N, N'-disubstituted Ureas as Novel Antiplatelet Agents: Synthesis, Pharmacological Evaluation and In Silico Studies.

INTRODUCTION: Thrombotic disorders are among the leading causes of morbidity and mortality worldwide. Drugs used in the prevention and treatment of atherothrombosis have pharmacokinetic limitations and adverse effects such as hemorrhagic conditions, highlighting the importance of developing more effective antiplatelet agents. ethod: In this work, we synthesized N,N'-disubstituted ureas 3a-3j and evaluated their antiplatelet profiles through in vitro, ex vivo, and in silico studies. The synthesized derivatives exhibited a selective inhibitory profile against platelet aggregation induced by arachidonic acid (AA) in vitro, without significantly affecting other aspects of primary hemostasis and blood coagulation. The compounds that showed inhibition greater than 85% were submitted to the analysis of their potency by calculating the concentration required to inhibit 50% of platelet aggregation induced by AA (IC50). Urea derivative 3a was the most potent with IC50 of 1.45 µM. Interestingly, this derivative inhibited more than 90% of platelet aggregation induced by AA ex vivo, with a similar effect to acetylsalicylic acid. In the hemolysis assay, most of the urea derivatives presented values below 10% suggesting good hemocompatibility. Additionally, the compounds tested at 100 µM also showed no cytotoxic effects in HepG2 and Vero cells. RESULT: The in silico results suggested that compound 3a may bind to the key residue of COX-1 similar to AA and known COX-1 inhibitors, and the results are also in agreement with our SAR, which suggests that the inhibition of this enzyme is the most likely mechanism of antiplatelet activity. CONCLUSION: Therefore, these results demonstrated that N,N'-disubstituted ureas are promising candidates for the development of novel antiplatelet agents.

Antiviral evaluation of 1,4-disubstituted-1,2,3-triazole derivatives against Chikungunya virus.

Aim: This work aimed to investigate the antiviral activity of two 1,4-disubstituted-1,2,3-triazole derivatives (1 and 2) against Chikungunya virus (CHIKV) replication. Materials & methods: Cytotoxicity was analyzed using colorimetric assays and the antiviral potential was evaluated using plaque assays and computational tools. Results: Compound 2 showed antiviral activity against CHIKV 181-25 in BHK-21 and Vero cells. Also, this compound presented a higher activity against CHIKV BRA/RJ/18 in Vero cells, like compound 1. Compound 2 exhibited virucidal activity and inhibited virus entry while compound 1 inhibited virus release. Molecular docking suggested that these derivatives inhibit nsP1 protein while compound 1 may also target capsid protein. Conclusion: Both compounds exhibit promising antiviral activity against CHIKV by blocking different steps of virus replication.

Structural insights into the inhibition of the nsP2 protease from Chikungunya virus by molecular modeling approaches.

Chikungunya virus (CHIKV) is the etiological agent of the Chikungunya fever which has spread worldwide. Clinically, this disease may lead to prolonged incapacitating joint pain that can compromise remarkably the patients' quality of life. However, there are no licensed vaccines or specific drugs to fight this infection yet, making the search for novel therapies an imperative need. In this scenario, the CHIKV nsP2 protease emerged as an attractive therapeutic target once this protein plays a pivotal role in viral replication and pathogenesis. Hence, we investigated the structural basis for the inhibition of this enzyme by using molecular docking and dynamics simulations. Compounds with inhibitory activities against CHIKV nsP2 protease determined experimentally were selected from the literature. Docking studies with a set of stereoisomers showed that trans isomers, but not cis ones, bound close to the catalytic dyad which may explain isomerism requirements to the enzyme's inhibition. Further, binding mode analyses of other known inhibitors revealed highly conserved contacts between inhibitors and enzyme residues like N1011, C1013, A1046, Y1079, N1082, W1084, L1205, and M1242. Molecular dynamics simulations reinforced the importance of some of these interactions and pointed to nonpolar interactions as the main forces for inhibitors' binding. Finally, we observed that true inhibitors exhibited lower structural fluctuation, higher ligand efficiency and did not induce significant changes in protein correlated motions. Collectively, our findings might allow discerning true inhibitors from false ones and can guide drug development efforts targeting the nsP2 protease to fight CHIKV infections in the future.

Pro-Apoptotic Antitumoral Effect of Novel Acridine-Core Naphthoquinone Compounds against Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC) is a global public health problem with high incidence and mortality. The chemotherapeutic agents used in the clinic, alone or in combination, usually lead to important side effects. Thus, the discovery and development of new antineoplastic drugs are essential to improve disease prognosis and reduce toxicity. In the present study, acridine-core naphthoquinone compounds were synthesized and evaluated for their antitumor activity in OSCC cells. The mechanism of action, pharmacokinetics, and toxicity parameters of the most promising compound was further analyzed using in silico, in vitro, and in vivo methods. Among the derivatives, compound 4e was highly cytotoxic (29.99 µM) and selective (SI 2.9) at levels comparable and generally superior to chemotherapeutic controls. Besides, compound 4e proved to be non-hemolytic, stable, and well tolerated in animals at all doses tested. Mechanistically, compound 4e promoted cell death by apoptosis in the OSCC cell, and molecular docking studies suggested this compound possibly targets enzymes important for tumor progression, such as RSK2, PKM2, and topoisomerase IIα. Importantly, compound 4e presented a pharmacological profile within desirable parameters for drug development, showing promise for future preclinical trials.

In vitro Studies on The Inhibition of Replication of Zika and Chikungunya Viruses by Dolastane Isolated from Seaweed Canistrocarpus cervicornis.

The lack of vaccines and antiviral treatment, along with the increasing number of cases of Zika virus (ZIKV) and Chikungunya virus (CHIKV) infections, emphasize the need for searching for new therapeutic strategies. In this context, the marine brown seaweed Canistrocarpus cervicornis has been proved to hold great antiviral potential. Hence, the aim of this work was to evaluate the anti-ZIKV and anti-CHIKV activity of a marine dolastane isolated from brown seaweed C. cervicornis and its crude extract. Vero cells were used in antiviral assays, submitted to ZIKV and CHIKV, and treated with different concentrations of C. cervicornis extract or dolastane. The crude extract of C. cervicornis showed inhibitory activities for both ZIKV and CHIKV, with EC50 values of 3.3 µg/mL and 3.1 µg/mL, respectively. However, the isolated dolastane showed a more significant and promising inhibitory effect (EC50 = 0.95 µM for ZIKV and 1.3 µM for CHIKV) when compared to both the crude extract and ribavirin, which was used as control. Also, the dolastane showed a very potent virucidal activity against CHIKV and was able to inhibit around 90% of the virus infectivity at 10 µM. For the ZIKV, the effects were somewhat lower, although interesting, at approximately 64% in this same concentration. Further, we observed that both the extract and the dolastane were able to inhibit the replication of ZIKV and CHIKV at different times of addition post-infection, remaining efficient even if added after 8 hours post-infection, but declining soon after. A synergistic effect using sub-doses of the extract and isolates was associated with ribavirin, inhibiting above 80% replication even at the lowest concentrations. Therefore, this work has unveiled the anti-ZIKV and CHIKV potential of C. cervicornis crude extract and an isolated dolastane, which, in turn, can be used as a preventive or therapeutic strategy in the future.

Targeting Chikungunya virus by computational approaches: from viral biology to the development of therapeutic strategies.

Introduction: Chikungunya virus (CHIKV) is the causative agent of Chikungunya fever, a reemerging infectious disease. This disease can cause severe manifestations that persist for months or years after acute infection and alas, there are no antiviral drugs or vaccines available. Hence, the development of new therapeutic approaches is necessary.Areas covered: We review how computational tools have provided insights on CHIKV proteins and examine the advances in the development of potential and novel antiviral drugs. A literature search was performed using PubMed and ScienceDirect databases up to April 2019.Expert opinion: Computational approaches are valuable for gaining insights into CHIKV proteins and for the design of new anti-CHIKV agents. The collaboration between computational and experimental researchers should be strengthened so that new agents can be developed in a more rational manner. Computer-aided tools could assist in the discovery and development of safer and more efficacious novel antiviral agents from unexplored chemical spaces. Technological advances dictate that this is likely to emerge soon, thus boosting interest and research on CHIKV biology and drug, vaccine and diagnostics development.

Mechanism of resistance to acyclovir in thymidine kinase mutants from Herpes simplex virus type 1: a computational approach.

Herpes simplex virus type 1 (HSV-1) infections affect about two-thirds of the world population, and the standard treatment consists of acyclovir (ACV) and its analogs, which interact with thymidine kinase (TK) blocking viral replication. Lately, the emergence of ACV-resistant strains has been reported, especially associated with TK mutations. In this context, ACV therapy fails against isolates encoding Y172C and Y53H/R163H TK mutants, but the molecular mechanism of drug resistance remains unclear. Thus, we examined the effects of these mutations on ACV and the cofactor ATP binding through molecular modeling approaches. We showed that Y172C prevents the anchoring of the aromatic ring of ACV through π-π stacking interactions, leading to an inversed binding mode and different interactions. On the other hand, Y53H/R163H remarkably affected the cofactor binding mode which shifted away from its binding site and also influenced the interaction network of ACV. This is likely due to the loss of polar interactions with R163 residue. Unlike what was observed in the wild-type complex, both drug and cofactor binding poses were not well positioned to allow the phosphorylation reaction which explains the resistance observed. Moreover, energy analysis corroborated the experimental data and showed lower theoretical affinity of ACV with mutant enzymes resulted from energetic loss in polar solvation in Y172C and electrostatic terms in Y53H/R163H mutant enzyme. Therefore, our study shed light on the resistance mechanism toward ACV of two mutant TKs identified in clinical HSV-1 strains and may further support the development of new anti-herpetic drugs to treat resistant infections. [Formula: see text] Communicated by Ramaswamy H. Sarma.

Virtual screening and drug repositioning as strategies for the discovery of new antifungal inhibitors of oxidosqualene cyclase.

Candidiasis is the most common fungal infection in immunocompromised patients, and Candida albicans is the fourth leading agent of nosocomial infections. Mortality from this infection is significant; however, the therapeutic treatment is limited, which demands the search for new drugs and new targets. In this context, oxidosqualene cyclase (OSC) catalyzes the cyclization of the 2,3-oxidosqualene to form lanosterol, an intermediate of ergosterol biosynthesis. Therefore, this enzyme constitutes an attractive therapeutic target. Thus, the aim of this study is to identify potential inhibitors of C. albicans OSC (CaOSC) from a marketed drugs database in order to discover new antifungal agents. The CaOSC 3D model was constructed using the Swiss-Model server and important features for CaOSC inhibition were identified by molecular docking of known inhibitors using Autodock Vina 1.1.2. Subsequently, virtual screening helped to identify calcitriol, the active form of vitamin D, and other four drugs, as potential inhibitors of CaOSC. The selected drugs presented an interesting pattern of interactions with this enzyme, including hydrogen bond with Asp450, a key residue in the active site. Thus, the antifungal activity of calcitriol was evaluated in vitro against Candida spp strains. Calcitriol showed antifungal activity against C. albicans and C. tropicalis, which reinforces the potential of this compound as candidate of CaOSC inhibitor. In short, the present study provides important insights for the development of new oxidosqualene cyclase inhibitors as antifungals.

Computational strategy for visualizing structures and teaching biochemistry.

Computational techniques have great potential to improve the teaching-learning. In this work, we used a computational strategy to visualize three-dimensional (3D) structures of proteins and DNA and help the student to comprehend biochemistry concepts such as protein structure and function, substrate, and inhibitors as well as DNA structural features. The practical classes included tutorials to be done in the computer using structures from Protein Data Bank and a free 3D structure visualization software, Swiss PDB Viewer. The activity was done with 76 students from biology and pharmacy undergraduate courses. Questionnaires were administered to evaluate the knowledge regarding specific biochemistry contents before and after the activity and the opinion of the students. An overall increased percentage of correct answers post-classes (75.91%) were observed in comparison to pre-classes (35.53%). All the students indicated that it could contribute to the learning of DNA and protein structure contents; approximately 90% stated that it enables structures visualization or makes the learning and understanding easier. Therefore, the strategy has shown to be effective, allowing the contextualization of biochemistry themes and may complement theoretical classes. © 2018 International Union of Biochemistry and Molecular Biology, 47(1):76-84, 2018.

Synthesis and evaluation of the cytotoxic activity of Furanaphthoquinones tethered to 1H-1,2,3-triazoles in Caco-2, Calu-3, MDA-MB231 cells.

Naphthoquinones and 1,2,3-triazoles are structural pharmacophore that is known to impart several cancer cells. This work shows a synthetic methodology to obtain hybrid molecules involving naphthoquinone and triazol scaffold as multiple ligands. A simple and efficient synthetic route was used to prepare a series of sixteen compounds being eight 2-(1-aryl-1H-1,2,3-triazol-4-yl)-2,3-dihydronaphtho[1,2 b]furan-4,5-diones and eight 2-(1-aryl-1H-1,2,3-triazol-4-yl)-2,3-dihydronaphtho[2,3-b]furan-4,9-diones. These compounds were tested in MDA-MB231, Caco-2 and Calu-3 human cancer cells, and among them 7a was the most selective compound on Caco-2 cells, the most sensitized cell line in this study. In silico study suggest that the blockage of topoisomerase I and IIα may be one of the mechanisms of action responsible for the cytotoxic effect of 7a in Caco-2 cells.

Class A ß-lactamases and inhibitors: In silico analysis of the binding mode and the relationship with resistance.

ß-lactams are one of the most common antimicrobials used to treat bacterial infections. However, bacterial resistance has compromised their efficacy, mainly due to the ß-lactamase enzyme production. To overcome this resistance, ß-lactamase inhibitors can be used in association with these antimicrobials. Herein, we analyzed the structural characteristics of ß-lactamases and their interactions with classical inhibitors, such as clavulanic acid (CA), sulbactam (SB) and tazobactam (TZ) to gain insights into resistance. The homology models of five class A ß-lactamases, namely CARB-3, IMI-1, SFO-1, SHV-5 and TEM-10, were constructed and validated and revealed an overall 3D structural conservation, but with significant differences in the electrostatic potential maps, especially at important regions in the catalytic site. Molecular dockings of CA, SB and TZ with these enzymes revealed a covalent bond with the S70 in all complexes, except Carb-3 which is in agreement with experimental data reported so far. This is likely related to the less voluminous active site of Carb-3 model. Although few specific contacts were observed in the ß-lactamase-inhibitor complexes, all compounds interacted with the residues in positions 73, 130, 132, 236 and 237. Therefore, this study provides new perspectives for the design of innovative compounds with broad-spectrum inhibitory profiles against ß-lactamases.

Design strategies of oxidosqualene cyclase inhibitors: Targeting the sterol biosynthetic pathway.

Targeting the sterol biosynthesis pathway has been explored for the development of new bioactive compounds. Among the enzymes of this pathway, oxidosqualene cyclase (OSC) which catalyzes lanosterol cyclization from 2,3-oxidosqualene has emerged as an attractive target. In this work, we reviewed the most promising OSC inhibitors from different organisms and their potential for the development of new antiparasitic, antifungal, hypocholesterolemic and anticancer drugs. Different strategies have been adopted for the discovery of new OSC inhibitors, such as structural modifications of the natural substrate or the reaction intermediates, the use of the enzyme's structural information to discover compounds with novel chemotypes, modifications of known inhibitors and the use of molecular modeling techniques such as docking and virtual screening to search for new inhibitors. This review brings new perspectives on structural insights of OSC from different organisms and reveals the broad structural diversity of OSC inhibitors which may help evidence lead compounds for further investigations with various therapeutic applications.