanosine 5'-monophosphate (Na25'-GMP). We found that under abyssal conditions, where temperatures as low as 5 °C and pressures up to 1 kbar are reached, the self-association of Na25'-GMP is most favoured. Beyond those conditions, the G-quartet stacks dissociate laterally into monomer stacks without significantly changing the longitudinal dimension. Among the tested alkali cations, K+ is the most efficient one to elevate the temperature as well as the pressure limits of GMP self-assembly.

Cyclic nucleotides mediate transient as well as plastic cellular responses. The most ultimate response is cell death. In the present study, we propose that an increase of intracellular cyclic guanosine monophosphate (cGMP) for at least 1h promotes cell death in the murine microglial cell line, BV-2 cells, as well as in primary murine microglia. Cells were exposed to ammonium, the guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), and to the membrane-permeable cGMP analogue, 8-Bromo-cGMP (8-Br-cGMP), respectively. Cell death was estimated using DAPI labelling and annexin-V labelling of exposed phosphatidylserine, and cGMP level was quantified by an immunoassay. Ammonium not only increased the number of apoptotic cells but also promoted a moderate increase in intracellular cGMP. Addition of ODQ suppressed ammonium-induced apoptosis. Furthermore, we found that 8-Br-cGMP significantly increased the number of BV-2 cells and primary microglia, respectively, containing nuclei with condensed chromatin accumulated at the nuclear periphery. Similarly, cells exposed to 8-Br-cGMP showed significantly more cells with exposed phosphatidylserine compared to control cells. Thus, according to the nuclear structure as well as to changes in the plasma membrane, chronic elevation of cGMP induces apoptosis in microglia.

The role of different vascular subtypes of phosphodiesterases (PDE) in cGMP compartmentalization was evaluated in human smooth muscle cells.

Background cGMP mediates numerous cardioprotective functions and is a potential therapeutic target for cardiovascular disease. Preclinical studies suggest that plasma cGMP is reflective of natriuretic peptide stimulation. Epidemiologic associations between cGMP and natriuretic peptide, as well as cardiovascular disease risk factors, are unknown. Methods and Results We measured plasma cGMP in 542 men and 496 women free of cardiovascular disease and heart failure in MESA (Multi-Ethnic Study of Atherosclerosis). Cross-sectional associations of N-terminal pro-B type natriuretic peptide, sex hormones, and cardiovascular disease/heart failure risk factors with log(cGMP) were analyzed using multivariable linear regression models. Mean (SD) cGMP was 4.7 (2.6) pmol/mL, with no difference between the sexes. After adjusting for cardiovascular risk factors, N-terminal pro-B type natriuretic peptide was significantly positively associated with cGMP (P<0.05). Higher blood pressure and lower estimated glomerular filtration rate were associated with higher cGMP (P<0.05). Triglyceride levels, total/high-density lipoprotein cholesterol ratio, presence of diabetes mellitus, and the homeostatic model assessment of insulin resistance were inversely associated with cGMP (P<0.05). Among women, free testosterone and dehydroepiandrosterone were inversely associated with cGMP, while sex hormone binding globulin was positively associated (P<0.05). Conclusions In a community-cohort, plasma cGMP was associated with natriuretic peptide signaling. Higher blood pressure and greater renal dysfunction were positively associated with cGMP, while adverse metabolic risk factors were inversely associated. Increased androgenicity in postmenopausal women was inversely associated with cGMP. These novel associations further our understanding of the role of cGMP in a general population.

Cyclic guanosine monophosphate (cGMP) signalling plays a fundamental role in many cell types, including platelets. cGMP has been implicated in platelet formation, but mechanistic detail about its spatio-temporal regulation in megakaryocytes (MKs) is lacking. Optogenetics is a technique which allows spatio-temporal manipulation of molecular events in living cells or organisms. We took advantage of this method and expressed a photo-activated guanylyl cyclase, Blastocladiella emersonii Cyclase opsin (BeCyclop), after viral-mediated gene transfer in bone marrow (BM)-derived MKs to precisely light-modulate cGMP levels. BeCyclop-MKs showed a significantly increased cGMP concentration after illumination, which was strongly dependent on phosphodiesterase (PDE) 5 activity. This finding was corroborated by real-time imaging of cGMP signals which revealed that pharmacological PDE5 inhibition also potentiated nitric oxide-triggered cGMP generation in BM MKs. In summary, we established for the first-time optogenetics in primary MKs and show that PDE5 is the predominant PDE regulating cGMP levels in MKs. These findings also demonstrate that optogenetics allows for the precise manipulation of MK biology.

1. Guanosine 3':5'-cyclic monophosphate (cyclic GMP) is an important second messenger mediating the effects of nitric oxide (NO) and natriuretic peptides. Cyclic GMP pathways regulate several aspects of lung pathophysiology in a number of airway cells. The regulation of this system has not been extensively studied in pulmonary epithelial tissue. 2. We have studied the production of cyclic GMP by suspensions of ovine tracheal epithelial cells in response to activators of soluble guanylyl cyclase (sodium nitroprusside (SNP) and S-nitroso-N-acetyl-penicillamine (SNAP) and particulate guanylyl cyclase (atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP) and E. coli heat stable enterotoxin (STa)). 3. Both 10(-7)-10(-3) M and 10(-7)-10(-3) M SNAP generated a concentration-dependent marked elevation in cyclic GMP production when incubated with 10(-3) M 3-isobutyl-l -methylxanthine (IBMX) (both greater than 25 x baseline values with highest drug concentration). 4. The increase in production of cyclic GMP in response to 10(-6) M SNP and 10(-5) M SNAP was markedly inhibited by both 5 x 10(-5) M haemoglobin (102% and 92% inhibition) and 5 x 10(-5) M methylene blue (82% and 84% inhibition). 5. The increase in cyclic GMP in response to 10(-3) M SNP was measured following co-incubation with the phosphodiesterase inhibitors 10(-7)-10(-3) M IBMX, 10(-7)-10(-4) M milrinone and 10(-7)-10(-4) M SKF 96231. Only 10(-4)-10(-3) M IBMX significantly increased cyclic GMP levels. 6. Cyclic GMP production was also significantly elevated from baseline by 10(-5) M ANP, 10(-5) M BNP, 10(-5) M CNP and 200 iu ml-3 of E. coli STa toxin in the presence of 10(-3) M IBMX. Increases with these natriuretic peptides and STa toxin were smaller in magnitude (2-4 fold) than those seen with SNP and SNAP. CNP was the most potent of the natriuretic peptides studied suggesting type B membrane bound guanylate cyclase is the predominant form expressed. 7. These results suggest that ovine tracheal epithelial cells contain active guanylyl cyclases. The more marked response to SNP and SNAP than to natriuretic peptides suggests that soluble guanylyl cyclase predominates.

Alzheimer's disease (AD) is a severe neurodegenerative disorder for which identification of differentially expressed genes is one way to find new therapeutic targets. Here, we conducted analysis to identify age-independent, AD-specific genes. We found that the MET, WIF1, and NPTX2 genes are downregulated in AD. WIF1 and MET are implicated in Wnt and MET signaling and regulate GSK3β activity and are thus linked with AD. Importantly, we found that the GMPR gene exhibited a gradual increase in AD progression. A logistic model based on GMPR has good ability to classify AD cases. GMPR's product GMPR1 is in the AMPK and adenosine receptor pathways and is thus associated with Tau phosphorylation in AD. This allows GMPR1 to be a therapeutic target. Therefore, we screened five possible inhibitors to GMPR1 by docking GMPR1 with 1,174 approved drugs. Among them, lumacaftor is ideal. We then tested the effects of lumacaftor on AD model mice. After 20 days of oral administration, we observed that β-Amyloid accumulation was slowed down, and phosphorylation of Tau was almost eliminated in the treated mice. We highlight the elevated expression level of GMPR in AD and propose a therapeutic strategy of inhibiting GMPR1 with lumacaftor.

The metabolic pathway of purine nucleotides in parasitic protozoa is a potent drug target for treatment of parasitemia. Guanosine 5'-monophosphate reductase (GMPR), which catalyzes the deamination of guanosine 5'-monophosphate (GMP) to inosine 5'-monophosphate (IMP), plays an important role in the interconversion of purine nucleotides to maintain the intracellular balance of their concentration. However, only a few studies on protozoan GMPR have been reported at present. Herein, we identified the GMPR in Trypanosoma brucei, a causative protozoan parasite of African trypanosomiasis, and found that the GMPR proteins were consistently localized to glycosomes in T. brucei bloodstream forms. We characterized its recombinant protein to investigate the enzymatic differences between GMPRs of T. brucei and its host animals. T. brucei GMPR was distinct in having an insertion of a tandem repeat of the cystathionine β-synthase (CBS) domain, which was absent in mammalian and bacterial GMPRs. The recombinant protein of T. brucei GMPR catalyzed the conversion of GMP to IMP in the presence of NADPH, and showed apparent affinities for both GMP and NADPH different from those of its mammalian counterparts. Interestingly, the addition of monovalent cations such as K+ and NH4+ to the enzymatic reaction increased the GMPR activity of T. brucei, whereas none of the mammalian GMPR's was affected by these cations. The monophosphate form of the purine nucleoside analog ribavirin inhibited T. brucei GMPR activity, though mammalian GMPRs showed no or only a little inhibition by it. These results suggest that the mechanism of the GMPR reaction in T. brucei is distinct from that in the host organisms. Finally, we demonstrated the inhibitory effect of ribavirin on the proliferation of trypanosomes in a dose-dependent manner, suggesting the availability of ribavirin to develop a new therapeutic agent against African trypanosomiasis.

The family of natriuretic peptides (NPs), including atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), and C-type natriuretic peptide (CNP), exert important and diverse actions for cardiovascular and renal homeostasis. The autocrine and paracrine functions of the NPs are primarily mediated through the cellular membrane bound guanylyl cyclase-linked receptors GC-A (NPR-A) and GC-B (NPR-B). As the ligands and receptors each contain disulfide bonds, a regulatory role for the cell surface protein disulfide isomerase (PDI) was investigated.

A key mechanism mediating cellular adaptive responses to hypoxia involves the activity of hypoxia-inducible factor 1 (HIF-1), a transcription factor composed of HIF-1α, and HIF-1β subunits. The classical mechanism of regulation of HIF-1 activity involves destabilisation of HIF-1α via oxygen-dependent hydroxylation of proline residues and subsequent proteasomal degradation. Studies from our laboratory revealed that nitric oxide (NO)-mediated activation of cyclic guanosine monophosphate (cGMP) signalling inhibits the acquisition of hypoxia-induced malignant phenotypes in tumour cells. The present study aimed to elucidate a mechanism of HIF-1 regulation involving NO/cGMP signalling. Using human DU145 prostate cancer cells, we assessed the effect of the NO mimetic glyceryl trinitrate (GTN) and the cGMP analogue 8-Bromo-cGMP on hypoxic accumulation of HIF-1α. Concentrations of GTN known to primarily activate the NO/cGMP pathway (100 nM-1 µM) inhibited hypoxia-induced HIF-1α protein accumulation in a time-dependent manner. Incubation with 8-Bromo-cGMP (1 nM-10 µM) also attenuated HIF-1α accumulation, while levels of HIF-1α mRNA remained unaltered by exposure to GTN or 8-Bromo-cGMP. Furthermore, treatment of cells with the calpain (Ca2+-activated proteinase) inhibitor calpastatin attenuated the effects of GTN and 8-Bromo-cGMP on HIF-1α accumulation. However, since calpain activity was not affected by incubation of DU145 cells with various concentrations of GTN or 8-Bromo-cGMP (10 nM or 1 µM) under hypoxic or well-oxygenated conditions, it is unlikely that NO/cGMP signalling inhibits HIF-1α accumulation via regulation of calpain activity. These findings provide evidence for a role of NO/cGMP signalling in the regulation of HIF-1α, and hence HIF-1-mediated hypoxic responses, via a mechanism dependent on calpain.

1. We investigated the effect of the NO-donor S-nitroso-N-acetyl-DL-penicillamine (SNAP) on cardiomyocytes isolated from control normotensive Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats. 2. Ventricular cardiomyocytes were isolated from SHR and WKY hearts and imaging analysis of fura-2-loaded cells was performed in order to evaluate calcium transient in electrical field paced (0.5 Hz) cells. 3. In WKY cardiomyocytes, 1 - 200 microM SNAP dose-dependently increased cyclic GMP content. In basal conditions, cyclic GMP content of SHR cardiomyocytes was significantly higher than in WKY, but SNAP failed to further increase cyclic GMP over the basal level. 4. In control conditions, the Delta F/F and decay time of the calcium transient were similar in both strains. In WKY cardiomyocytes, SNAP (1 - 100 microM) reduced the decay time. In SHR cardiomyocytes, SNAP was ineffective. Dibutyryl cyclic GMP (10(-6) - 10(-8) M), a membrane permeable cyclic GMP analogue, behaved similarly to SNAP. 5. In WKY and SHR cardiomyocytes, 10(-8) M isoprenaline similarly increased Delta F/F and decreased the decay time. SNAP and dibutyryl cyclic GMP prevented the effect of isoprenaline in WKY, whereas both molecules were ineffective in SHR cardiomyocytes. In WKY, SNAP effects were blocked by pretreating cells with the cGK inhibitor KT-5823. 6. Western blotting analysis of cGK type I showed that the enzyme was expressed in WKY isolated cardiomyocytes, but absent in four out of five SHR preparations. 7. We concluded that the low expression of cGKI may determine the lack of NO/cyclic GMP-dependent regulation on calcium transient in SHR cardiomyocytes. This alteration may contribute to the development of heart hypertrophy in hypertensive status.

In mammals, the well-organized activation of quiescent primordial follicles is pivotal for female reproductive reserve. In the present study, we examined the mechanisms underlying primordial follicle activation in mice. We found that endothelial nitric oxide synthase (eNOS) and its downstream effectors, cyclic guanosine monophosphate (cGMP) and cGMP-dependent protein kinase G (PKG), were expressed in pre-granulosa cells and promoted primordial follicle activation, oocyte growth and granulosa cell proliferation in neonatal ovaries. Mammalian target of rapamycin (mTOR) colocalized with PKG in pre-granulosa cells and was essential for eNOS/cGMP/PKG pathway-induced primordial follicle activation. The eNOS/cGMP/PKG pathway was found to stabilize mTOR protein. The mRNA levels of F-box and WD repeat domain containing 7 (FBXW7), an E3 ubiquitin ligase, correlated negatively with mTOR protein levels in neonatal ovaries. FBXW7 bound to and destabilized mTOR protein in pre-granulosa cells in a ubiquitin/proteasome-dependent manner. However, agonists of the eNOS/cGMP/PKG pathway reduced FBXW7 mRNA levels. FBXW7 overexpression suppressed primordial follicle activation and prevented the eNOS/cGMP/PKG pathway from activating primordial follicles and stabilizing mTOR protein. These findings demonstrate that the eNOS/cGMP/PKG pathway activates primordial follicles by suppressing FBXW7-induced ubiquitination of mTOR in mice.

  We have previously demonstrated a principal role for nitric oxide (NO) in the endothelium/shear-dependent regulation of contractility in rat thoracic duct (TD). In this study we tested the hypothesis that cyclic guanosine monophosphate (cGMP) and the dependent protein kinase (PKG) are central to the intrinsic and extrinsic flow-dependent modulation of lymphatic contractility. Lymphatic diameters and indices of pumping in isolated, cannulated and pressurized segments of rat TD were measured. The influences of increased transmural pressure (1-5 cmH2O) and imposed flow (1-5 cm H2O transaxial pressure gradients) on lymphatic function were studied before and after: (1) inhibition of guanylate cyclase (GC) with and without a NO donor; (2) application of stable cGMP analogue; and (3) inhibition of the cGMP activation of PKG. Additionally, Western blotting and immunofluorescent tissue staining were used to analyse the PKG isoforms expressed in TD. We found that the GC inhibitor ODQ induced changes in TD contractility similar to NO synthase blockade and prevented the relaxation induced by the NO donor S-nitroso-N-acetylpenicillamine. The cGMP analogue, 8-(4-Chlorophenylthio)-guanosine 3,5-cyclic monophosphate sodium salt (8pCPTcGMP), mimicked the extrinsic flow-induced relaxation in a dose-dependent manner, whereas treatment with the cGMP/PKG inhibitor, guanosine 3,5-cyclic monophosphorothioate, 8-(4-chlorophenylthio)-, Rp-isomer, triethylammonium salt (Rp-8-Br-PETcGMPS), eliminated intrinsic flow-dependent relaxation, and largely inhibited extrinsic flow-dependent relaxation. Western blotting demonstrated that both PKG-Iα and -Iβ isoforms are found in TD, with ∼10 times greater expression of the PKG-Iα protein in TD compared with the aorta and vena cava. The PKG-Iβ isoform expressed equally in TD and vena cava, both being ∼2 times higher than that in the aorta. Immunofluorescent labelling of PKG-Iα protein in the wall of rat thoracic duct confirmed its localization inside TD muscle cells. These findings demonstrate that cGMP is critical to the flow-dependent regulation of TD contractility; they also indicate an important involvement of PKG, especially PKG-Iα in these processes and identifies PKG protein as a potential therapeutic target.

The guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase II (cGKII) serine/threonine kinase relays signaling through guanylyl cyclase C (GCC) to control intestinal fluid homeostasis. Here, we report the discovery of small-molecule inhibitors of cGKII. These inhibitors were imidazole-aminopyrimidines, which blocked recombinant human cGKII at submicromolar concentrations but exhibited comparatively little activity toward the phylogenetically related protein kinases cGKI and cAMP-dependent protein kinase (PKA). Whereas aminopyrimidyl motifs are common in protein kinase inhibitors, molecular modeling of these imidazole-aminopyrimidines in the ATP-binding pocket of cGKII indicated an unconventional binding mode that directs their amine substituent into a narrow pocket delineated by hydrophobic residues of the hinge and the αC-helix. Crucially, this set of residues included the Leu-530 gatekeeper, which is not conserved in cGKI and PKA. In intestinal organoids, these compounds blocked cGKII-dependent phosphorylation of the vasodilator-stimulated phosphoprotein (VASP). In mouse small intestinal tissue, cGKII inhibition significantly attenuated the anion secretory response provoked by the GCC-activating bacterial heat-stable toxin (STa), a frequent cause of infectious secretory diarrhea. In contrast, both PKA-dependent VASP phosphorylation and intestinal anion secretion were unaffected by treatment with these compounds, whereas experiments with T84 cells indicated that they weakly inhibit the activity of cAMP-hydrolyzing phosphodiesterases. As these protein kinase inhibitors are the first to display selective inhibition of cGKII, they may expedite research on cGMP signaling and may aid future development of therapeutics for managing diarrheal disease and other pathogenic syndromes that involve cGKII.

Background Cyclic guanosine monophosphate (cGMP) is a second messenger regulated through natriuretic peptide and nitric oxide pathways. Stimulation of cGMP signaling is a potential therapeutic strategy for heart failure with preserved ejection fraction (HFpEF) and atherosclerotic cardiovascular disease (ASCVD). We hypothesized that plasma cGMP levels would be associated with lower risk for incident HFpEF, any HF, ASCVD, and coronary heart disease (CHD). Methods and Results We conducted a case-cohort analysis nested in the ARIC (Atherosclerosis Risk in Communities) study. Plasma cGMP was measured in 875 participants at visit 4 (1996-1998), with oversampling of incident HFpEF cases. We used Cox proportional hazard models to assess associations of cGMP with incident HFpEF, HF, ASCVD (CHD+stroke), and CHD. The mean (SD) age was 62.4 (5.6) years and median (interquartile interval) cGMP was 3.4 pmol/mL (2.4-4.6). During a median follow-up of 9.9 years, there were 283 incident cases of HFpEF, 329 any HF, 151 ASCVD, and 125 CHD. In models adjusted for CVD risk factors, the hazard ratios (95% CI) associated with the highest cGMP tertile compared with lowest for HFpEF, HF, ASCVD, and CHD were 1.88 (1.17-3.02), 2.18 (1.18-4.06), 2.84 (1.44-5.60), and 2.43 (1.19-5.00), respectively. In models further adjusted for N-terminal-proB-type natriuretic peptide, associations were attenuated for HFpEF and HF but remained statistically significant for ASCVD (2.56 [1.26-5.20]) and CHD (2.25 [1.07-4.71]). Conclusions Contrary to our hypothesis, higher cGMP levels were associated with incident CVD in a community-based cohort. The associations of cGMP with HF or HFpEF may be explained by N-terminal-proB-type natriuretic peptide, but not for ASCVD and CHD.

Cyclic diguanosine monophosphate (c-di-GMP) is widely used by bacteria to control biological functions in response to diverse signals or cues. A previous study showed that potential c-di-GMP metabolic enzymes play a role in the regulation of biofilm formation and motility in Acinetobacter baumannii. However, it was unclear whether and how A. baumannii cells use c-di-GMP signaling to modulate biological functions. Here, we report that c-di-GMP is an important intracellular signal in the modulation of biofilm formation, motility, and virulence in A. baumannii. The intracellular level of c-di-GMP is principally controlled by the diguanylate cyclases (DGCs) A1S_1695, A1S_2506, and A1S_3296 and the phosphodiesterase (PDE) A1S_1254. Intriguingly, we revealed that A1S_2419 (an elongation factor P [EF-P]), is a novel c-di-GMP effector in A. baumannii. Response to a c-di-GMP signal boosted A1S_2419 activity to rescue ribosomes from stalling during synthesis of proteins containing consecutive prolines and thus regulate A. baumannii physiology and pathogenesis. Our study presents a unique and widely conserved effector that controls bacterial physiology and virulence by sensing the second messenger c-di-GMP.

The development of a high-performance liquid chromatography (HPLC)-based method, for guanosine monophosphate kinase activity assays, is presented. The method uses the intrinsic UV absorption (at 260 nm) of substrates and products of the enzymatic reaction (GMP, ATP, ADP and GDP) to unambiguously determine percent conversion of substrate into product. It uses a commercially available C18 column which can separate reaction samples by elution under isocratic conditions in 12 min per run. The kinetics of the forward reaction catalyzed by Plasmodium vivax guanylate kinase (PvGK), a potential drug target against malaria, was determined. The relative concentrations of the two substrates (GMP and ATP) have a distinct effect on reaction velocity. Kinetic analyses showed the PvGK-catalyzed reaction to be associated with atypical kinetics, where substrate inhibition kinetics and non-Michaelis-Menten (sigmoidal) kinetics were found with respect to GMP and ATP, respectively. Additionally, the method was used in inhibition assays to screen twenty fragment-like compounds. The assays were robust and reproducible, with a signal window of 3.8 and a Z' factor of 0.6. For the best inhibitor, an IC50 curve was generated.

The Multidrug Resistance Protein ABCC11/MRP8 is expressed in physiological barriers and tumor breast tissues in which it secretes various substrates including cGMP (cyclic guanosine monophosphate) and 5FdUMP (5-fluoro-2'-deoxyuridine-5'-monophosphate), the active metabolite of the anticancer drug 5-FluoroUracil (frequently included to anticancer therapy).Previously, we described that ABCC11 high levels are associated to the estrogen receptor (ER) expression level in breast tumors and in cell lines resistant to tamoxifen. Consequently, by lowering the intracellular concentration of anticancer drugs, ABCC11 likely promotes a multidrug resistance (MDR) phenotype and decreases efficiency of anticancer therapy of 5FdUMP. Since no experimental data about binding sites of ABCC11 substrate are available, we decided to in silico localize putative substrate interaction sites of the nucleotide derivatives. Taking advantage of molecular dynamics simulation, we also analysed their evolution under computational physiological conditions and during the time.

The hierarchical process of guanosine (G) self-assembly, leading in aqueous solution and in the presence of metal cations to the formation of G-quadruplexes, represents an intriguing topic both for the biological correlation with telomerase activity and for the nano-technological applications, as demonstrated by the current measured in a quadruplex wire 100 nm long. Similar to G-rich DNA sequences and G-oligonucleotides, the guanosine 5'-monophosphate (GMP) self-aggregates in water to form quadruplexes. However, due to the absence of a covalent axial backbone, this system can be very useful to understand the chemical-physical conditions that govern the guanosine supramolecular aggregation. We have then investigated by in-solution Synchrotron Small Angle X-ray Scattering technique the role of different cations in promoting the quadruplex formation as a function of concentration and temperature. Results show how potassium, with its peculiar biological traits, favours the G-quadruplex elongation process in respect to other cations (Na + , but also NH 4 + and Li + ), determining the longest particles in solution. Moreover, the formation and the elongation of G-quadruplexes have been demonstrated to be controlled by both GMP concentration and excess cation content, even if they specifically contribute to these processes in different ways. The occurrence of condensed liquid crystalline phases was also detected, proving that excess cations play also unspecific effects on the effective charges on the G-quadruplex surface.