Thiazine-derived compounds in inhibiting efflux pump in Staphylococcus aureus K2068, mepA gene expression, and membrane permeability alteration.

Staphylococcus aureus is a bacterium responsible for resistance to multiple drugs and the efflux system is widely studied among the resistance mechanisms developed by this species. The present study evaluates the inhibition of the MepA efflux pump by thiadiazine-derived compounds. For this purpose, thiadiazine-derived compounds (IJ-14 to IJ-20) were tested against S. aureus K2068 strains. Microdilution tests were initially conducted to assess the Minimum Inhibitory Concentration (MIC) of the compounds and their efflux pump inhibition activity. In addition, fluorimetry tests were performed using BrEt emission and tests were conducted to inhibit the expression of the mepA gene. This involved comparing the bacterial gene expression with the antibiotic alone to the gene expression after combining compounds (IJ-17 and IJ-20) with the antibiotic. Furthermore, membrane permeability assessment tests and in silico molecular docking tests were performed. It was observed that the IJ17 and IJ20 compounds exhibited direct activity against the tested strain. The IJ17 compound produced significant results in the gene inhibition tests, which was also evidenced through the membrane permeability alteration test. These findings suggest that thiadiazine-derived compounds have promising effects against one of the main resistance mechanisms, with the IJ17 compound presenting observable mechanisms of action.

Synthesis, Antileishmanial Activity and in silico Studies of Aminoguanidine Hydrazones (AGH) and Thiosemicarbazones (TSC) Against Leishmania chagasi Amastigotes.

BACKGROUND: Leishmaniasis is a worldwide health problem, highly endemic in developing countries. Among the four main clinical forms of the disease, visceral leishmaniasis is the most severe, fatal in 95% of cases. The undesired side-effects from first-line chemotherapy and the reported drug resistance search for effective drugs that can replace or supplement those currently used in an urgent need. Aminoguanidine hydrazones (AGH's) have been explored for exhibiting a diverse spectrum of biological activities, in particular the antileishmanial activity of MGBG. The bioisosteres thiosemicarbazones (TSC's) offer a similar biological activity diversity, including antiprotozoal effects against Leishmania species and Trypanosoma cruzi. OBJECTIVES: Considering the impact of leishmaniasis worldwide, this work aimed to design, synthesize, and perform a screening upon L. chagasi amastigotes and for the cytotoxicity of the small "inhouse" library of both AGH and TSC derivatives and their structurally-related compounds. METHODS: A set of AGH's (3-7), TSC's (9, 10), and semicarbazones (11) were initially synthesized. Subsequently, different semi-constrained analogs were designed and also prepared, including thiazolidines (12), dihydrothiazines (13), imidazolines (15), pyrimidines (16, 18) azines (19, 20), and benzotriazepinones (23-25). All intermediates and target compounds were obtained with satisfactory yields and exhibited spectral data consistent with their structures. All final compounds were evaluated against L. chagasi amastigotes and J774.A1 cell line. Molecular docking was performed towards trypanothione reductase using GOLD® software. RESULTS: The AGH's 3i, 4a, and 5d, and the TSC's 9i, 9k, and 9o were selected as valuable hits. These compounds presented antileishmanial activity compared with pentamidine, showing IC50 values ranged from 0.6 to 7.27 µM, maximal effects up to 55.3%, and satisfactory SI values (ranged from 11 to 87). On the other hand, most of the resulting semi-constrained analogs were found cytotoxic or presented reduced antileishmanial activity. In general, TSC class is more promising than its isosteric AGH analogs, and the beneficial aromatic substituent effects are not similar in both series. In silico studies have suggested that these hits are capable of inhibiting the trypanothione reductase from the amastigote forms. CONCLUSION: The promising antileishmanial activity of three AGH's and three TSC's was characterized. These compounds presented antileishmanial activity compared with PTD, showing IC50 values ranged from 0.6 to 7.27 µM, and satisfactory SI values. Further pharmacological assays involving other Leishmania strains are in progress, which will help choose the best hits for in vivo experiments.

Targeting Chikungunya Virus Entry: Alternatives for New Inhibitors in Drug Discovery.

Chikungunya virus (CHIKV) is an Alphavirus (Togaviridae) responsible for Chikungunya fever (CHIKF) that is mainly characterized by a severe polyarthralgia, in which it is transmitted by the bite of infected Aedes aegypti and Ae. albopictus mosquitoes. Nowadays, there are no licensed vaccines or approved drugs to specifically treat this viral disease. Structural viral proteins participate in key steps of its replication cycle, such as viral entry, membrane fusion, nucleocapsid assembly, and virus budding. In this context, envelope E3-E2-E1 glycoproteins complex could be targeted for designing new drug candidates. In this review, aspects of the CHIKV entry mechanism are discussed to provide insights into assisting the drug discovery process. Moreover, several naturals, naturebased and synthetic compounds, as well as repurposed drugs and virtual screening are also explored as alternatives for developing CHIKV entry inhibitors. Finally, we provided a complementary analysis of studies involving inhibitors that were not explored by in silico methods. Based on this, Phe118, Val179, and Lys181 were found to be the most frequent residues, being present in 89.6, 82.7, and 93.1% of complexes, respectively. Lastly, some chemical aspects associated with interactions of these inhibitors and mature envelope E3- E2-E1 glycoproteins' complex were discussed to provide data for scientists worldwide, supporting their search for new inhibitors against this emerging arbovirus.

Tau Protein Aggregation in Alzheimer's Disease: Recent Advances in the Development of Novel Therapeutic Agents.

Major research in Alzheimer's disease (AD) related to disease-modifying agents is concentrated on pharmacological approaches related to diagnostic markers, neurofibrillary tangles and amyloid plaques. Although most studies focus on anti-amyloid strategies, investigations on tau protein have produced significant advances in the modulation of the pathophysiology of several neurodegenerative diseases. Since the discovery of phenothiazines as tau protein aggregation inhibitors (TAGIs), many additional small molecule inhibitors have been discovered and characterized in biological model systems, which exert their interaction effects by covalent and noncovalent means. In this paper, we summarize the latest advances in the discovery and development of tau aggregation inhibitors using a specialized approach in their chemical classes. The design of new TAGIs and their encouraging use in in vivo and clinical trials support their potential therapeutic use in AD.

Indole-3-guanylhydrazone hydrochloride mitigates long-term cognitive impairment in a neonatal sepsis model with involvement of MAPK and NFκB pathways.

BACKGROUND: Neonatal sepsis is defined as a systemic inflammatory response caused by a suspected or proven infection, occurring in the first month of life, and remains one of the main causes of morbidity and mortality in newborn and preterm infants. Frequently, survivors of neonatal sepsis have serious long-term cognitive impairment and adverse neurologic outcomes. There is currently no specific drug treatment for sepsis. Indole-3-guanylhydrazone hydrochloride (LQM01) is an aminoguanidine derivative that has been described as an anti-inflammatory, antihypertensive and antioxidant with potential applicability in inflammatory diseases. METHODS: We used a LPS-challenged neonatal sepsis rodent model to investigate the effect of LQM01 on cognitive impairment and anxiety-like behavior in sepsis mice survivors, and examined the possible molecular mechanisms involved. RESULTS: It was found that LQM01 exposure during the neonatal period reduces anxiety-like behavior and cognitive impairment caused by lipopolysaccharides (LPS) in adult life. Additionally, treatment with LQM01 decreased pro-inflammatory cytokine levels and reduced NFκB, COX-2, MAPK and microglia activation in hippocampus of neonatal mice. Furthermore, LQM01 was also able to prevent oxidative damage in hippocampus of neonatal mice and preserve brain barrier integrity. CONCLUSIONS: LQM01 attenuated inflammatory reactions in an LPS-challenged neonatal sepsis mice model through the MAPK and NFκB signaling pathways and microglia activation suppression. All these findings are associated with mitigated cognitive impairment in 70 days-old LQM01 treated-mice. GENERAL SIGNIFICANCE: We revealed the effect of LQM01 as an anti-septic agent, and the role of crucial molecular pathways in mitigating the potential damage caused by neonatal sepsis.

Evaluation of guanylhydrazone derivatives as inhibitors of Candida rugosa digestive lipase: Biological, biophysical, theoretical studies and biotechnological application.

This work aimed to evaluate the inhibition of Candida rugosa lipase by five guanylhydrazone derivatives through biological, biophysical and theoretical studies simulating physiologic conditions. The compound LQM11 (IC50 = 14.70 µM) presented the highest inhibition against the enzyme. Therefore, for a better understanding of the interaction process, spectroscopic and theoretical studies were performed. Fluorescence and UV-vis assays indicate a static quenching mechanism with non-fluorescent supramolecular complex formation and changing the native protein structure. The binding process was spontaneous (ΔG < 0) and electrostatic forces (ΔH < 0 and ΔS > 0) played a preferential role in stabilizing the complex ligand-lipase. The compounds were classified as non-competitive inhibitors using orlistat as a reference in competition studies. Based on the 1H NMR assays it was possible to propose the sites of ligand (epitope) that bind preferentially to the enzyme and the theoretical studies were consistent with the experimental results. Finally, LQM11 was efficient as a lipase inhibitor of the crude intestinal extract of larvae of Rhynchophorus palmarum, an important agricultural plague, showing potential for control of this pest. Within this context, the real potential of this biotechnological application deserves further studies.

Correlation between DNA/HSA-interactions and antimalarial activity of acridine derivatives: Proposing a possible mechanism of action.

Acridines are considered an important class of compounds due to their wide variety of biological activities. In this work, we synthesized four acridine derivatives (1-4) and evaluated their biological activity against the Plasmodium falciparum W2 line, as well as studied the interaction with ctDNA and HSA using spectroscopic techniques and molecular docking. The acridine derivative 2 (IC50 = 0.90 ±â€¯0.08 µM) was more effective against P. falciparum than primaquine (IC50 = 1.70 ±â€¯0.10 µM) and similar to amsacrine (IC50 = 0.80 ±â€¯0.10 µM). In the fluorescence and UV-vis assays, it was verified that the acridine derivatives interact with ctDNA and HSA leading to a non-fluorescent supramolecular complex formation. The non-covalent binding constants ranged from 2.09 to 7.76 × 103 M-1, indicating moderate interaction with ctDNA. Through experiments with KI, fluorescence contact energy transfer and competition assays were possible to characterize the main non-covalent binding mode of the acridines evaluated with ctDNA as intercalation. The binding constants obtained showed a high linear correlation with the IC50 values against the antimalarial activity, suggesting that DNA may be the main biological target of these molecules. Finally, HSA interaction studies were performed and all evaluated compounds bind to the site II of the protein. The less active compounds (1 and 3) presented the highest affinity to HSA, indicating that the interaction with carrier protein can affect the (bio)availability of these compounds to the biological target.

Highly functionalized piperidines: Free radical scavenging, anticancer activity, DNA interaction and correlation with biological activity.

Twenty-five piperidines were studied as potential radical scavengers and antitumor agents. Quantitative interaction of compounds with ctDNA using spectroscopic techniques was also evaluated. Our results demonstrate that the evaluated piperidines possesses different abilities to scavenge the radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) and the anion radical superoxide (•O2-). The piperidine 19 was the most potent radical DPPH scavenger, while the most effective to •O2- scavenger was piperidine 10. In general, U251, MCF7, NCI/ADR-RES, NCI-H460 and HT29 cells were least sensitive to the tested compounds and all compounds were considerably more toxic to the studied cancer cell lines than to the normal cell line HaCaT. The binding mode of the compounds and ctDNA was preferably via intercalation. In addition, these results were confirmed based on theoretical studies. Finally, a linear and exponential correlation between interaction constant (Kb) and GI50 for several human cancer cell was observed.

Evidence for the involvement of TNF-α, IL-1ß and IL-10 in the antinociceptive and anti-inflammatory effects of indole-3-guanylhydrazone hydrochloride, an aromatic aminoguanidine, in rodents.

BACKGROUND: Indole-3-guanylhydrazone hydrochloride (LQM01) is a new derivative of aminoguanidine hydrochloride, an aromatic aminoguanidine. METHODS: Mice were treated with LQM01 (5, 10, 25 or 50 mg/kg, i.p.), vehicle (0.9% saline i.p.) or a standard drug. The mice were subjected to carrageenan-induced pleurisy, abdominal writhing induced by acetic acid, the formalin test and the hot-plate test. The model of non-inflammatory chronic muscle pain induced by saline acid was also used. Mice from the chronic protocol were assessed for withdrawal threshold, muscle strength and motor coordination. LQM01 or vehicle treated mice were evaluated for Fos protein. RESULTS: LQM01 inhibits TNF-α and IL-1ß production, as well as leukocyte recruitment during inflammation process. The level of IL-10 in LQM01-treated mice increased in pleural fluid. In addition, LQM01 decreased the nociceptive behavior in the acetic acid induced writhing test, the formalin test (both phases) and increased latency time on the hot-plate. LQM01 treatment also decreased mechanical hyperalgesia in mice with chronic muscle pain, with no changes in muscle strength and motor coordination. LQM01 reduced the number of Fos positive cells in the superficial dorsal horn. This compound exhibited antioxidant properties in in vitro assays. CONCLUSIONS: LQM01 has an outstanding anti-inflammatory and analgesic profile, probably mediated through a reduction in proinflammatory cytokines release, increase in IL-10 production and reduction in neuron activity in the dorsal horn of the spinal cord in mice. GENERAL SIGNIFICANCE: Beneficial effects of LQM01 suggest that it has some important clinical features and can play a role in the management of 'dysfunctional pain' and inflammatory diseases.

Annonalide and derivatives: Semisynthesis, cytotoxic activities and studies on interaction of annonalide with DNA.

The cytotoxic activity of the pimarane diterpene annonalide (1) and nine of its semisynthetic derivatives (2-10) was investigated against the human tumor cell lines HL-60 (leukemia), PC-3 (prostate adenocarcinoma), HepG2 (hepatocellular carcinoma), SF-295 (glioblastoma) and HCT-116 (colon cancer), and normal mouse fibroblast (L929) cells. The preparation of 2-10 involved derivatization of the side chain of 1 at C-13. Except for 2, all derivatives are being reported for the first time. Most of the tested compounds presented IC50s below 4.0 µM, being considered potential antitumor agents. The structures of all new compounds were elucidated by spectroscopic analyses including 2D NMR and HRMS. Additionally, the interaction of annonalide (1) with ctDNA was evaluated using spectroscopic techniques, and the formation of a supramolecular complex with the macromolecule was confirmed. Competition assays with fluorescent probes (Hoechst and ethidium bromide) and theoretical studies confirmed that 1 interacts preferentially via DNA intercalation with stoichiometric ratio of 1:1 (1:ctDNA). The ΔG value was calculated as -28.24 kJ mol-1, and indicated that the interaction process occurs spontaneously. Docking studies revealed that van der Walls is the most important interaction in 1-DNA and EB-DNA complexes, and that both ligands (1 and EB) interact with the same DNA residues (DA6, DA17 and DT19).

The medicinal chemistry of Chikungunya virus.

Arthropod-borne viruses (arboviruses) are an important threat to human and animal health globally. Among these, zoonotic diseases account for billions of cases of human illness and millions of deaths every year, representing an increasing public health problem. Chikungunya virus belongs to the genus Alphavirus of the family Togariridae, and is transmitted mainly by the bite of female mosquitoes of the Aedes aegypti and/or A. albopictus species. The focus of this review will be on the medicinal chemistry of Chikungunya virus, including synthetic and natural products, as well as rationally designed compounds.

Inclusion complex between ß-cyclodextrin and hecogenin acetate produces superior analgesic effect in animal models for orofacial pain.

Hecogenin acetate (HA) is a steroidal sapogenin-acetylated with pharmacological properties which have already been described in the literature such as, anti-inflammatory, anti-hyperalgesic and antinociceptive, but it has low solubility in aqueous media. Therefore, in an attempt to overcome this, we set out to create inclusion complexes between HA and b-cyclodextrin (b-CD) and evaluate the antinociceptive effects in the orofacial nociception in mice. The complexes were prepared using different methods in the molar ratios 1:1 and 1:2 and characterized physicochemically. The results of the physicochemical characterization elucidated inclusion complexes formation between b-CD and HA by freeze drying method in the molar ratio 1:2, which obtained a complexation efficiency of 92% and produced superior analgesic effect in animal models for orofacial pain at a lower dose when compared to HA alone.

Dynamic Simulation, Docking and DFT Studies Applied to a Set of Anti-Acetylcholinesterase Inhibitors in the enzyme ß-Secretase (BACE-1): An Important Therapeutic Target in Alzheimer`s disease.

BACKGROUND: Alzheimer`s disease (AD) affects mainly elderly people over 60 years of age. Currently, there are more than 35 million people with this disease worldwide. The enzyme ß-secretase is involved in the processing of the amyloid precursor protein and plays a key role in the physiopathology of AD. The action of some acetylcholinesterase inhibitors (AChEI) as ß-secretase inhibitors has been reported. OBJECTIVE: The aim of this study was to highlight the modes of the binding of acetylcholinesterase ligands onto the active site of the ß-secretase enzyme. METHODS: Molecular dynamics and docking were used in order to identify pivotal interactions that favor the inhibitory activity and provide a rational basis for planning novel ß-secretase inhibitors. Additionally, density functional theory (DFT) was used to provide accurate energy values for the complexes. A mechanistic study of the amide hydrolysis was also performed at the M06/6-31G(d) basis set. RESULTS: Of the 100 AChE inhibitors, 10 were able to interact with Asp32 and/or Asp228 residues from the enzyme BACE-1, suggesting that these could act as multi-target compounds. These inhibitors were selected for DFT studies in order to provide more accurate energy values. Interestingly, the range of energy values (-27.01 to -8.64 kJ mol-1) obtained was in agreement with the anti-AChE activity. The results obtained in the mechanistic study of compound 93 using DFT are in agreement with theoretical studies described in the literature. CONCLUSION: The results reported in this study will advance our understanding of the influence of the distinct chemical structures of inhibitors at the active site and aid the development of new virtual screening protocols to design novel AChE multi-target inhibitors.

Preliminary in vitro evaluation of the anti-proliferative activity of guanylhydrazone derivatives.

Guanylhydrazones have shown promising antitumor activity in preclinical tumor models in several studies. In this study, we aimed at evaluating the cytotoxic effect of a series of synthetic guanylhydrazones. Different human tumor cell lines, by including HCT-8 (colon carcinoma), MDA-MB-435 (melanoma) and SF-295 (glioblastoma) were continuous exposed to guanylhydrazone derivatives for 72 hours and growth inhibition of tumor cell lines and macrophages J774 was measured using tetrazolium salt (MTT) assay. Compounds 7, 11, 16 and 17 showed strong cytotoxic activity with IC50 values lower than 10 µmol L(-1) against four tumor cell lines. Among them, 7 was less toxic to non-tumor cells. Finally, obtained data suggest that guanylhydrazones may be regarded as potential lead compounds for the design of novel anticancer agents.