Selectivity of Hydroxamate- and Difluoromethyloxadiazole-Based Inhibitors of Histone Deacetylase 6 In Vitro and in Cells.

Histone deacetylase 6 (HDAC6) is a unique member of the HDAC family of enzymes due to its complex domain organization and cytosolic localization. Experimental data point toward the therapeutic use of HDAC6-selective inhibitors (HDAC6is) for use in both neurological and psychiatric disorders. In this article, we provide side-by-side comparisons of hydroxamate-based HDAC6is frequently used in the field and a novel HDAC6 inhibitor containing the difluoromethyl-1,3,4-oxadiazole function as an alternative zinc-binding group (compound 7). In vitro isotype selectivity screening uncovered HDAC10 as a primary off-target for the hydroxamate-based HDAC6is, while compound 7 features exquisite 10,000-fold selectivity over all other HDAC isoforms. Complementary cell-based assays using tubulin acetylation as a surrogate readout revealed approximately 100-fold lower apparent potency for all compounds. Finally, the limited selectivity of a number of these HDAC6is is shown to be linked to cytotoxicity in RPMI-8226 cells. Our results clearly show that off-target effects of HDAC6is must be considered before attributing observed physiological readouts solely to HDAC6 inhibition. Moreover, given their unparalleled specificity, the oxadiazole-based inhibitors would best be employed either as research tools in further probing HDAC6 biology or as leads in the development of truly HDAC6-specific compounds in the treatment of human disease states.

Establishment of a Rapid Detection System for ISG20-Dependent SARS-CoV-2 Subreplicon RNA Degradation Induced by Interferon-α.

Inhaled nebulized interferon (IFN)-α and IFN-ß have been shown to be effective in the management of coronavirus disease 2019 (COVID-19). We aimed to construct a virus-free rapid detection system for high-throughput screening of IFN-like compounds that induce viral RNA degradation and suppress the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We prepared a SARS-CoV-2 subreplicon RNA expression vector which contained the SARS-CoV-2 5'-UTR, the partial sequence of ORF1a, luciferase, nucleocapsid, ORF10, and 3'-UTR under the control of the cytomegalovirus promoter. The expression vector was transfected into Calu-3 cells and treated with IFN-α and the IFNAR2 agonist CDM-3008 (RO8191) for 3 days. SARS-CoV-2 subreplicon RNA degradation was subsequently evaluated based on luciferase levels. IFN-α and CDM-3008 suppressed SARS-CoV-2 subreplicon RNA in a dose-dependent manner, with IC50 values of 193 IU/mL and 2.54 µM, respectively. HeLa cells stably expressing SARS-CoV-2 subreplicon RNA were prepared and treated with the IFN-α and pan-JAK inhibitor Pyridone 6 or siRNA-targeting ISG20. IFN-α activity was canceled with Pyridone 6. The knockdown of ISG20 partially canceled IFN-α activity. Collectively, we constructed a virus-free rapid detection system to measure SARS-CoV-2 RNA suppression. Our data suggest that the SARS-CoV-2 subreplicon RNA was degraded by IFN-α-induced ISG20 exonuclease activity.

Novel 1,3,4-thiadiazole/oxadiazole-linked honokiol derivatives suppress cancer via inducing PI3K/Akt/mTOR-dependent autophagy.

Honokiol is a bioactive biphenolic component derived from Magnoliae officinalis Cortex (known as "Hou Po" in Chinese), a traditional Chinese herbal medicine. A series of novel 1,3,4-thiadiazole/oxadiazole-linked honokiol derivatives were synthesized and tested for anticancer activity against seven human cancer cell lines in this study. Among all derivatives, 8a had the most potent cytotoxic effect on all tested cancer cells, with IC50 values ranging from 1.62 ± 0.19 to 4.61 ± 0.51 µM, which were 10.38-34.36 folds more potent than the parental honokiol (IC50 values of 30.96 ± 1.81-55.67 ± 0.31 µM). On A549, HCT116, and MDA-MB-231 cell lines, 8a demonstrated 5.69-fold, 5.65-fold, and 4.83-fold greater cytotoxicity than cisplatin, respectively. Compound 8a also had higher selectivity (SI values of 8.41-49.38) towards seven cancer cell lines over the normal cell lines than cisplatin (SI values of 1.24-2.52). The analysis of structure-activity relationships (SARs) revealed that honokiol derivatives bearing 1,3,4-thiadiazoles (8a-j) possessed stronger anticancer activity than those containing 1,3,4-oxadiazoles. Further mechanistic investigation indicated that 8a induced cytotoxic autophagy in cancer cells in a time- and dose-independent manner via suppressing the PI3K/Akt/mTOR pathway. Molecular docking suggested that 8a could bind to the PI3Kα active sites. Additionally, 8a inhibited the migration and invasion of A549 and MDA-MB-231 cancer cells.

Structure-based screening for the discovery of 1,2,4-oxadiazoles as promising hits for the development of new anti-inflammatory agents interfering with eicosanoid biosynthesis pathways.

The multiple inhibition of biological targets involved in pro-inflammatory eicosanoid biosynthesis represents an innovative strategy for treating inflammatory disorders in light of higher efficacy and safety. Herein, following a multidisciplinary protocol involving virtual combinatorial screening, chemical synthesis, and in vitro and in vivo validation of the biological activities, we report the identification of 1,2,4-oxadiazole-based eicosanoid biosynthesis multi-target inhibitors. The multidisciplinary scientific approach led to the identification of three 1,2,4-oxadiazole hits (compounds 1, 2 and 5), all endowed with IC50 values in the low micromolar range, acting as 5-lipoxygenase-activating protein (FLAP) antagonists (compounds 1 and 2), and as a multi-target inhibitor (compound 5) of arachidonic acid cascade enzymes, namely cyclooxygenase-1 (COX-1), 5-lipoxygenase (5-LO) and microsomal prostaglandin E2 synthase-1 (mPGES-1). Moreover, our in vivo results demonstrate that compound 5 is able to attenuate leukocyte migration in a model of zymosan-induced peritonitis and to modulate the production of IL-1ß and TNF-α. These results are of interest for further expanding the chemical diversity around the 1,2,4-oxadiazole central core, enabling the identification of novel anti-inflammatory agents characterized by a favorable pharmacological profile and considering that moderate interference with multiple targets might have advantages in re-adjusting homeostasis.

Azilsartan ameliorates ox-LDL-induced endothelial dysfunction via promoting the expression of KLF2.

BACKGROUND: Oxidized LDL(Ox-LDL) mediated endothelial dysfunction is involved in the pathogenesis of various cardiovascular diseases, including atherosclerosis. Azilsartan is a potent agent for the treatment of hypertension as the antagonist of the angiotensin II receptor. This study will investigate whether Azilsartan possesses a beneficial effect against endothelial cell dysfunction induced by ox-LDL and explore the underlying preliminary mechanism. METHODS: Ox-LDL was applied to construct an in vitro endothelial dysfunction model in human umbilical vascular endothelial cells (HUVECs). The expression of lectin-type oxidized LDL receptor 1 (LOX-1), endothelial nitric oxide synthase (eNOS), tight junction protein occludin, and transcriptional factor Krüppel-like factor 2 (KLF2) was detected using qRT-PCR and Western blot. ELISA and qRT-PCR were utilized to evaluate the production of chemokine monocyte chemotactic protein 1 (MCP-1) and chemokine (C-X-C motif) Ligand 1 Protein (CXCL1) in treated HUVECs. The generation of nitro oxide (NO) was determined using DAF-FM DA staining assay. KLF2 was silenced by transfecting the cells with specific Small interfering RNA (siRNA). FITC-dextran permeation assay was used to check the endothelial monolayer permeability of treated HUVECs. RESULTS: Firstly, the elevated expressions of LOX-1, MCP-1, and CXCL-1 induced by stimulation with ox-LDL were significantly suppressed by Azilsartan. The downregulated eNOS and reduced production of NO induced by ox-LDL were reversed by the introduction of Azilsartan. Secondly, enlarged endothelial monolayer permeability and decreased expression of occludin stimulated with ox-LDL were greatly reversed by treatment with Azilsartan but were abolished by silencing the expression of KLF2. Lastly, the inhibited expression of KLF2 induced by ox-LDL was significantly elevated by the introduction of Azilsartan. CONCLUSION: Azilsartan might ameliorate ox-LDL-induced endothelial damage via elevating the expression of KLF2.

Synthesis of oxadiazole-2-oxide derivatives as potential drug candidates for schistosomiasis targeting SjTGR.

BACKGROUND: Schistosomiasis is a chronic parasitic disease that affects millions of people's health worldwide. Because of the increasing drug resistance to praziquantel (PZQ), which is the primary drug for schistosomiasis, developing new drugs to treat schistosomiasis is crucial. Oxadiazole-2-oxides have been identified as potential anti-schistosomiasis reagents targeting thioredoxin glutathione reductase (TGR). METHODS: In this work, one of the oxadiazole-2-oxides derivatives furoxan was used as the lead compound to exploit a series of novel furoxan derivatives for studying inhibitory activity against both recombinant Schistosoma japonicum TGR containing selenium (rSjTGR-Sec) and soluble worm antigen protein (SWAP) containing wild-type Schistosoma japonicum TGR (wtSjTGR), in order to develop a new leading compound for schistosomiasis. Thirty-nine novel derivatives were prepared to test their activity toward both enzymes. The docking method was used to detect the binding site between the active molecule and SjTGR. The structure-activity relationship (SAR) of these novel furoxan derivatives was preliminarily analyzed. RESULTS: It was found that several new derivatives, including compounds 6a-6d, 9ab, 9bd and 9be, demonstrated greater activity toward rSjTGR-Sec or SWAP containing wtSjTGR than did furoxan. Interestingly, all intermediates bearing hydroxy (6a-6d) showed excellent inhibitory activity against both enzymes. In particular, compound 6d with trifluoromethyl on a pyridine ring was found to have much higher inhibition toward both rSjTGR-Sec (half-maximal inhibitory concentration, IC50,7.5nM) and SWAP containing wtSjTGR (IC50 55.8nM) than furoxan. Additionally, the docking method identified the possible matching sites between 6d and Schistosoma japonicum TGR (SjTGR), which theoretically lends support to the inhibitory activity of 6d. CONCLUSION: The data obtained herein showed that 6d with trifluoromethyl on a pyridine ring could be a valuable leading compound for further study.

Beyond direct Nrf2 activation; reinvestigating 1,2,4-oxadiazole scaffold as a master key unlocking the antioxidant cellular machinery for cancer therapy.

Harnessing the antioxidant cellular machinery has sparked considerable interest as an efficient anticancer strategy. Activating Nrf2, the master switch of the cellular redox system, suppresses ROS, alleviates oxidative stress, and halts cancer progression. 1,2,4-oxadiazoles are iconic direct Nrf2 activators that disrupt Nrf2 interaction with its endogenous repressor Keap1. This study introduces rationally designed 1,2,4-oxadiazole derivatives that inhibit other Nrf2 suppressors (TrxR1, IKKα, and NF-kB) thus enhancing Nrf2 activation for preventing oxidative stress and carcinogenesis. Preliminary screening showed that the phenolic oxadiazoles 11, 15, and 19 were comparable to ascorbic acid (ROS scavenging) and EDTA (iron chelation), and superior to doxorubicin against HepG-2, MDA-MB231, and Caco-2 cells. They suppressed ROS by 3 folds and activated Nrf2 by 2 folds in HepG-2 cells. Mechanistically, they inhibited TrxR1 (IC50; 13.19, 17.89, and 9.21 nM) and IKKα (IC50; 11.0, 15.94, and 19.58 nM), and downregulated NF-κB (7.6, 1.4 and 1.9 folds in HepG-2), respectively. They inhibited NADPH oxidase (IC50; 16.4, 21.94, and 10.71 nM, respectively) that potentiates their antioxidant activities. Docking studies predicted their important structural features. Finally, they recorded drug-like in silico physicochemical properties, ADMET, and ligand efficiency metrics.

Antihypertensive and vasorelaxant effect of leucodin and achillin isolated from Achillea millefolium through calcium channel blockade and NO production: In vivo, functional ex vivo and in silico studies.

ETHNOPHARMACOLOGICAL RELEVANCE: Achillea millefolium L. (Asteraceae), known as yarrow (milenrama), is a plant used in Mexican traditional medicine for the treatment of hypertension, diabetes, and related diseases. AIM: To determine the vasorelaxant and antihypertensive effect of A. millefollium and to isolate the main bioactive antihypertensive agents. MATERIALS AND METHODS: Organic (hexane, dichloromethane and methanol) and hydro-alcohol (Ethanol-H2O: 70:30) extracts obtained from flowers, leaves and stems were evaluated on isolated aorta rat rings with and without endothelium to determine their vasorelaxant effect. Hexane extract from flowers (HEAmF) was studied to evaluate its antihypertensive effect on spontaneously hypertensive rats (SHR). From HEAmF, bioactive compounds were obtained by bio-guided phytochemical separation through chromatography. RESULTS: Organic extracts showed the best vasorelaxant activity. Hexane extract from flowers was the most potent and efficient ex vivo vasorelaxant agent, showing significant decrease of systolic and diastolic blood pressure in SHR (p < 0.05). Phytochemical separation of HEAmF yielded two epimeric sesquiterpene lactones: leucodin (1) and achillin (2), the major components of the extract. Both 1 and 2 showed similar vasorelaxant action ex vivo (p < 0.05), and their effects where modified by L-NAME (10 µM, nitric oxide synthase inhibitor), by ODQ (1 µM, soluble guanylyl cyclase inhibitor), and also relaxed the contraction induced by KCl (80 mM). Finally, 1 and 2 intragastric administration (50 mg/kg) decreased systolic and diastolic blood pressure in SHR. CONCLUSIONS: Achillea millefolium showed antihypertensive and vasorelaxant effects, due mainly to leucodin and achillin (epimers). Both compounds showed antihypertensive activity by vasorelaxation putatively by endothelium-dependent NO release and cGMP increase, as well as by calcium channels blockade.

Evaluation and Docking Study of Pyrazine Containing 1, 3, 4-Oxadiazoles Clubbed with Substituted Azetidin-2-one: A New Class of Potential Antimicrobial and Antitubercular.

BACKGROUND: Tuberculosis (TB) caused by Mycobacterium tuberculosis is one of the main killers of people all over the world. The major hurdles with existing therapy are the lengthy regimen and appearance of multi drug resistant (MDR) and extensively drug resistant (XDR) strains of M.tuberculosis. AIMS: The present work was aimed to synthesize and determine antitubercular and antimicrobial potential of some novel 3-chloro-4-aryl-1-[4-(5-pyrazin-2-yl[1,3,4]oxadiazole-2-ylmethoxy)-phenyl]-azetidin-2-one derivatives 7: (A: -H: ) from pyrazinoic acid as precursor, which is a well-established antitubercular agent. Here we report the synthesis of a new class of heterocyclic molecules in which pyrazine, 1, 3, 4-oxadiazole and azetidinone moieties were present in one frame work. METHODS: Pyrazinoic acid (1: ) was esterified first (2: ) followed by amination to produce hydrazide (3: ) which was refluxed with POCl3 to obtain 2-chloromethyl-5pyrazino-1, 3, 4-oxadiazole (4: ). This was then further reacted with 4-amino phenol to obtain 4-[5-pyrazino-1, 3, 4-oxadiazol-2-yl-methoxy]-phenyl amine (5: ) which on condensation with various aromatic aldehydes afforded a series Schiff's bases 6(A-H): . Dehydrative annulations of 6(A-H): in the presence of chloroacetyl chloride and triethylamine yielded 3-chloro-4-aryl-1-[4-(5-pyrazin-2-yl-[1, 3, 4]oxadiazole-2-ylmethoxy)-phenyl]-azetidin-2-one derivatives 7(A-H): . Antibacterial, antifungal and antitubercular potential of all the synthesized compounds were assessed. Docking study was performed using the software VLife Engine tools of Vlifemds 4.6 on the protein lumazine synthase of M. tuberculosis (PDB entry code 2C92). RESULTS: The present studies demonstrated that synthesized oxadiazole derivatives have good antimicrobial activity against the various microorganisms. Among the synthesized derivative, 7B: and 7G: were found to be prominent compounds which have potential antibacterial, antifungal and antitubercular activity (with MIC 3.12 µg/ml and high dock score ranging from -59.0 to -54.0) against Mycobacterium tuberculosis. CONCLUSIONS: Derivatives 7B: and 7G: would be effective lead candidates for tuberculosis therapy.

Evaluation of Biological Activity of Derivatives of 1,3,4-Thiadiazole, 1,3,4-Oxadiazole and 1,2,4-Triazole.

OBJECTIVE: 1,3,4-thiadiazole (A), 1,3,4-oxadiazole (B) and 1,2,4-triazole (C) derivatives have been known for their immense pharmacotherapeutic potential. The current research article attempts to further explore and understand the probable biochemical mechanism related to antiinflammatory activity of derivatives. METHODS: The screened A, B and C derivatives were investigated for both in-vitro (Erythrocyte Membrane stabilization activity, Proteinase enzyme inhibitory activities) and in-vivo correlation using acute and chronic anti-inflammatory potential by carrageenan induced rats paw edema and cotton pellet granuloma methods, respectively. The activity was studied after interpreting acute toxicity studies results. RESULTS: In vitro studies in the case of Erythrocyte Membrane stability and Proteinase enzyme inhibitory activities exhibited by A, B, and C at 100 ppm were found to be 48.89%, 51.08% and 50.08% and 66.78%, 76.91% and 57.41%, respectively. The maximum toxic dose was found to be 2000 mg/kg. The derivatives were studied for two-dose levels viz; Lower (100 mg/kg) and higher dose (200 mg/kg). In rat paw edema, maximum decrease was obtained for A (50.05%), B (50.05%) and C (51.06%) at lower and higher dose at 68.76%, 55.61%, and 65.26%, respectively for effect up to 24 h. In the chronic model of cotton pellet granuloma viz; higher and lower doses of A, B and C exhibited 38.15%, 33.19% and 30.25 % and 19.45%, 18.55% and 17.55 %, respectively. CONCLUSION: The studied models depicted that derivatives A, B and C have the probable potential as anti-inflammatory agents. Further studies need to be undertaken to explore their potential in the different therapeutic areas.

Furoxan derivatives demonstrated in vivo efficacy by reducing Mycobacterium tuberculosis to undetectable levels in a mouse model of infection.

OBJECTIVES: The most recent survey conducted by the World Health Organization described Tuberculosis (TB) as one of the top 10 causes of death and the leading cause of death from a single infectious agent. The increasing number of TB-resistant cases has contributed to this scenario. In light of this, new strategies to control and treat the disease are necessary. Our research group has previously described furoxan derivatives as promising scaffolds to be explored as new antitubercular drugs. RESULTS: Two of these furoxan derivatives, (14b) and (14c), demonstrated a high selectivity against Mycobacterium tuberculosis. The compounds (14b) and (14c) were also active against a latent M. tuberculosis strain, with MIC90 values of 6.67 µM and 9.84 µM, respectively; they were also active against monoresistant strains (MIC90 values ranging from 0.61 to 20.42 µM) and clinical MDR strains (MIC90 values ranging from 3.09 to 42.95 µM). Time-kill experiments with compound (14c) showed early bactericidal effects that were superior to those of the first- and second-line anti-tuberculosis drugs currently used in therapy. The safety of compounds (14b) and (14c) was demonstrated by the Ames test because these molecules were not mutagenic under the tested conditions. Finally, we confirmed the safety, and high efficacy of compounds (14b) and (14c), which reduced M. tuberculosis to undetectable levels in a mouse aerosol model of infection. CONCLUSION: Altogether, we have identified two advanced lead compounds, (14b) and (14c), as novel promising candidates for the treatment of TB infection.

Synthesis, Characterization, and Biologic Activity of New Acyl Hydrazides and 1,3,4-Oxadiazole Derivatives.

Starting from isoniazid and carboxylic acids as precursors, thirteen new hydrazides and 1,3,4-oxadiazoles of 2-(4-substituted-phenoxymethyl)-benzoic acids were synthesized and characterized by appropriate means. Their biological properties were evaluated in terms of apoptosis, cell cycle blocking, and drug metabolism gene expression on HCT-8 and HT-29 cell lines. In vitro antimicrobial tests were performed by the microplate Alamar Blue assay for the anti-mycobacterial activities and an adapted agar disk diffusion technique for other non-tubercular bacterial strains. The best antibacterial activity (anti-Mycobacterium tuberculosis effects) was proved by 9. Compounds 7, 8, and 9 determined blocking of G1 phase. Compound 7 proved to be toxic, inducing apoptosis in 54% of cells after 72 h, an effect that can be predicted by the increased expression of mRNA caspases 3 and 7 after 24 h. The influence of compounds on gene expression of enzymes implicated in drug metabolism indicates that synthesized compounds could be metabolized via other pathways than NAT2, spanning adverse effects of isoniazid. Compound 9 had the best antibacterial activity, being used as a disinfectant agent. Compounds 7, 8, and 9, seemed to have antitumor potential. Further studies on the action mechanism of these compounds on the cell cycle may bring new information regarding their biological activity.

The preclinical discovery and development of opicapone for the treatment of Parkinson's disease.

INTRODUCTION: Opicapone (OPC) is a well-established catechol-O-methyltransferase (COMT) inhibitor that is approved for the treatment of Parkinson's disease (PD) associated with L-DOPA/L-amino acid decarboxylase inhibitor (DDI) therapy allowing for prolonged activity due to a more continuous supply of L-DOPA in the brain. Thus, OPC decreases fluctuation in L-DOPA plasma levels and favors more constant central dopaminergic receptor stimulation, thus improving PD symptomatology. AREAS COVERED: This review evaluates the preclinical development, pharmacology, pharmacokinetics and safety profile of OPC. Data was extracted from published preclinical and clinical studies published on PUBMED and SCOPUS (Search period: 2000-2019). Clinical and post-marketing data are also evaluated. EXPERT OPINION: OPC is a third generation COMT inhibitor with a novel structure. It has an efficacy and tolerability superior to its predecessors, tolcapone (TOL) and entacapone (ENT). It also provides a safe and simplified drug regimen that allows neurologists to individually adjust the existing daily administration of L-DOPA. OPC is indicated as an adjunctive therapy to L-DOPA/DDI in patients with PD and end-of-dose motor fluctuations who cannot be stabilized on those combinations.

Fragment-Based Optimized EthR Inhibitors with in Vivo Ethionamide Boosting Activity.

Killing more than one million people each year, tuberculosis remains the leading cause of death from a single infectious agent. The growing threat of multidrug-resistant strains of Mycobacterium tuberculosis stresses the need for alternative therapies. EthR, a mycobacterial transcriptional regulator, is involved in the control of the bioactivation of the second-line drug ethionamide. We have previously reported the discovery of in vitro nanomolar boosters of ethionamide through fragment-based approaches. In this study, we have further explored the structure-activity and structure-property relationships in this chemical family. By combining structure-based drug design and in vitro evaluation of the compounds, we identified a new oxadiazole compound as the first fragment-based ethionamide booster which proved to be active in vivo, in an acute model of tuberculosis infection.

Synthesis and evaluation of novel S-benzyl- and S-alkylphthalimide- oxadiazole -benzenesulfonamide hybrids as inhibitors of dengue virus protease.

Direct acting antiviral drugs (DAADs) are becoming therapeutics of choice for the treatment of viral infections. Successful development of anti HIV and HCV drugs by targeting the viral proteases has provided impetus for discovering newer DAADs. Dengue virus (DENV) protease, which is composed of two nonstructural proteins, NS2B and NS3pro, can be likewise exploited for discovering new anti-dengue therapeutics. In this study, we have linked together two pharmaceutically interesting motifs, namely 1,3,4-oxadiazole and benzenesulfonamide in two alternative series to develop novel S-benzylated and S-alkylphthalimidated hybrids. For the first series of hybrids, 4-aminobenzoic acid (1) was reacted with substituted benzenesulfonyl chlorides via its amino group, whereas the carboxylic acid side was elaborated to sulfonamido-1,3,4-oxadiazole-2-thiols (6a/b) in three steps. At this stage, the intermediates 6a/b were bifurcated to either S-alkylphthalimidated (8a-j) or S-benzylated (9a-c) hybrids by reacting with corresponding halides. For the alternative series of hybrids, the carboxylic acid group of probenecid (10) was similarly elaborated to sulfonamido-1,3,4-oxadiazole-2-thiols (13), and diverged to S-alkylphthalimidated (14a-f) and S-benzylated hybrids (15a-e). Bioactivity assays demonstrated that 8g and 8h are the most potent inhibitors among the synthesized analogs, exhibiting the IC50 values of 13.9 µM and 15.1 µM, respectively. Computational assessment predicted the binding of the inhibitors at an allosteric site developed in the open conformation of DENV2 NS2B/NS3pro. Taken together these findings point out that the synthesized hybrid inhibitors possess a great potential for further antiviral drug development.

Uncovering the Pharmacological Mechanism of Stemazole in the Treatment of Neurodegenerative Diseases Based on a Network Pharmacology Approach.

Stemazole exerts potent pharmacological effects against neurodegenerative diseases and protective effects in stem cells. However, on the basis of the current understanding, the molecular mechanisms underlying the effects of stemazole in the treatment of Alzheimer's disease and Parkinson's disease have not been fully elucidated. In this study, a network pharmacology-based strategy integrating target prediction, network construction, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses, and molecular docking was adopted to predict the targets of stemazole relevant to the treatment of neurodegenerative diseases and to further explore the involved pharmacological mechanisms. The majority of the predicted targets were highly involved in the mitogen-activated protein kinase (MAPK) signaling pathway. RAC-alpha serine/threonine-protein kinase (AKT1), caspase-3 (CASP3), caspase-8 (CASP8), mitogen-activated protein kinase 8 (MAPK8), and mitogen-activated protein kinase 14 (MAPK14) are the core targets regulated by stemazole and play a central role in its anti-apoptosis effects. This work provides a scientific basis for further elucidating the mechanism underlying the effects of stemazole in the treatment of neurodegenerative diseases.

Fast relaxation and desensitization of angiotensin II contraction in the pulmonary artery via AT1R and Akt-mediated phosphorylation of muscular eNOS.

Angiotensin II (AngII) triggers a transient contraction of pulmonary arteries (PAs) followed by protracted desensitization. Based on the unconventional eNOS expression in PA smooth muscle cells (PASMCs), we hypothesized that activation of smooth muscle eNOS by AngII might be responsible for fast relaxation and tachyphylaxis. Using dual-wire myograph, mechanically endothelium-denuded rat PA [E(-)PA] showed AngII concentration-dependent transient contractions (ΔTAngII, 95% decay within 1 min), which were abolished by losartan (AT1R antagonist). Neither PD123319 (AT2R antagonist) nor A779 (MasR antagonist) affected ΔTAngII. When the vessels were pretreated with L-NAME (NOS inhibitor), ODQ (guanylate cyclase inhibitor), or KT5823 (PKG inhibitor), ΔTAngII of E(-)PA became larger and sustained, whereas nNOS or iNOS inhibitors had no such effect. Immunoblotting of human PASMCs (hPASMCs) also showed eNOS expression, and AngII treatment induced activating phosphorylations of Ser1177 in eNOS and of Ser473 in Akt (Ser/Thr protein kinase B), an upstream signal of eNOS phosphorylation. In addition, L-NAME co-treatment promoted AngII-induced Ser19 phosphorylation of myosin light chain. In hPASMCs, AngII abolished plasma membrane expression of AT1R, and recovery by washout took more than 1 h. Consistent with the data from hPASMCs, the second application of AngII to E(-)PA did not induce contraction, and significant recovery of ΔTAngII required prolonged washout (> 2 h) in the myography study. L-NAME treatment before the second application facilitated recovery of ΔTAngII. Muscular eNOS plays an auto-inhibitory role in ΔTAngII of PAs. The molecular changes investigated in hPASMCs revealed eNOS phosphorylation and internalization of AT1R by AngII. We propose that the rat PA smooth muscle eNOS-induced lusitropy and slow recovery of AT1R from tachyphylaxis might counterbalance the excessive contractile response to AngII, contributing to the distinctive low-pressure pulmonary circulation.

Antidiarrheal, vasorelaxant, and neuropharmacological actions of the diterpene tilifodiolide.

Salvia tiliifolia is used in folk medicine as a relaxant agent and for the treatment of diarrhea and neurodegenerative diseases. Tilifodiolide (TFD) is a diterpene obtained from this plant. The purpose of this work was to evaluate the antidiarrheal, vasorelaxant, and neuropharmacological actions of TFD. These effects were selected based on the folk medicinal use of S. tiliifolia. The antidiarrheal activity of 1-50 mg/kg p.o. TFD was assessed with the castor oil related tests. The vasorelaxant effect of TFD (0.9-298 µM) was performed with smooth muscle tissues from rats, and its mechanism of action was evaluated using different inhibitors. The sedative, anxiolytic, and antidepressant effects of 1-100 mg/kg TFD were assessed. The possible mechanisms of action of the anxiolytic and antidepressant effects of TFD were evaluated using inhibitors. TFD exhibited antidiarrheal (ED50 = 10.62 mg/kg) and vasorelaxant (EC50 = 48 ± 3.51 µM) effects. The coadministration of TFD with N(ω)-nitro-L-arginine methyl ester (L-NAME) or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), reverted the vasorelaxant action showed by TFD alone. TFD exerted anxiolytic actions (ED50 = 20 mg/kg) in the cylinder exploratory test, whereas TFD (50 mg/kg) showed antidepressant actions in the tail suspension test by 44%. The pretreatment with 2 mg/kg flumazenil partially reverted the anxiolytic actions of TFD, whereas the pretreatment with 1 mg/kg yohimbine abolished the antidepressant effects of TFD. In summary, TFD exerted antidiarrheal activity by decreasing the intestinal fluid accumulation and vasorelaxant effects mediated by nitric oxide and cyclic guanosine monophosphate. TFD showed anxiolytic and antidepressant effects by the partial involvement of gamma-Aminobutyric acid (GABA) receptors and the possible participation of α2-adrenoreceptors, respectively.

Antifungal activity of two oxadiazole compounds for the paracoccidioidomycosis treatment.

Paracoccidioidomycosis (PCM) is a neglected disease present in Latin America with difficulty in treatment and occurrence of serious sequelae. Thus, the development of alternative therapies is imperative. In the current work, two oxadiazole compounds (LMM5 and LMM11) presented fungicidal activity against Paracoccidioides spp. The minimum inhibitory and fungicidal concentration values ranged from 1 to 32 µg/mL, and a synergic effect was observed for both compounds when combined with Amphotericin B. LMM5 and LMM11 were able to reduce CFU counts (≥2 log10) on the 5th and 7th days of time-kill curve, respectively. The fungicide effect was confirmed by fluorescence microscopy (FUN-1/FUN-2). The hippocratic screening and biochemical analysis were performed in Balb/c male mice that received a high dose of each compound, and the compounds showed no in vivo toxicity. The treatment of experimental PCM with the new oxadiazoles led to significant reduction in CFU (≥1 log10). Histopathological analysis of the groups treated exhibited control of inflammation, as well as preserved lung areas. These findings suggest that LMM5 and LMM11 are promising hits structures, opening the door for implementing new PCM therapies.