Photochemistry of 2-thiooxazole: a plausible prebiotic precursor to RNA nucleotides.

Potentially prebiotic chemical reactions leading to RNA nucleotides involve periods of UV irradiation, which are necessary to promote selectivity and destroy biologially irrelevant side products. Nevertheless, UV light has only been applied to promote specific stages of prebiotic reactions and its effect on complete prebiotic reaction sequences has not been extensively studied. Here, we report on an experimental and computational investigation of the photostability of 2-thiooxazole (2-TO), a potential precursor of pyrimidine and 8-oxopurine nucleotides on early Earth. Our UV-irradiation experiments resulted in rapid decomposition of 2-TO into unidentified small molecule photoproducts. We further clarify the underlying photochemistry by means of accurate ab initio calculations and surface hopping molecular dynamics simulations. Overall, the computational results show efficient rupture of the aromatic ring upon the photoexcitation of 2-TO via breaking of the C-O bond. Consequently, the initial stage of the divergent prebiotic synthesis of pyrimidine and 8-oxopurine nucleotides would require periodic shielding from UV light either with sun screening chromophores or through a planetary scenario that would protect 2-TO until it is transformed into a more stable intermediate compound, e.g. oxazolidinone thione.

Stereo-Defined Acyclic Nucleoside Phosphonates are Selective and Potent Inhibitors of Parasite 6-Oxopurine Phosphoribosyltransferases.

Pathogens such as Plasmodium and Trypanosoma spp. are unable to synthesize purine nucleobases. They rely on the salvage of these purines and their nucleosides from the host cell to synthesize the purine nucleotides required for DNA/RNA production. The key enzymes in this pathway are purine phosphoribosyltransferases (PRTs). Here, we synthesized 16 novel acyclic nucleoside phosphonates, 12 with a chiral center at C-2', and eight bearing a second chiral center at C-6'. Of these, bisphosphonate (S,S)-48 is the most potent inhibitor of the Plasmodium falciparum and P. vivax 6-oxopurine PRTs and the most potent inhibitor of two Trypanosoma brucei (Tbr) 6-oxopurine PRTs yet discovered, with Ki values as low as 2 nM. Crystal structures of (S,S)-48 in complex with human and Tbr 6-oxopurine PRTs show that the inhibitor binds to the enzymes in different conformations, providing an explanation for its potency and selectivity (i.e., 35-fold in favor of the parasite enzymes).

A Toxoplasma gondii Oxopurine Transporter Binds Nucleobases and Nucleosides Using Different Binding Modes.

Toxoplasma gondii is unable to synthesize purines de novo, instead salvages them from its environment, inside the host cell, for which they need high affinity carriers. Here, we report the expression of a T. gondii Equilibrative Nucleoside Transporter, Tg244440, in a Trypanosoma brucei strain from which nucleobase transporters have been deleted. Tg244440 transported hypoxanthine and guanine with similar affinity (Km ~1 µM), while inosine and guanosine displayed Ki values of 4.05 and 3.30 µM, respectively. Low affinity was observed for adenosine, adenine, and pyrimidines, classifying Tg244440 as a high affinity oxopurine transporter. Purine analogues were used to probe the substrate-transporter binding interactions, culminating in quantitative models showing different binding modes for oxopurine bases, oxopurine nucleosides, and adenosine. Hypoxanthine and guanine interacted through protonated N1 and N9, and through unprotonated N3 and N7 of the purine ring, whereas inosine and guanosine mostly employed the ribose hydroxy groups for binding, in addition to N1H of the nucleobase. Conversely, the ribose moiety of adenosine barely made any contribution to binding. Tg244440 is the first gene identified to encode a high affinity oxopurine transporter in T. gondii and, to the best of our knowledge, the first purine transporter to employ different binding modes for nucleosides and nucleobases.

Can zaprinast and avanafil induce the levels of angiogenesis, bone morphogenic protein 2, 4 and 7 in kidney of ovariectomised rats?

OBJECTIVE: This study investigated effects of zaprinast and avanafil on angiogenesis, vascular endothelial growth factor (VEGF), bone morphogenic protein (BMP) 2, 4 and 7. METHODS: Female rats were randomly divided into four groups (n = 6). Sham; abdomen was approximately 2 cm opened and closed. Ovariectomised (OVX); abdomen was opened 2 cm and the ovaries were cut. OVX + zaprinast and OVX + avanafil groups; after the same procedure with OVX, 10 mg/kg zaprinast and avanafil were orally administered for 2 month, respectively. Angiogenesis and the levels of VEGF, BMP2, 4 and 7 were determined. RESULTS: VEGF, BMP2, 4 and 7 levels in OVX + zaprinast and especially OVX + avanafil groups were higher than the sham and OVX (p < .05). However, only VEGF and BMP2 levels in OVX + zaprinast group were significant according to sham (p < .05). Also, angiogenesis in OVX + zaprinast and OVX + avanafil groups was dominant according to sham and OVX (p < .05). CONCLUSIONS: Zaprinast and avanafil induced BMP2, 4 and 7 levels synergistically with increased VEGF and angiogenesis in renal tissue.

Zaprinast and avanafil increase the vascular endothelial growth factor, vitamin D3, bone morphogenic proteins 4 and 7 levels in the kidney tissue of male rats applied the glucocorticoid.

Objective: This study investigated effect of zaprinast and avanafil on vascular endothelial growth factor (VEGF), bone morphogenic protein (BMP) 4 and 7, and vitamin D3 levels against the negative effect of dexamethazone.Method: Rats were randomly divided into four groups (n = 6). Control: Empty a syringe was immersed and removed subcutaneously. Dexamethasone (DEX): 120 µg/kg DEX was injected subcutaneously once a day for 28 days. DEX + zaprinast and DEX + avanafil groups: 10 mg/kg zaprinast and avanafil were administrated to rats in addition to the same procedure in the DEX, respectively. VitaminD3, VEGF, BMP4 and 7 levels by enzyme linked immunosorbent assay (ELISA) and angiogenesis by histopathological/immunohistochemical were evaluated.Results: BMP4 values in the DEX were lower than the other groups (p < .05). DEX + zaprinast and DEX + avanafil exhibited an increase in all the parameters compared to the control and DEX (p < .05). However, these were not significant for the DEX + zaprinast (p > .05). Also, there was a significant increase in angiogenesis in the DEX + zaprinast and DEX + avanafil.Conclusion: Zaprinast and significantly avanafil induced vitamin D3, BMP4 and 7 levels by increasing angiogenesis in renal.

A High-Throughput Chemical Screen in DJ-1ß Mutant Flies Identifies Zaprinast as a Potential Parkinson's Disease Treatment.

Dopamine replacement represents the standard therapy for Parkinson's disease (PD), a common, chronic, and incurable neurological disorder; however, this approach only treats the symptoms of this devastating disease. In the search for novel disease-modifying therapies that target other relevant molecular and cellular mechanisms, Drosophila has emerged as a valuable tool to study neurodegenerative diseases due to the presence of a complex central nervous system, the blood-brain barrier, and a similar neurotransmitter profile to humans. Human PD-related genes also display conservation in flies; DJ-1ß is the fly ortholog of DJ-1, a gene for which mutations prompt early-onset recessive PD. Interestingly, flies mutant for DJ-1ß exhibit PD-related phenotypes, including motor defects, high oxidative stress (OS) levels and metabolic alterations. To identify novel therapies for PD, we performed an in vivo high-throughput screening assay using DJ-1ß mutant flies and compounds from the Prestwick® chemical library. Drugs that improved motor performance in DJ-1ß mutant flies were validated in DJ-1-deficient human neural-like cells, revealing that zaprinast displayed the most significant ability to suppress OS-induced cell death. Zaprinast inhibits phosphodiesterases and activates GPR35, an orphan G-protein-coupled receptor not previously associated with PD. We found that zaprinast exerts its beneficial effect in both fly and human PD models through several disease-modifying mechanisms, including reduced OS levels, attenuated apoptosis, increased mitochondrial viability, and enhanced glycolysis. Therefore, our results support zaprinast as a potential therapeutic for PD in future clinical trials.

GPR35 regulates osteogenesis via the Wnt/GSK3ß/ß-catenin signaling pathway.

It is well known that osteoporosis is a significant chronic disease with the increase of the aging population. Here, we report that expression of G protein-coupled receptor 35 (GPR35) in bone marrow mesenchymal stem cells (BMSCs) is suppressed in diagnosed osteoporosis patients and osteoporotic mice. The expression of GPR35 on BMSCs is enhanced during osteogenic differentiation. GPR35 knockout suppresses the proliferation and osteogenesis of BMSCs and deteriorates bone mass in both sham-treated and ovariectomized mice. Moreover, GPR35 deficiency reduces ß-catenin activity in BMSCs. In contrast, the overexpression of GPR35 contributes to these processes in BMSCs. Finally, using zaprinast, a synthetic GPR35 agonist, we show that zaprinast rescues OVX-induced bone loss and promotes bone generation in mice. Thus, GPR35 may as a new target and its agonist zaprinast may serve as a novel treatment for osteoporosis.

UV-induced hydrogen transfer in DNA base pairs promoted by dark nπ* states.

Dark nπ* states were shown to have substantial contribution to the destructive photochemistry of pyrimidine nucleobases. Based on quantum-chemical calculations, we demonstrate that the characteristic hydrogen bonding pattern of the GC base pair could facilitate the formation of a wobble excited-state charge-transfer complex. This entails a barrierless electron-driven proton transfer (EDPT) process which enables damageless photodeactivation of the base pair. These photostabilizing properties are retained even when guanine is exchanged to hypoxanthine. The inaccessibility of this process in the AT base pair sheds further light on the reasons why cytosine is less susceptible to the formation of photodimers in double-stranded DNA.

Phosphodiesterase 5 inhibitors as novel agents for the treatment of Alzheimer's disease.

Alzheimer's disease (AD), characterized by a progressive impairment of memory and cognition, is a major health problem in both developing and developed countries. Currently, no drugs can reverse the progression of AD. Phosphodiesterase 5 (PDE5) is a critical component of the cyclic guanosine monophosphate/protein kinase G (cGMP/PKG) signaling pathway in neurons, the inhibition of which has produced neuroprotective effects, and PDE5 inhibitors have recently been thought to be potential therapeutic agents for AD. In this paper, we summarized the outstanding progress that has been made in PDE5 inhibitors as anti-AD agents with encouraging results in animal studies, clinical trials and the investigations on the underlying mechanisms. The novel PDE5 inhibitors reported recently in the treatment of AD were also reviewed and discussed.

Tracking the origin of photostability in purine nucleobases: the photophysics of 2-oxopurine.

This work scrutinizes the relaxation mechanism of 2-oxopurine. Contrary to its ancestor, purine, which is a UVC chromophore, 2-oxopurine shows a red-shifted absorption spectrum centered in the UVA region. In 2-oxopurine, relaxation along the ππ* spectroscopic state directs the population from the Franck-Condon (FC) region towards a minimum, which acts as a crossroad for the further decay of the system either to triplet states or, alternatively, to the ground state through a C6-puckered S1/S0 funnel. A comparison of the optical properties and excited state potential energy surfaces of purine, 2-oxopurine, 2-aminopurine, 6-oxopurine and adenine, allows establishing how the position and nature of substituent tune the photophysics of purine. For this series, we conclude that both C2 and C6 substitution redshift the absorption spectrum of purine, with 2-oxo substitution exhibiting the largest shift. An important exception is the canonical nucleobase adenine, which presents a blue shifted absorption spectrum. The topography of purine's ππ* potential energy surface experiences major changes when functionalized at the C6 position. In particular, the disappearance of the minimum along the ππ* potential energy surface efficiently funnels the excited state population from the FC region to the ground state and increases the photostability of 6-aminopurine (adenine) and 6-oxopurine (hypoxanthine) nucleobases.

Design, synthesis of novel purin-6-one derivatives as phosphodiesterase 2 (PDE2) inhibitors: The neuroprotective and anxiolytic-like effects.

Phosphodiesterase 2 (PDE2) has received much attention for the potential treatment of the central nervous system (CNS) disorders. Herein, based on the existing PDE2 inhibitors and their binding modes, a series of purin-6-one derivatives were designed, synthesized and evaluated for PDE2 inhibitory activities, which led to the discovery of the best compounds 6p and 6s with significant inhibitory potency (IC50: 72 and 81 nM, respectively). Docking simulation was performed to insert compound 6s into the crystal structure of PDE2 at the active site to determine the binding mode. Furthermore, compound 6s significantly protected HT-22 cells against corticosterone-induced cytotoxicity and rescued corticosterone-induced decreases in cAMP and cGMP levels. It also produced anxiolytic-like effect in the elevated plus-maze test and exhibited favorable pharmacokinetic properties in vivo. These results might bring significant instruction for further development of potent PDE2 inhibitors.

Kynurenic acid and zaprinast diminished CXCL17-evoked pain-related behaviour and enhanced morphine analgesia in a mouse neuropathic pain model.

BACKGROUND: The G protein-coupled receptor 35 (GPR35), is considered important for nociceptive transmission, as suggested by accumulating evidence. This receptor was discovered in 1998; however, a lack of pharmacological tools prevented a complete understanding of its function and how to exploit it therapeutically. We studied the influence of CXCL17, kynurenic acid and zaprinast on nociceptive transmission in naïve and neuropathic mice. Additionally, we investigated the influence of kynurenic acid and zaprinast on morphine effectiveness in neuropathic pain. METHODS: The chronic constriction injury (CCI) of the sciatic nerve in Swiss mice was performed. The CXCL17, kynurenic acid, zaprinast and morphine were injected intrathecally into naive and CCI-exposed mice at day 14. To evaluate tactile and thermal hypersensitivity, the von Frey and cold plate tests were used, respectively. RESULTS: Our results have shown, for the first time, that administration of CXCL17 in naïve mice induced strong pain-related behaviours, as measured by von Frey and cold plate tests. Moreover, we demonstrated that kynurenic acid and zaprinast diminished CXCL17-evoked pain-related behaviours in both tests. Kynurenic acid and zaprinast reduced thermal and tactile hypersensitivity developed by sciatic nerve injury and strongly enhanced the effectiveness of morphine in neuropathy. CONCLUSIONS: Our study highlights the importance of GPR35 as a receptor involved in neuropathic pain development. Therefore, these results suggest that the modulation of GPR35 could become a potential strategy for the treatment of neuropathic pain.

Zaprinast diminished pain and enhanced opioid analgesia in a rat neuropathic pain model.

The mechanism of neuropathic pain is complex and unclear. Based on our results, we postulate that an intensification of the kynurenine pathway occurs as a consequence of nerve injury. The G protein-coupled receptor 35 (GPR35) is important for kynurenine pathway activation. Cyclic GMP-specific phosphodiesterase inhibitors have also been shown to have beneficial effects on neuropathic pain. Therefore, the aims of our research were to elucidate how a substance that acts as both an agonist of GPR35 and an inhibitor of phosphodiesterase influences neuropathic pain in a rat model. Here, we demonstrated that preemptive and repeated intrathecal (i.t.) administration (16 h and 1 h before injury and then after nerve ligation daily for 7 days) of zaprinast (1 µg/5 µl) significantly attenuated mechanical (von Frey test) and thermal (cold plate test) hypersensitivity measured on day 7 after chronic constriction injury, and the effect of even a single injection lasted up to 24 h. Our data indicate that zaprinast diminished the number of IBA1-positive cells and consequently attenuated the levels of IL-1beta, IL-6, IL-18, and NOS2 in the lumbar spinal cord and/or dorsal root ganglia. Our results also demonstrated that zaprinast potentiated the analgesic properties of morphine and buprenorphine. In summary, in a neuropathic pain model, zaprinast significantly reduced pain symptoms and enhanced the effectiveness of opioids. Our data provide new evidence that modulation of both GPR35 and phosphodiesterase could be an important strategy for innovative pharmacological treatments designed to decrease hypersensitivity evoked by nerve injury.

Purin-6-one and pyrrolo[2,3-d]pyrimidin-4-one derivatives as potentiating agents of doxorubicin cytotoxicity.

AIM: DNA damage response plays an eminent role in patients' response to conventional chemotherapy and radiotherapy. Its inhibition is of great interest as it can overcome cancer cell resistance and reduce the effective doses of DNA damaging agents. Results & methodology: We have focused our research on phosphatidylinositol 3-kinase-related kinases and prepared 35 novel compounds through a scaffold hopping approach. The newly synthesized inhibitors were tested on a panel of nine cancer and one healthy cell lines alone and in combination with appropriate doses of doxorubicin. CONCLUSION: Five novel compounds 4f, 10b, 15g, 7e and 15f in combination with doxorubicin showed significant antiproliferative effect on seven cancer cell lines while not affecting the cell growth alone.

PKG-Dependent Cell Death in 661W Cone Photoreceptor-like Cell Cultures (Experimental Study).

In humans cone photoreceptors are responsible for high-resolution colour vision. A variety of retinal diseases can compromise cone viability, and, at present, no satisfactory treatment options are available. Here, we present data towards establishing a reliable, high-throughput assay system that will facilitate the search for cone neuroprotective compounds using the murine-photoreceptor cell line 661 W. To further characterize 661 W cells, a retinal marker study was performed, followed by the induction of cell death using paradigms over-activating cGMP-dependent protein kinase G (PKG). We found that 661 W cells may be used to mimic specific aspects of cone degeneration and may thus be valuable for future compound screening studies.

Antidepressant-like effects of translocator protein (18 kDa) ligand ZBD-2 in mouse models of postpartum depression.

The 18 kDa translocator protein (TSPO) is primarily localized in the outer mitochondrial membrane of steroid-synthesizing cells in the central and peripheral nervous systems. One of the protein's main functions is transporting substrate cholesterol into the mitochondria in a prerequisite process for steroid synthesis. Clinical trials have indicated that TSPO ligands might be valuable in treating some neuropathies and psychopathies. However, limited information is known about the role of TSPO in postpartum depression (PPD). The TSPO ligand ZBD-2, a derivative of XBD173, was synthesized in our laboratory. Behavioral tests, enzyme linked immunosorbent assay, and Western blot were employed to evaluate ZBD-2's efficacy against PPD and to elucidate the potential underlying molecular mechanism. The TSPO levels significantly decreased in the basolateral amygdala of PPD models. After treatment for 2 weeks, ZBD-2 alleviated depression-like behaviors and enhanced the TSPO level in a PPD animal model. The underlying mechanisms of ZBD-2 were related to regulate the hypothalamic-pituitary-adrenal axis, enhance 5-HT and BDNF secretion, and maintain the excitatory and inhibitory synaptic protein expression to normal levels. Our results directly confirm that ZBD-2 exerts a therapeutic effect on PPD, which provides a new target for anti-PPD drug development.

Evaluation of the Trypanosoma brucei 6-oxopurine salvage pathway as a potential target for drug discovery.

Due to toxicity and compliance issues and the emergence of resistance to current medications new drugs for the treatment of Human African Trypanosomiasis are needed. A potential approach to developing novel anti-trypanosomal drugs is by inhibition of the 6-oxopurine salvage pathways which synthesise the nucleoside monophosphates required for DNA/RNA production. This is in view of the fact that trypanosomes lack the machinery for de novo synthesis of the purine ring. To provide validation for this approach as a drug target, we have RNAi silenced the three 6-oxopurine phosphoribosyltransferase (PRTase) isoforms in the infectious stage of Trypanosoma brucei demonstrating that the combined activity of these enzymes is critical for the parasites' viability. Furthermore, we have determined crystal structures of two of these isoforms in complex with several acyclic nucleoside phosphonates (ANPs), a class of compound previously shown to inhibit 6-oxopurine PRTases from several species including Plasmodium falciparum. The most potent of these compounds have Ki values as low as 60 nM, and IC50 values in cell based assays as low as 4 µM. This data provides a solid platform for further investigations into the use of this pathway as a target for anti-trypanosomal drug discovery.

Novel butanehydrazide derivatives of purine-2,6-dione as dual PDE4/7 inhibitors with potential anti-inflammatory activity: Design, synthesis and biological evaluation.

A novel butanehydrazide derivatives of purine-2,6-dione designed using a ligand-based approach were synthesized and their in vitro activity against both PDE4B and PDE7A isoenzymes was assessed. The 7,8-disubstituted purine-2,6-dione derivatives 31, 34, 37, and 40 appeared to be the most potent PDE4/7 inhibitors with IC50 values in the range of that of the reference rolipram and BRL-50481, respectively. Moreover, docking studies explained the importance of N-(2,3,4-trihydroxybenzylidene)butanehydrazide substituent in position 7 of purine-2,6-dione core for dual PDE4/7 inhibitory properties. The inhibition of both the cAMP-specific PDE isoenzymes resulted in a strong anti-TNF-α effect. Compounds 31, 34, and 37 in the in vivo study in rats with LPS-induced endotoxemia decreased the maximum concentration of this proinflammatory cytokine by 53, 84 and 88%, respectively.

The bradykinin-cGMP-PKG pathway augments insulin sensitivity via upregulation of MAPK phosphatase-5 and inhibition of JNK.

Bradykinin (BK) promotes insulin sensitivity and glucose uptake in adipocytes and other cell types. We demonstrated that in rat adipocytes BK enhances insulin-stimulated glucose transport via endothelial nitric oxide synthase, nitric oxide (NO) generation, and decreased activity of the mitogen-activated protein kinase (MAPK) JNK (c-Jun NH2-terminal kinase). In endothelial cells, NO increases soluble guanylate cyclase (sGC) activity, which, in turn, activates protein kinase G (PKG) by increasing cGMP levels. In this study, we investigated whether BK acts via the sGC-cGMP-PKG pathway to inhibit the negative effects of JNK on insulin signaling and glucose uptake in rat adipocytes. BK augmented cGMP concentrations. The BK-induced enhancement of insulin-stimulated glucose uptake was mimicked by the sGC activator YC-1 and a cell-permeable cGMP analog, CPT-cGMP, and inhibited by the sGC inhibitor ODQ and the PKG inhibitor KT 5823. Transfection of dominant-negative PKG reduced the BK augmentation of insulin-induced Akt phosphorylation. The activation of JNK and ERK1/2 by insulin was attenuated by BK, which was mediated by the sGC-cGMP-PKG pathway. Whereas insulin-stimulated phosphorylation of upstream activators of JNK and ERK, i.e., MKK4 and MEK1/2, was unaffected, BK augmented insulin-mediated induction of MKP-5 mRNA and protein levels. Furthermore, zaprinast, a phosphodiesterase inhibitor, enhanced cGMP and MKP-5 and prolonged the action of BK. These data indicate that BK enhances insulin action by inhibition of negative feedback by JNK and ERK via upregulation of MKP-5, mediated by the sGC-cGMP-PKG signaling pathway.