Protective Effects of Aminooxyacetic Acid on Colitis Induced in Mice with Dextran Sulfate Sodium

As a known inhibitor of pyridoxal phosphate-dependent transaminase glutamic-oxaloacetic transaminase 1 (GOT1), aminooxyacetic acid (AOAA) has been pointed out to have potential pharmacological effects in antiepileptic, anticonvulsant, antibacterial, cancer cell proliferation inhibition, and acute myocardial infarction (MI) relief. However, its role in inflammatory bowel disease (IBD) has not been reported. Through the in vivo experiment of dextran sulfate sodium- (DSS-) induced colitis in mice, it was found that AOAA significantly attenuated the symptoms, signs, and pathological changes of colitis. In addition, AOAA treatment prevented gut barrier damages by enhancing the expression of zona occludens- (ZO-) 1, occludin, claudin-1, and E-cadherin and recovering the upregulation of the most abundant intermediate filament protein (vimentin). Moreover, the release of interleukin- (IL-) 1β, IL-6, and tumour necrosis factor- (TNF-) α was suppressed, yet the level of IL-10 was upregulated by AOAA treatment compared to the model group. Furthermore, it was shown that AOAA administration boosted M2-like phenotype and effectively reduced M1 macrophage phenotype in the lamina propria of mouse colonic epithelium. Similarly, the effect of AOAA was verified in vitro. AOAA effectively inhibited the classically activated M1 macrophage phenotype and proinflammatory cytokine (IL-1β, TNF-α, and IL-6) expression induced by lipopolysaccharide (LPS) and promoted M2-like phenotype. Collectively, this study reveals for the first time that short-term treatment of AOAA can significantly alleviate DSS-induced acute colitis by regulating intestinal barrier function and macrophage polarization, which provides a theoretical basis for the potential use of AOAA in the treatment of IBD.

Efficacy of Novel Aminooxyacetic Acid Prodrugs in Colon Cancer Models: Towards Clinical Translation of the Cystathionine β-Synthase Inhibition Concept

Upregulation of hydrogen sulfide (H2S) biosynthesis, at least in part related to the upregulation of cystathionine β-synthetase (CBS) in cancer cells, serves as a tumor-promoting factor and has emerged as a possible molecular target for antitumor drug development. To facilitate future clinical translation, we have synthesized a variety of novel CBS-targeting, esterase-cleavable prodrugs based on the structure of the prototypical CBS inhibitor aminooxyacetic acid (AOAA). The pharmacological properties of these compounds were evaluated in cell-free assays with recombinant human CBS protein, the human colon cancer cell line HCT116, and in vivo using various tumor-bearing mice models. The prodrug YD0251 (the isopropyl ester derivative of AOAA) was selected for detailed characterization. YD0251 exhibits improved antiproliferative efficacy in cell culture models when compared to AOAA. It is up to 18 times more potent than AOAA at suppressing HCT116 tumor growth in vivo and is effective when administered to tumor-bearing mice either via subcutaneous injection or oral gavage. Patient-derived xenografts (PDTXs) with higher levels of CBS protein grew significantly larger than tumors with lower levels, and YD0251 treatment inhibited the growth of PDTXs with elevated CBS, whereas it had no significant effect on PDTXs with low CBS protein levels. The toxicity of YD0251 was assessed in mice subjected to subchronic administration of supratherapeutic doses the inhibitor; no significant alteration in circulating markers of organ injury or histopathological alterations were noted, up to 60 mg/kg/day × 5 days. In preparation to a future theranostic concept (to match CBS inhibitor therapy to high-CBS expressors), we identified a potential plasma marker of CBS-expressing tumors. Colon cancer cells produced significant levels of lanthionine, a rare metabolic intermediate of CBS-mediated H2S biosynthesis; forced expression of CBS into non-transformed epithelial cells increased lanthionine biogenesis in vitro and in vivo (measured in the urine of tumor-bearing mice). These current results may be useful to facilitate the translation of a CBS inhibition-based antitumor concept into the clinical space.

Ανάπτυξη ενός μοντέλου για την μελέτη της επίδρασης της έλλειψης της ενδογενούς παραγωγής υδρόθειου στην ανθεκτικότητα πολυανθεκτικών μικροοργανισμών (Pseudomonas aeruginosa & Klebsiella pneumoniae) ενάντια στην αντιμικροβιακή αγωγή

Το υδρόθειο (H2S) έχει πρόσφατα αναγνωριστεί ως ένας νέος αέριος διαβιβαστής μεαντιφλεγμονώδεις δράσεις. Ο ρόλος του παραγόμενου από τον ξενιστή H2S σελοιμώξεις από αεριογόνο ψευδομονάδα Pseudomonas (P.) aeruginosa διερευνήθηκεσε ένα κλινικό και ζωικό μοντέλο. Μελετήθηκαν οι συγκεντρώσεις του H2S και ηεπιβίωση σηπτικών ασθενών με λοιμώξεις του κατώτερου αναπνευστικού. Στησυνέχεια αναπτύχθηκε ένα ζωικό μοντέλο σοβαρής συστηματικής λοίμωξης απόπολυανθεκτική P. aeruginosa iχρησιμοποιώντας μύες με έλλειψη του γονιδίου της γ-λυάσης της κυσταθειονίνης (Cse) (Cse-/- μύες) και μύες με φυσιολογική έκφραση τουγονιδίου (Cse+/+ μύες). Πειράματα επαναλήφθηκαν σε μύες μετά από α) θεραπεία μεκυκλοφωσφαμίδη, β) μεταμόσχευση μυελού των οστών από δότη Cse+/+, γ) θεραπείαμε τους αναστολείς της σύνθεσης H2S αμινοοξεϊκό οξύ (aminooxyacetic acid, ΑΟΑΑ)ή προπαργυλγλυκίνη (propargylglycine, PAG) και δ) τους δότες H2S θειοθειικό νάτριο(sodium thiosulfate, STS) ή το GYY3147. Το μικροβιακό φορτίο και ημυελουπεροξειδάση προσδιορίστηκαν σε δείγματα ιστών. Η έκφραση του τωνγόνιδίων του συτήματος quorum sensing (QS) της ψευδομονάδας προσδιορίσθηκε invivo και in vitro. Μετρήθηκαν οι συγκεντρώσεις κυτταροκινών στον ορό και διεγερμένασπληνοκύτταρα. Οι ασθενείς που επιβίωσαν 28 ημέρες μετά τη λοίμωξη είχανσημαντικά υψηλότερα επίπεδα H2S ορού σε σύγκριση με ασθενείς που δεν επιβίωσαν.Επίπεδα H2S ορού 5.3 μΜ διαχωρίζουν τους επιβιώσαντες με ευαισθησία 92.3%. Ηθνητότητα στις 28 ημέρες ήταν 30.9% και 93.7% σε ασθενείς με επίπεδα H2Sυψηλότερα ή χαμηλότερα από 5.3 μΜ αντίστοιχα (p= 7 x 10-6). Η φυσιολογική έκφρασητου Cse καθώς και η θεραπεία με δότη H2S δρα προστατευτικά ενάντια σε λοίμωξη μεπολυανθεκτική P. aeruginosa. Η προθεραπεία με κυκλοφωσφαμίδη εξάλειψε τοπλεονέκτημα επιβίωσης των Cse+/+ μυών, ενώ η μεταμόσχευση μυελού των οστώναύξησε την επιβίωση των Cse-/- μυών. Οι Cse-/- μύες εμφάνισαν αυξημένα μικροβιακάφορτία σε σύγκριση με τους Cse+/+ μύες. Η φαγοκυτταρική δράση των λευκοκυττάρωντων Cse-/- μυών ήταν μειωμένη ενώ αποκαταστάθηκε μετά από προσθήκη H2S. Μίασχετιζόμενη με το H2S μείωση της έκφρασης των γονιδίων του QS P.aeruginosaαναδείχθηκε in vivo και in vitro. Η ενδογενής παραγωγή H2S είναι ένας πιθανόςανεξάρτητος προστατευτικός παράγοντας για την έκβαση λοίμωξης από P.aeruginosa. Το H2S παρέχει προστασία ενάντια σε πολυανθεκτικά βακτηριακάπαθογόνα.

Host cystathionine-γ lyase derived hydrogen sulfide protects against Pseudomonas aeruginosa sepsis

Hydrogen sulfide (H2S) has recently been recognized as a novel gaseous transmitter with several anti-inflammatory properties. The role of host- derived H2S in infections by Pseudomonas aeruginosa was investigated in clinical and mouse models. H2S concentrations and survival was assessed in septic patients with lung infection. Animal experiments using a model of severe systemic multidrug-resistant P. aeruginosa infection were performed using mice with a constitutive knock-out of cystathionine-γ lyase (Cse) gene (Cse-/-) and wild-type mice with a physiological expression (Cse+/+). Experiments were repeated in mice after a) treatment with cyclophosphamide; b) bone marrow transplantation (BMT) from a Cse+/+ donor; c) treatment with H2S synthesis inhibitor aminooxyacetic acid (ΑΟΑΑ) or propargylglycine (PAG) and d) H2S donor sodium thiosulfate (STS) or GYY3147. Bacterial loads and myeloperoxidase activity were measured in tissue samples. The expression of quorum sensing genes (QS) was determined in vivo and in vitro. Cytokine concentration was measured in serum and incubated splenocytes. Patients survivors at day 28 had significantly higher serum H2S compared to non-survivors. A cut- off point of 5.3 μΜ discriminated survivors with sensitivity 92.3%. Mortality after 28 days was 30.9% and 93.7% in patients with H2S higher and less than 5.3 μΜ (p = 7 x 10−6). In mice expression of Cse and application of STS afforded protection against infection with multidrug-resistant P. aeruginosa. Cyclophosphamide pretreatment eliminated the survival benefit of Cse+/+ mice, whereas BMT increased the survival of Cse-/- mice. Cse-/- mice had increased pathogen loads compared to Cse+/+ mice. Phagocytic activity of leukocytes from Cse-/- mice was reduced but was restored after H2S supplementation. An H2S dependent down- regulation of quorum sensing genes of P.aeruginosa could be demonstrated in vivo and in vitro. Endogenous H2S is a potential independent parameter correlating with the outcome of P. aeruginosa. H2S provides resistance to infection by MDR bacterial pathogens.

The Ethylene Biosynthesis Genes ACS2 and ACS6 Modulate Disease Severity of Verticillium dahliae

Verticillium dahliae is one of the most destructive soilborne plant pathogens since it has a broad host range and there is no chemical disease management. Therefore, there is a need to unravel the molecular interaction between the pathogen and the host plant. For this purpose, we examined the role of 1-aminocyclopropane-1-carboxylic acid synthases (ACSs) of Arabidopsis thaliana upon V. dahliae infection. We observed that the acs2, acs6, and acs2/6 plants are partially resistant to V. dahliae, since the disease severity of the acs mutants was lower than the wild type (wt) Col-0 plants. Quantitative polymerase chain reaction analysis revealed that acs2, acs6, and acs2/6 plants had lower endophytic levels of V. dahliae than the wt. Therefore, the observed reduction of the disease severity in the acs mutants is rather associated with resistance than tolerance. It was also shown that ACS2 and ACS6 were upregulated upon V. dahliae infection in the root and the above ground tissues of the wt plants. Furthermore, the addition of 1-aminocyclopropane-1-carboxylic acid (ACC) and aminooxyacetic acid (AOA), the competitive inhibitor of ACS, in wt A. thaliana, before or after V. dahliae inoculation, revealed that both substances decreased Verticillium wilt symptoms compared to controls irrespectively of the application time. Therefore, our results suggest that the mechanism underpinning the partial resistance of acs2 and acs6 seem to be ethylene depended rather than ACC related, since the application of ACC in the wt led to decreased disease severity compared to control.

Cystathionine-β-synthase: Molecular Regulation and Pharmacological Inhibition

Cystathionine-β-synthase (CBS), the first (and rate-limiting) enzyme in the transsulfuration pathway, is an important mammalian enzyme in health and disease. Its biochemical functions under physiological conditions include the metabolism of homocysteine (a cytotoxic molecule and cardiovascular risk factor) and the generation of hydrogen sulfide (H2S), a gaseous biological mediator with multiple regulatory roles in the vascular, nervous, and immune system. CBS is up-regulated in several diseases, including Down syndrome and many forms of cancer; in these conditions, the preclinical data indicate that inhibition or inactivation of CBS exerts beneficial effects. This article overviews the current information on the expression, tissue distribution, physiological roles, and biochemistry of CBS, followed by a comprehensive overview of direct and indirect approaches to inhibit the enzyme. Among the small-molecule CBS inhibitors, the review highlights the specificity and selectivity problems related to many of the commonly used “CBS inhibitors” (e.g., aminooxyacetic acid) and provides a comprehensive review of their pharmacological actions under physiological conditions and in various disease models.

In-silico comparative study of three (3) bioactive compounds from methanol extracts of Combretum micranthum leaf, and diazepam with Gabaa receptor molecule

Stress affects monoamine neurotransmitter in the central nervous system such as GABA (a major inhibitory neurotransmitter in the brain). GABAA receptor is hetero-oligomeric Cl-channel that is elective blocked by the alkaloid, bicuculline and modulated by steroids, barbiturates and benzodiazepines. The anticonvulsant activity of Diazepam may be mediated by enhancement of inhibition involving gamma-aminobutyric acid (GABA). Combretum micranthum is one of the maximum effective medicinal plants of therapeutic importance. Thus this study is to examine the effect of Combretum micranthum methanol leaf extract on GABAA Receptor via In-Silico analysis. Combretum micranthum methanol leaf extract was found using GC-MS to contain bioactive compounds (3,5-dichlorophenylhydrazine, guanidine and aminooxyacetic acid) with GABAergic functions. And the popular docking programs PatchDock and AutoDockVina were then used to predict computationally binding modes of these compounds with GABAA receptor. The molecular docking analyses indicated highly and effectively interactions (binding energy in kcal/mol) between GABAA receptor and the Combretum micranthum compounds (ligands): 3,5-dichlorophenylhydrazine (-193.85 and-5.6), guanidine (-87.63 and -3.3) and aminooxyacetic acid (-85.3 and -3.2) for both PatchDock and AutoDock Vina respectively. Results shows that 3,5-dichlorophenylhydrazine has a close binding energy in kcal/mol to that of Diazepam (-200.68 and -6.1 respectively). Findings of the study shows that the interaction between Combretum micranthum compounds (3,5-dichlorophenylhydrazine, guanidine and aminooxyacetic acid) with GABAA receptor can be explore for the development of new therapeutics to manage mental disorders. Keywords: Gamma-aminobutyric acid, Combretum micranthum , AutoDockVina, PatchDock