Soluble Guanylyl Cyclase Activator BI 685509 Reduces Portal Hypertension and Portosystemic Shunting in a Rat Thioacetamide-Induced Cirrhosis Model.

Portal hypertension (PT) commonly occurs in cirrhosis. Nitric oxide (NO) imbalance contributes to PT via reduced soluble guanylyl cyclase (sGC) activation and cGMP production, resulting in vasoconstriction, endothelial cell dysfunction, and fibrosis. We assessed the effects of BI 685509, an NO-independent sGC activator, on fibrosis and extrahepatic complications in a thioacetamide (TAA)-induced cirrhosis and PT model. Male Sprague-Dawley rats received TAA twice-weekly for 15 weeks (300-150 mg/kg i.p.). BI 685509 was administered daily for the last 12 weeks (0.3, 1, and 3 mg/kg p.o.; n = 8-11 per group) or the final week only (Acute, 3 mg/kg p.o.; n = 6). Rats were anesthetized to measure portal venous pressure. Pharmacokinetics and hepatic cGMP (target engagement) were measured by mass spectrometry. Hepatic Sirius Red morphometry (SRM) and alpha-smooth muscle actin (αSMA) were measured by immunohistochemistry; portosystemic shunting was measured using colored microspheres. BI 685509 dose-dependently increased hepatic cGMP at 1 and 3 mg/kg (3.92 ± 0.34 and 5.14 ± 0.44 versus 2.50 ± 0.19 nM in TAA alone; P < 0.05). TAA increased hepatic SRM, αSMA, PT, and portosystemic shunting. Compared with TAA, 3 mg/kg BI 685509 reduced SRM by 38%, αSMA area by 55%, portal venous pressure by 26%, and portosystemic shunting by 10% (P < 0.05). Acute BI 685509 reduced SRM and PT by 45% and 21%, respectively (P < 0.05). BI 685509 improved hepatic and extrahepatic cirrhosis pathophysiology in TAA-induced cirrhosis. These data support the clinical investigation of BI 685509 for PT in patients with cirrhosis. SIGNIFICANCE STATEMENT: BI 685509 is an NO-independent sGC activator that was tested in a preclinical rat model of TAA-induced nodular, liver fibrosis, portal hypertension, and portal systemic shunting. BI 685509 reduced liver fibrosis, portal hypertension, and portal-systemic shunting in a dose-dependent manner, supporting its clinical assessment to treat portal hypertension in patients with cirrhosis.

Hybrid Chemoenzymatic Synthesis of C7 -Sugars for Molecular Evidence of in†vivo Shikimate Pathway Inhibition.

The design of distinctive chemical synthesis strategies aims for the most efficient routes towards versatile compounds in drug target studies. Here, we establish a powerful hybrid synthetic approach of total chemical and chemoenzymatic synthesis to efficiently obtain various 7-deoxy-sedoheptulose (7dSh, 1) analogues, unique C7 sugars, for structure-activity relationship studies. 7dSh (1) is a rare microbial sugar with in planta herbicidal activity. As natural antimetabolite of 3-dehydroquinate synthase (DHQS), 7dSh (1) inhibits the shikimate pathway, which is essential for the synthesis of aromatic amino acids in bacteria, fungi, and plants, but absent in mammals. As glyphosate, the most used chemical herbicide faces restrictions worldwide, DHQS has gained more attention as valid target of herbicides and antimicrobial agents. In vitro and in vivo analyses of the C7 -deoxysugars confirm DHQS as enzymatic target, highlight the crucial role of uptake for inhibition and add molecular aspects to target mechanism studies of C7 -sugars as our contribution to global efforts for alternative weed-control strategies.

In vivo Inhibition of the 3-Dehydroquinate Synthase by 7-Deoxysedoheptulose Depends on Promiscuous Uptake by Sugar Transporters in Cyanobacteria.

7-Deoxysedoheptulose (7dSh) is a bioactive deoxy-sugar actively excreted by the unicellular cyanobacterium Synechococcus elongatus PCC 7942 (S. elongatus) but also Streptomyces setonensis. In our previous publications we have shown that in S. elongatus, 7dSh is exclusively synthesized by promiscuous enzyme activity from an inhibitory by-product of radical SAM enzymes, without a specific gene cluster being involved. Additionally, we showed that 7dSh inhibits the growth of cyanobacteria, but also the growth of plants and fungi, presumably by inhibiting the 3-dehydroquinate synthase (DHQS), the second enzyme of the shikimate pathway, as the substrate of this enzyme strongly accumulates in cells treated with 7dSh. In this study, by using purified DHQS of Anabaena variabilis ATCC 29413 (A. variabilis) we biochemically confirmed that 7dSh is a competitive inhibitor of this enzyme. By analyzing the effect of 7dSh on a subset of cyanobacteria from all the five subsections, we identified different species whose growth was inhibited by 7dSh. We also found that in some of the susceptible cyanobacteria import of 7dSh is mediated by structurally different and promiscuous transporters: 7dSh can be taken up by the fructose ABC-transporter in A. variabilis and via the glucose permease in Synechocystis sp. PCC 6803 (Synechocystis sp.). In both cases, an effective uptake and thereby intracellular enrichment of 7dSh was essential for the inhibitory activity. Importantly, spontaneous mutations in the sugar transporters of A. variabilis and Synechocystis sp. not only disabled growth of the two strains on fructose and glucose, respectively, but also almost abolished their sensitivity to 7dSh. Although we have clearly shown in these examples that the effective uptake plays an essential role in the inhibitory effect of 7dSh, questions remain about how 7dSh resistance works in other (cyano)bacteria. Also, the involvement of a putative ribokinase in 7dSh resistance in the producer strain S. elongatus remained to be further investigated. Overall, these data establish 7dSh as the first allelochemical targeting the shikimate pathway in other cyanobacteria and plants and suggest a role of 7dSh in niche competition.

A bioactive molecule made by unusual salvage of radical SAM enzyme byproduct 5-deoxyadenosine blurs the boundary of primary and secondary metabolism.

5-Deoxyadenosine (5dAdo) is the byproduct of many radical S-adenosyl-l-methionine enzyme reactions in all domains of life. 5dAdo is also an inhibitor of the radical S-adenosyl-l-methionine enzymes themselves, making it necessary for cells to construct pathways to recycle or dispose of this toxic metabolite. However, the specific pathways involved have long remained unexplored. Recent research demonstrated a growth advantage in certain organisms by using 5dAdo or intermediates as a sole carbon source and elucidated the corresponding salvage pathway. We now provide evidence using supernatant analysis by GC-MS for another 5dAdo recycling route. Specifically, in the unicellular cyanobacterium Synechococcus elongatus PCC 7942 (S. elongatus), the activity of promiscuous enzymes leads to the synthesis and excretion first of 5-deoxyribose and subsequently of 7-deoxysedoheptulose. 7-Deoxysedoheptulose is an unusual deoxy-sugar, which acts as an antimetabolite of the shikimate pathway, thereby exhibiting antimicrobial and herbicidal activity. This strategy enables organisms with small genomes and lacking canonical gene clusters for the synthesis of secondary metabolites, like S. elongatus, to produce antimicrobial compounds from primary metabolism and enzymatic promiscuity. Our findings challenge the view of bioactive molecules as sole products of secondary metabolite gene clusters and expand the range of compounds that microorganisms can deploy to compete for their ecological niche.

How glyphosate and its associated acidity affect early development in zebrafish (Danio rerio).

BACKGROUND: Glyphosate is among the most extensively used pesticides worldwide. Following the ongoing highly controversial debate on this compound, its potential impact on non-target organisms is a fundamental scientific issue. In its pure compound form, glyphosate is known for its acidic properties. METHODS: We exposed zebrafish (Danio rerio) embryos to concentrations between 10 µM and 10 mM glyphosate in an unbuffered aqueous medium, as well as at pH 7, for 96 hours post fertilization (hpf). Furthermore, we investigated the effects of aqueous media in the range of pH 3 to 8, in comparison with 1 mM glyphosate treatment at the respective pH levels. Additionally, we exposed zebrafish to 7-deoxy-sedoheptulose (7dSh), another substance that interferes with the shikimate pathway by a mechanism analogous to that of glyphosate, at a concentration of one mM. The observed endpoints included mortality, the hatching rate, developmental delays at 24 hpf, the heart rate at 48 hpf and the malformation rate at 96 hpf. LC10/50, EC10 and, if reasonable, EC50 values were determined for unbuffered glyphosate. RESULTS: The results revealed high mortalities in all treatments associated with low pH, including high concentrations of unbuffered glyphosate (>500 µM), low pH controls and glyphosate treatments with pH < 3.4. Sublethal endpoints like developmental delays and malformations occurred mainly at higher concentrations of unbuffered glyphosate. In contrast, effects on the hatching rate became particularly prominent in treatments at pH 7, showing that glyphosate significantly accelerates hatching compared with the control and 7dSh, even at the lowest tested concentration. Glyphosate also affected the heart rate, resulting in alterations both at pH 7 and, even more pronounced, in the unbuffered system. In higher concentrations, glyphosate tended to accelerate the heart rate in zebrafish embryos, again, when not masked by the decelerating influence of its low pH. At pH > 4, no mortality occurred, neither in the control nor in glyphosate treatments. At 1 mM, 7dSh did not induce any mortality, developmental delays or malformations; only slightly accelerated hatching and a decelerated heart rate were observed. Our results demonstrate that lethal impacts in zebrafish embryos can be attributed mainly to low pH, but we could also show a pH-independent effect of glyphosate on the development of zebrafish embryos on a sublethal level.

Cyanobacterial antimetabolite 7-deoxy-sedoheptulose blocks the shikimate pathway to inhibit the growth of prototrophic organisms.

Antimetabolites are small molecules that inhibit enzymes by mimicking physiological substrates. We report the discovery and structural elucidation of the antimetabolite 7-deoxy-sedoheptulose (7dSh). This unusual sugar inhibits the growth of various prototrophic organisms, including species of cyanobacteria, Saccharomyces, and Arabidopsis. We isolate bioactive 7dSh from culture supernatants of the cyanobacterium Synechococcus elongatus. A chemoenzymatic synthesis of 7dSh using S. elongatus transketolase as catalyst and 5-deoxy-D-ribose as substrate allows antimicrobial and herbicidal bioprofiling. Organisms treated with 7dSh accumulate 3-deoxy-D-arabino-heptulosonate 7-phosphate, which indicates that the molecular target is 3-dehydroquinate synthase, a key enzyme of the shikimate pathway, which is absent in humans and animals. The herbicidal activity of 7dSh is in the low micromolar range. No cytotoxic effects on mammalian cells have been observed. We propose that the in vivo inhibition of the shikimate pathway makes 7dSh a natural antimicrobial and herbicidal agent.

Down-Regulation of the Alternative Sigma Factor SigJ Confers a Photoprotective Phenotype to Anabaena PCC 7120.

Alternative sigma factors belonging to Group 3 are thought to play an important role in the adaptation of cyanobacteria to environmental challenges by altering expression of genes needed for coping with such stresses. In this study, the role of an alternative sigma factor, SigJ, was analyzed in the filamentous nitrogen-fixing cyanobacterium, Anabaena sp. PCC 7120 by knocking down the expression of the sigJ gene (alr0277) employing an antisense RNA-mediated approach. In the absence of any stress, the knock-down (KD0277) or the wild-type strain both grew similarly. Upon exposure to high-intensity light, KD0277 showed substantially reduced bleaching of its pigments, higher photosynthetic activity and consequently better survival than the wild type. KD0277 also showed an enhanced accumulation of two carotenoids, which were identified as myxoxanthophyll and keto-myxoxanthophyll. Further, KD0277 was more tolerant to ammonium-triggered photodamage than the wild type. Moreover, PSII was better protected against photodamage in KD0277 than in the wild type. Down-regulation of sigJ in Anabaena PCC 7120, however, reduced its ability to cope with desiccation. This study demonstrates that down-regulation of the sigJ gene in Anabaena PCC 7120 differentially affects its ability to tolerate two environmentally relevant stresses, i.e. high-intensity light and desiccation.