Peptidylarginine Deiminases Inhibitors Decrease Endothelial Cells Angiogenic Potential by Affecting Akt Signaling and the Expression and Secretion of Angiogenic Factors.

BACKGROUND/AIMS: Endothelial cells (ECs) play a crucial role in various physiological processes, particularly those related to the cardiovascular system, but also those affecting the entire organism. The biology of ECs is regulated by multiple biochemical stimuli and epigenetic drivers that govern gene expression. We investigated the angiogenic potential of ECs from a protein citrullination perspective, regulated by peptidyl-arginine deiminases (PADs) that modify histone and non-histone proteins. Although the involvement of PADs has been demonstrated in several physiological processes, inflammation-related disorders and cancer, their role in angiogenesis remains unclear. METHODS: To elucidate the role of PADs in endothelial angiogenesis, we used two human EC models: primary vein (HUVECs) and microvascular endothelial cells (HMEC-1). PADs activity was inhibited using irreversible inhibitors: BB-Cl-amidine, Cl-amidine and F-amidine. We analyzed all three steps of angiogenesis in vitro : proliferation, migration, and capillary-like tube formation, as well as secretory activities, gene expression and signaling in ECs. RESULTS: All used PAD inhibitors reduced the histone H3 citrullination (H3cit) mark, inhibited endothelial cell migration and capillary-like tube formation, and favored an angiostatic activity in HMEC-1 cells, by increasing PEDF (pigment epithelium-derived factor) and reducing VEGF (vascular endothelial growth factor) mRNA expression and protein secretion. Additionally, BB-Cl-amidine reduced the total activity of MMPs (Matrix metalloproteinases). The observed effects were underlined by the inhibition of Akt phosphorylation.>. CONCLUSION: Our findings suggest that pharmacological inhibitors of citrullination are promising therapeutic agents to target angiogenesis.

Inhibiting Inducible Nitric Oxide Synthase with 1400W Reduces Soman (GD)-Induced Ferroptosis in Long-Term Epilepsy-Associated Neuropathology: Structural and Functional Magnetic Resonance Imaging Correlations with Neurobehavior and Brain Pathology.

Organophosphate (OP) nerve agent (OPNA) intoxication leads to long-term brain dysfunctions. The ineffectiveness of current treatments for OPNA intoxication prompts a quest for the investigation of the mechanism and an alternative effective therapeutic approach. Our previous studies on 1400W, a highly selective inducible nitric oxide synthase (iNOS) inhibitor, showed improvement in epilepsy and seizure-induced brain pathology in rat models of kainate and OP intoxication. In this study, magnetic resonance imaging (MRI) modalities, behavioral outcomes, and biomarkers were comprehensively investigated for brain abnormalities following soman (GD) intoxication in a rat model. T1 and T2 MRI robustly identified pathologic microchanges in brain structures associated with GD toxicity, and 1400W suppressed those aberrant alterations. Moreover, functional network reduction was evident in the cortex, hippocampus, and thalamus after GD exposure, and 1400W rescued the losses except in the thalamus. Behavioral tests showed protection by 1400W against GD-induced memory dysfunction, which also correlated with the extent of brain pathology observed in structural and functional MRIs. GD exposure upregulated iron-laden glial cells and ferritin levels in the brain and serum, 1400W decreased ferritin levels in the epileptic foci in the brain but not in the serum. The levels of brain ferritin also correlated with MRI parameters. Further, 1400W mitigated the overproduction of nitroxidative markers after GD exposure. Overall, this study provides direct evidence for the relationships of structural and functional MRI modalities with behavioral and molecular abnormalities following GD exposure and the neuroprotective effect of an iNOS inhibitor, 1400W. SIGNIFICANT STATEMENT: Our studies demonstrate the MRI microchanges in the brain following GD toxicity, which strongly correlate with neurobehavioral performances and iron homeostasis. The inhibition of iNOS with 1400W mitigates GD-induced cognitive decline, iron dysregulation, and aberrant brain MRI findings.

The effect of novel aromatic heterocycle substituted aminamidine derivatives on Necator americanus.

BACKGROUND: The efficacy of current drugs against hookworms at a single dose is highly variable across regions, age groups and infection intensity. Extensive and repeated use of these drugs also leads to potential drug resistance. Therefore, novel drugs are required for sustained disease control. OBJECTIVES: Novel aromatic heterocycle substituted aminamidine derivatives (AADs) were synthesized based on tribendimine (TBD), and their in vivo potency against Necator americanus was tested. METHODS: The efficacy of the AADs was tested in male hamsters. Oral and IV pharmacokinetic parameters were determined in male Sprague-Dawley rats. The proteomic profiles of N. americanus samples treated with AADs were compared using tandem mass tag-based quantitative proteomic analyses. RESULTS: Most AADs exhibited better anthelmintic activity than TBD at a single oral dose. Compound 3c exhibited improved solubility (>50×), and the curative dose was as low as 25 mg/kg. Similar to TBD, 3c was rapidly metabolized after oral administration and transformed into p-(1-dimethylamino ethylimino)aniline (dADT), an active metabolite against intestinal nematodes. dADT from 3c had better pharmacokinetic profiles than that from TBD and achieved an oral bioavailability of 99.5%. Compound 3c possessed rapid anthelmintic activity, clearing all worms within 24 h after an oral dose of 50 mg/kg. Quantitative proteomic analysis indicated that it might be related to ATP metabolism and cuticle protein synthesis. CONCLUSIONS: Compound 3c is a novel and promising compound against N. americanus in vivo.

Mechanistic insights into antifungal potential of Alexidine dihydrochloride and hexachlorophene in Candida albicans: a drug repurposing approach.

Candida albicans has been listed in the critical priority group by the WHO in 2022 depending upon its contribution in invasive candidiasis and increased resistance to conventional drugs. Drug repurposing offers an efficient, rapid, and cost-effective solution to develop alternative therapeutics against pathogenic microbes. Alexidine dihydrochloride (AXD) and hexachlorophene (HCP) are FDA approved anti-cancer and anti-septic drugs, respectively. In this study, we have shown antifungal properties of AXD and HCP against the wild type (reference strain) and clinical isolates of C. albicans. The minimum inhibitory concentrations (MIC50) of AXD and HCP against C. albicans ranged between 0.34 and 0.69 µM and 19.66-24.58 µM, respectively. The biofilm inhibitory and eradication concentration of AXD was reported comparatively lower than that of HCP for the strains used in the study. Further investigations were performed to understand the antifungal mode of action of AXD and HCP by studying virulence features like cell surface hydrophobicity, adhesion, and yeast to hyphae transition, were also reduced upon exposure to both the drugs. Ergosterol content in cell membrane of the wild type strain was upregulated on exposure to AXD and HCP both. Biochemical analyses of the exposed biofilm indicated reduced contents of carbohydrate, protein, and e-DNA in the extracellular matrix of the biofilm when compared to the untreated control biofilm. AXD exposure downregulated activity of tissue invading enzyme, phospholipase in the reference strain. In wild type strain, ROS level, and activities of antioxidant enzymes were found elevated upon exposure to both drugs. FESEM analysis of the drug treated biofilms revealed degraded biofilm. This study has indicated mode of action of antifungal potential of alexidine dihydrochloride and hexachlorophene in C. albicans.

Synthesis of 6,8-diaminopurines via acid-induced cascade cyclization of 5-aminoimidazole precursors and preliminary anticancer evaluation.

Inspired by the pharmacological interest generated by 6-substituted purine roscovitine for cancer treatment, 5-aminoimidazole-4-carboxamidine precursors containing a cyanamide unit were prepared by condensation of 5-amino-N-cyanoimidazole-4-carbimidoyl cyanides with a wide range of primary amines. When these amidine precursors were combined with acids, a fast cascade cyclization occurred at room temperature, affording new 6,8-diaminopurines with the N-3 and N-6 substituents changed relatively to the original positions they occupied in the amidine and imidazole moieties of precursors. The efficacy and wide scope of this method was well demonstrated by an easy and affordable synthesis of 22 6,8-diaminopurines decorated with a wide diversity of substituents at the N-3 and N-6 positions of the purine ring. Preliminary in silico and in vitro assessments of these 22 compounds were carried out and the results showed that 13 of these tested compounds not only exhibited IC50 values between 1.4 and 7.5 µM against the colorectal cancer cell line HCT116 but also showed better binding energies than known inhibitors in docking studies with different cancer-related target proteins. In addition, good harmonization observed between in silico and in vitro results strengthens and validates this preliminary evaluation, suggesting that these novel entities are good candidates for further studies as new anticancer agents.

Novel Amidine Derivative K1586 Sensitizes Colorectal Cancer Cells to Ionizing Radiation by Inducing Chk1 Instability.

Checkpoint kinase 1 (Chk1) is a key mediator of the DNA damage response that regulates cell cycle progression, DNA damage repair, and DNA replication. Small-molecule Chk1 inhibitors sensitize cancer cells to genotoxic agents and have shown preclinical activity as single agents in cancers characterized by high levels of replication stress. However, the underlying genetic determinants of Chk1-inhibitor sensitivity remain unclear. Although treatment options for advanced colorectal cancer are limited, radiotherapy is effective. Here, we report that exposure to a novel amidine derivative, K1586, leads to an initial reduction in the proliferative potential of colorectal cancer cells. Cell cycle analysis revealed that the length of the G2/M phase increased with K1586 exposure as a result of Chk1 instability. Exposure to K1586 enhanced the degradation of Chk1 in a time- and dose-dependent manner, increasing replication stress and sensitizing colorectal cancer cells to radiation. Taken together, the results suggest that a novel amidine derivative may have potential as a radiotherapy-sensitization agent that targets Chk1.

The investigation of cytotoxic and apoptotic activity of Cl-amidine on the human U-87 MG glioma cell line.

BACKGROUND: Peptidyl (protein) arginine deiminases (PADs) provide the transformation of peptidyl arginine to peptidyl citrulline in the presence of calcium with posttranslational modification. The dysregulated PAD activity plays an important role on too many diseases including also the cancer. In this study, it has been aimed to determine the potential cytotoxic and apoptotic activity of chlorine-amidine (Cl-amidine) which is a PAD inhibitor and whose effectiveness has been shown in vitro and in vivo studies recently on human glioblastoma cell line Uppsala 87 malignant glioma (U-87 MG) forming an in vitro model for the glioblastoma multiforme (GBM) which is the most aggressive and has the highest mortality among the brain tumors. METHODS: In the study, the antiproliferative and apoptotic effects of Cl-amidine on GBM cancer model were investigated. The antiproliferative effects of Cl-amidine on U-87 MG cells were determined by 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate method at the 24th and 48th hours. The apoptotic effects were analyzed by Annexin V and Propidium iodide staining, caspase-3 activation, and mitochondrial membrane polarization (5,5', 6,6'-tetrachloro-1,1', 3,3' tetraethyl benzimidazolyl carbocyanine iodide) methods in the flow cytometry. RESULTS: It has been determined that Cl-amidine exhibits notable antiproliferative properties on U-87 MG cell line in a time and concentration-dependent manner, as determined through the 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate assay. Assessment of apoptotic effects via Annexin V and Propidium iodide staining and 5,5', 6,6'-tetrachloro-1,1', 3,3' tetraethyl benzimidazolyl carbocyanine iodide methods has revealed significant efficacy, particularly following a 24-hour exposure period. It has been observed that Cl-amidine induces apoptosis in cells by enhancing mitochondrial depolarization, independently of caspase-3 activation. Furthermore, regarding its impact on healthy cells, it has been demonstrated that Cl-amidine shows lower cytotoxic effects when compared to carmustine, an important therapeutic agent for glioblastoma. CONCLUSION: The findings of this study have shown that Cl-amidine exhibits significant potential as an anticancer agent in the treatment of GBM. This conclusion is based on its noteworthy antiproliferative and apoptotic effects observed in U-87 MG cells, as well as its reduced cytotoxicity toward healthy cells in comparison to existing treatments. We propose that the antineoplastic properties of Cl-amidine should be further investigated through a broader spectrum of cancer cell types. Moreover, we believe that investigating the synergistic interactions of Cl-amidine with single or combination therapies holds promise for the discovery of novel anticancer agents.

Synthesis and evaluation of arylamidine derivatives for new antimicrobial and cytotoxic activities

ABSTRACT A series of arylamidines 3a-j was designed, synthesized and investigated for antimicrobial activity. Structures of the compounds were confirmed by IR, 1H-NMR and 13C-NMR and a 2D spectroscopic study was performed. A preliminary screening of the antimicrobial tests clearly showed that three out of ten arylamidines, viz, 3f, 3g and 3i, were effective against all the gram-negative bacteria: Klebsiella pneumoniae, Pseudomonas aeruginosa and Salmonella enteric; and against the yeast, candida albicans. Further, the Minimum Inhibitory Concentrations (MIC) against the bacteria and yeast were determined. All compounds 3a-d, 3f, 3g, 3i and 3j were also investigated for their low cytotoxic effects on tested cell lines. Compounds 3d and 3f were the most effective derivatives against HL-60 and HEp-2 cells, respectively, with IC50 value (2µg/mL), and low normal cells toxicity.

Apontamentos acerca da Cadeia do Ser e o lugar dos negros na filosofia natural na Europa setecentista / Notes on the Chain of Being and the place of blacks in eighteenth-century European natural philosophy

Por meio da análise de obras acadêmicas produzidas por filósofos naturais no século XVIII, pretendemos discutir algumas ideias recorrentes acerca da Grande Cadeia do Ser. Para tal, analisamos as relações entre filosofia e teologia natural no período. Reavaliamos ainda alguns elementos da Cadeia do Ser, investigando autores que discorreram sobre o tema em seus escritos. Por fim, elencamos um ponto específico das discussões setecentistas sobre a scala naturae, qual seja, as diversas e nem sempre convergentes ideias de que, a partir de características específicas, haveria diferenças entre os homens, bem como seu consequente lugar na Cadeia do Ser.

This examination of academic works produced by eighteenth-century natural philosophers discusses some recurring ideas about the Chain of Being. To this end, the article analyzes the relations between natural philosophy and theology during the period. It also re-evaluates some elements of the Chain of Being through an exploration of authors who addressed the topic in their writings. Lastly, it identifies a specific element within eighteenth-century discussions of scala naturae, to wit, the various and not always convergent ideas about whether there are differences between humans based on specific characteristics and, consequently, about the places they occupy in the chain of being.

Antioxidant effect of 4-nerolidylcatechol and -tocopherol in erythrocyte ghost membranes and phospholipid bilayers

4-Nerolidylcatechol (4-NC) is found in Pothomorphe umbellata root extracts and is reported to have a topical protective effect against UVB radiation-induced skin damage, toxicity in melanoma cell lines, and antimalarial activity. We report a comparative study of the antioxidant activity of 4-NC and α-tocopherol against lipid peroxidation initiated by two free radical-generating systems: 2,2′-azobis(2-aminopropane) hydrochloride (AAPH) and FeSO4/H2O2, in red blood cell ghost membranes and in egg phosphatidylcholine (PC) vesicles. Lipid peroxidation was monitored by membrane fluidity changes assessed by electron paramagnetic resonance spectroscopy of a spin-labeled lipid and by the formation of thiobarbituric acid-reactive substances. When lipoperoxidation was initiated by the hydroxyl radical in erythrocyte ghost membranes, both 4-NC and α-tocopherol acted in a very efficient manner. However, lower activities were observed when lipoperoxidation was initiated by the peroxyl radical; and, in this case, the protective effect of α-tocopherol was lower than that of 4-NC. In egg PC vesicles, malondialdehyde formation indicated that 4-NC was effective against lipoperoxidation initiated by both AAPH and FeSO4/H2O2, whereas α-tocopherol was less efficient in protecting against lipoperoxidation by AAPH, and behaved as a pro-oxidant for FeSO4/H2O2. The DPPH (2,2-diphenyl-1-picrylhydrazyl) free-radical assay indicated that two free radicals were scavenged per 4-NC molecule, and one free radical was scavenged per α-tocopherol molecule. These data provide new insights into the antioxidant capacity of 4-NC, which may have therapeutic applications for formulations designed to protect the skin from sunlight irradiation.

Ascorbate protects (+)-catechin from oxidation both in a pure chemical system and human plasma

We evaluated the interaction between ascorbic acid (AA) and (+)-catechin (CTCH) in potassium phosphate solution, pH 7.4 (PPS) and in human plasma. In both systems, the oxidation was started by adding 2,2'-azobis-(2-amidinopropane) clorhidrate (AAPH). The concentrations of AA and CTCH were determined by HPLC using electrochemical detection. In PPS, CTCH was oxidized by AAPH (50 mM), in either the absence or presence of different initial concentrations of AA (25-200 microM). In the presence of AA, CTCH depletion was delayed, an effect that was dependent upon the initial concentration of AA. When 100 microM AA was added after the oxidation had begun, CTCH depletion was arrested for 30 min. The kinetics of AA oxidation by AAPH was also characterized in PPS. AA (100 microM) was completely consumed after 60 min of reaction at 37 degrees C, in both the absence and presence of 100 mM CTCH. When human plasma was incubated with 50 mM AAPH in the absence of added CTCH, AA was completely consumed after 45-60 min. CTCH did not prevent AA depletion in human plasma at the concentrations tested (10, 50 100 microM). The results point out that AA is able to protect other aqueous soluble antioxidants, e.g.: CTCH.