New Inhibitors of Bcr-Abl Based on 2,6,9-Trisubstituted Purine Scaffold Elicit Cytotoxicity in Chronic Myeloid Leukemia-Derived Cell Lines Sensitive and Resistant to TKIs.

Bcr-Abl is an oncoprotein with aberrant tyrosine kinase activity involved in the progression of chronic myeloid leukemia (CML) and has been targeted by inhibitors such as imatinib and nilotinib. However, despite their efficacy in the treatment of CML, a mechanism of resistance to these drugs associated with mutations in the kinase region has emerged. Therefore, in this work, we report the synthesis of 14 new 2,6,9-trisubstituted purines designed from our previous Bcr-Abl inhibitors. Here, we highlight 11b, which showed higher potency against Bcr-Abl (IC50 = 0.015 µM) than imatinib and nilotinib and exerted the most potent antiproliferative properties on three CML cells harboring the Bcr-Abl rearrangement (GI50 = 0.7-1.3 µM). In addition, these purines were able to inhibit the growth of KCL22 cell lines expressing Bcr-AblT315I, Bcr-AblE255K, and Bcr-AblY253H point mutants in micromolar concentrations. Imatinib and nilotinib were ineffective in inhibiting the growth of KCL22 cells expressing Bcr-AblT315I (GI50 > 20 µM) compared to 11b-f (GI50 = 6.4-11.5 µM). Molecular docking studies explained the structure-activity relationship of these purines in Bcr-AblWT and Bcr-AblT315I. Finally, cell cycle cytometry assays and immunodetection showed that 11b arrested the cells in G1 phase, and that 11b downregulated the protein levels downstream of Bcr-Abl in these cells.

Comparative study on Antibacterial efficacy of a series of chromone sulfonamide derivatives against drug-resistant and MDR-isolates.

Sulfonamide derivatives have numerous pharmaceutical applications having antiviral, antibacterial, antifungal, antimalarial, anticancer, and antidepressant activities. The structural flexibility of sulfonamide derivatives makes them an excellent candidate for the development of new multi-target agents, although long-time exposure to sulfonamide drugs results in many toxic impacts on human health. However, sulfonamides may be functionalized for developing less toxic and more competent drugs. In this work, sulfonamides including Sulfapyridine (a), Sulfathiazole (b), Sulfamethoxazole (c), and Sulfamerazine (d) are used to synthesize Schiff bases of 7-hydroxy-4-methyl-2-oxo-2H-chromene-8-carbalde-hyde (1a-1d). The synthesized compounds were spectroscopically characterized and tested against hospital isolates of three Gram-positive (Methicillin-resistant Staphylococcus aureus PH217, Ampicillin-resistant Coagulase-negative Staphylococcus aureus, multidrug-resistant (MDR) Enterococcus faecalis PH007R) and two Gram-negative bacteria (multidrug-resistant Escherichia coli, and Salmonella enterica serovar Typhi), compared to the quality control strains from ATCC (S. aureus 29213, E. faecalis 25922, E. coli 29212) and MTCC (S. Typhi 734). Two of the four Schiff bases 1a and 1b are found to be more active than their counterpart 1c and 1d; while 1a have showed significant activity by inhibiting MRSA PH217 and MDR isolates of E. coli at the minimum inhibitory concentration (MIC) of 150 µg/mL and 128 µg/mL with MBC of 1024 µg/mL, respectively. On the other hand, the MIC of 1b was 150 µg/mL against both S. aureus ATCC 29213 and Salmonella Typhi MTCC 734, compared to the control antibiotics Ampicillin and Gentamycin. Scanning electron microscopy demonstrated the altered surface structure of bacterial cells as a possible mechanism of action, supported by the in-silico molecular docking analysis.

In Silico Screening of Drugs That Target Different Forms of E Protein for Potential Treatment of COVID-19.

Recently the E protein of SARS-CoV-2 has become a very important target in the potential treatment of COVID-19 since it is known to regulate different stages of the viral cycle. There is biochemical evidence that E protein exists in two forms, as monomer and homopentamer. An in silico screening analysis was carried out employing 5852 ligands (from Zinc databases), and performing an ADMET analysis, remaining a set of 2155 compounds. Furthermore, docking analysis was performed on specific sites and different forms of the E protein. From this study we could identify that the following ligands showed the highest binding affinity: nilotinib, dutasteride, irinotecan, saquinavir and alectinib. We carried out some molecular dynamics simulations and free energy MM-PBSA calculations of the protein-ligand complexes (with the mentioned ligands). Of worthy interest is that saquinavir, nilotinib and alectinib are also considered as a promising multitarget ligand because it seems to inhibit three targets, which play an important role in the viral cycle. On the other side, saquinavir was shown to be able to bind to E protein both in its monomeric as well as pentameric forms. Finally, further experimental assays are needed to probe our hypothesis derived from in silico studies.

In Vivo and In Silico Studies of the Hepatoprotective Activity of Tert-Butylhydroquinone.

Tert-butylhydroquinone (TBHQ) is a synthetic food antioxidant with biological activities, but little is known about its pharmacological benefits in liver disease. Therefore, this work aimed to evaluate TBHQ during acute liver damage induced by CCl4 (24 h) or BDL (48 h) in Wistar rats. It was found that pretreatment with TBHQ prevents 50% of mortality induced by a lethal dose of CCl4 (4 g/kg, i.p.), and 80% of BDL+TBHQ rats survived, while only 50% of the BDL group survived. Serum markers of liver damage and macroscopic and microscopic (H&E staining) observations suggest that TBHQ protects from both hepatocellular necrosis caused by the sublethal dose of CCl4 (1.6 g/kg, i.p.), as well as necrosis/ductal proliferation caused by BDL. Additionally, online databases identified 49 potential protein targets for TBHQ. Finally, a biological target candidate (Keap1) was evaluated in a proof-of-concept in silico molecular docking assay, resulting in an interaction energy of -5.5491 kcal/mol, which was higher than RA839 and lower than monoethyl fumarate (compounds known to bind to Keap1). These findings suggest that TBHQ increases the survival of animals subjected to CCl4 intoxication or BDL, presumably by reducing hepatocellular damage, probably due to the interaction of TBHQ with Keap1.

In Silico Structural Analysis of Serine Carboxypeptidase Nf314, a Potential Drug Target in Naegleria fowleri Infections.

Naegleria fowleri, also known as the "brain-eating" amoeba, is a free-living protozoan that resides in freshwater bodies. This pathogenic amoeba infects humans as a casual event when swimming in contaminated water. Upon inhalation, N. fowleri invades the central nervous system and causes primary amoebic meningoencephalitis (PAM), a rapidly progressive and often fatal disease. Although PAM is considered rare, reducing its case fatality rate compels the search for pathogen-specific proteins with a structure-function relationship that favors their application as targets for discovering new or improved drugs against N. fowleri infections. Herein, we report a computational approach to study the structural features of Nf314 (a serine carboxypeptidase that is a virulence-related protein in N. fowleri infections) and assess its potential as a drug target, using bioinformatics tools and in silico molecular docking experiments. Our findings suggest that Nf314 has a ligand binding site suitable for the structure-based design of specific inhibitors. This study represents a further step toward postulating a reliable therapeutic target to treat PAM with drugs specifically aimed at blocking the pathogen proliferation by inhibiting protein function.

Substituted Purines as High-Affinity Histamine H3 Receptor Ligands.

Continuing with our program to obtain new histamine H3 receptor (H3R) ligands, in this work we present the synthesis, H3R affinity and in silico studies of a series of eight new synthetically accessible purine derivatives. These compounds are designed from the isosteric replacement of the scaffold presented in our previous ligand, pyrrolo[2,3-d]pyrimidine ring, by a purine core. This design also considers maintaining the fragment of bipiperidine at C-4 and aromatic rings with electron-withdrawing groups at N-9, as these fragments are part of the proposed pharmacophore. The in vitro screening results show that two purine derivatives, 3d and 3h, elicit high affinities to the H3R (Ki values of 2.91 and 5.51 nM, respectively). Both compounds are more potent than the reference drug pitolisant (Ki 6.09 nM) and show low toxicity with in vitro models (IC50 > 30 µM on HEK-293, SH-SY5Y and HepG2 cell lines). Subsequently, binding modes of these ligands are obtained using a model of H3R by docking and molecular dynamics studies, thus determining the importance of the purine ring in enhancing affinity due to the hydrogen bonding of Tyr374 to the N-7 of this heterocycle. Finally, in silico ADME properties are predicted, which indicate a promising future for these molecules in terms of their physical−chemical properties, absorption, oral bioavailability and penetration in the CNS.

Computer Aided Drug Design Methodologies with Natural Products in the Drug Research Against Alzheimer's Disease.

Natural products are compounds isolated from plants that provide a variety of lead structures for the development of new drugs by the pharmaceutical industry. The interest in these substances increases because of their beneficial effects on human health. Alzheimer's disease (AD) affects occur in about 80% of individuals aged 65 years. AD, the most common cause of dementia in elderly people, is characterized by progressive neurodegenerative alterations, as decrease of cholinergic impulse, increased toxic effects caused by reactive oxygen species and the inflammatory process that the amyloid plaque participates. In silico studies is relevant in the process of drug discovery; through technological advances in the areas of structural characterization of molecules, computational science and molecular biology have contributed to the planning of new drugs used against neurodegenerative diseases. Considering the social impairment caused by an increased incidence of disease and that there is no chemotherapy treatment effective against AD; several compounds are studied. In the researches for effective neuroprotectants as potential treatments for Alzheimer's disease, natural products have been extensively studied in various AD models. This study aims to carry out a literature review with articles that address the in silico studies of natural products aimed at potential drugs against Alzheimer's disease (AD) in the period from 2015 to 2021.

Exploring the antileishmanial activity of N1,N2-disubstituted-benzoylguanidines: synthesis and molecular modeling studies.

In this report, we describe the synthesis and evaluation of nine N1,N2-disubstituted-benzoylguanidines against promastigotes and amastigotes forms of Leishmania amazonensis. The derivatives 2g and 2i showed low IC50 values against promastigote form (90.8 ± 0.05 µM and 68.4 ± 0.03 µM, respectively), low cytotoxicity profile (CC50 396 ± 0.02 µM and 857.9 ± 0.06 µM) for peritoneal macrophages cells and SI of 5.5 and 12.5, respectively. Investigations about the mechanism of action of 2g and 2i showed that both compounds cause mitochondrial depolarization, increase in ROS levels, and generation of autophagic vacuoles on free promastigotes forms. These compounds were also capable of reducing the number of infected macrophages with amastigotes forms (59.5% ± 0.08% and 98.1% ± 0.46%) and the number of amastigotes/macrophages (79.80% ± 0.05% and 96.0% ± 0.16%), through increasing induction of microbicide molecule NO. Additionally, ADMET-Tox in silico predictions showed drug-like features and free of toxicological risks. The molecular docking studies with arginase and gp63 showed that relevant intermolecular interactions could explain the experimental results. Therefore, these results reinforce that benzoylguanidines could be a starting scaffold for the search for new antileishmanial drugs.Communicated by Ramaswamy H. Sarma.

Enhanced Solubility of Albendazole in Cyclodextrin Inclusion Complex: A Molecular Modeling Approach and Physicochemical Evaluation.

BACKGROUND: Albendazole (ABZ) is the drug of choice for the treatment of a variety of human and veterinary parasites. However, it has low aqueous solubility and low bioavailability. Cyclodextrins (CD) are pharmaceutical excipients with the ability to modulate the solubilization property of hydrophobic molecules. OBJECTIVE: The aim of the study was to analyze through in vitro and in silico studies (Autodock Vina software and CycloMolder platform) the formation of inclusion complexes between ABZ, ß-cyclodextrin (ß-CD) and its derivatives Methyl-ß-cyclodextrin (M-ß-CD) and Hydroxypropyl-ß-cyclodextrin (HP-ß-CD). METHODS: The most stable inclusion complexes were produced by the kneading method and characterized by Fourier Transform Infrared Spectroscopy (FTIR), Differential Scanning Calorimetry (DSC), X-Ray Diffraction (XRD), determination of the ABZ content and in vitro dissolution profile. RESULTS: Molecular modeling revealed that inclusion complexes between HP-ß-CD:ABZ (in the proportion 1:1 and 2:1) presented the lowest formation energy and the highest number of intermolecular interactions, showing that the use of more cyclodextrins does not generate gains in the stability of the complex. On the characterization tests, the complexes experimentally obtained by the kneading method demonstrated highly suggestive parameters, including ABZ in HP-ß-CD in both molar proportions, suppression of bands in the infrared spectrum, displacement of the drug's melting temperature in DSC, crystallinity halos instead of the characteristic peaks of ABZ crystals in the XRD and a release of more than 80% of ABZ in less than 5 minutes, dissolution efficiency of up to 92%. CONCLUSION: In silico studies provided a rational selection of the appropriate complexes of cyclodextrin, enabling the elaboration of more targeted complexes, decreasing time and costs for elaboration of new formulations, thereby increasing the oral biodisponibility of ABZ.

Synthesis, Structural Characterization, and In Vitro and In Silico Antifungal Evaluation of Azo-Azomethine Pyrazoles (PhN2(PhOH)CHN(C3N2(CH3)3)PhR, R = H or NO2).

The azo-azomethine imines, R1-N=N-R2-CH=N-R3, are a class of active pharmacological ligands that have been prominent antifungal, antibacterial, and antitumor agents. In this study, four new azo-azomethines, R1 = Ph, R2 = phenol, and R3 = pyrazol-Ph-R' (R = H or NO2), have been synthesized, structurally characterized using X-ray, IR, NMR and UV-Vis techniques, and their antifungal activity evaluated against certified strains of Candida albicans and Cryptococcus neoformans. The antifungal tests revealed a high to moderate inhibitory activity towards both strains, which is regulated as a function of both the presence and the location of the nitro group in the aromatic ring of the series. These biological assays were further complemented with molecular docking studies against three different molecular targets from each fungus strain. Molecular dynamics simulations and binding free energy calculations were performed on the two best molecular docking results for each fungus strain. Better affinity for active sites for nitro compounds at the "meta" and "para" positions was found, making them promising building blocks for the development of new Schiff bases with high antifungal activity.

Modelling of the Citrus CCD4 Family Members: In Silico Analysis of Membrane Binding and Substrate Preference.

Coloring is one of the most important characteristics in commercial flowers and fruits, generally due to the accumulation of carotenoid pigments. Enzymes of the CCD4 family in citrus intervene in the generation of ß-citraurin, an apocarotenoid responsible for the reddish-orange color of mandarins. Citrus CCD4s enzymes could be capable of interacting with the thylakoid membrane inside chloroplasts. However, to date, this interaction has not been studied in detail. In this work, we present three new complete models of the CCD4 family members (CCD4a, CCD4b, and CCD4c), modeled with a lipid membrane. To identify the preference for substrates, typical carotenoids were inserted in the active site of the receptors and the protein-ligand interaction energy was evaluated. The results show a clear preference of CCD4s for xanthophylls over aliphatic carotenes. Our findings indicate the ability to penetrate the membrane and maintain a stable interaction through the N-terminal α-helical domain, spanning a contact surface of 2250 to 3250 Å2. The orientation and depth of penetration at the membrane surface suggest that CCD4s have the ability to extract carotenoids directly from the membrane through a tunnel consisting mainly of hydrophobic residues that extends up to the catalytic center of the enzyme.

Different Classes of Antidepressants Inhibit the Rat α7 Nicotinic Acetylcholine Receptor by Interacting within the Ion Channel: A Functional and Structural Study.

Several antidepressants inhibit nicotinic acetylcholine receptors (nAChRs) in a non-competitive and voltage-dependent fashion. Here, we asked whether antidepressants with a different structure and pharmacological profile modulate the rat α7 nAChR through a similar mechanism by interacting within the ion-channel. We applied electrophysiological (recording of the ion current elicited by choline, ICh, which activates α7 nAChRs from rat CA1 hippocampal interneurons) and in silico approaches (homology modeling of the rat α7 nAChR, molecular docking, molecular dynamics simulations, and binding free energy calculations). The antidepressants inhibited ICh with the order: norfluoxetine ~ mirtazapine ~ imipramine < bupropion ~ fluoxetine ~ venlafaxine ~ escitalopram. The constructed homology model of the rat α7 nAChR resulted in the extracellular vestibule and the channel pore is highly negatively charged, which facilitates the permeation of cations and the entrance of the protonated form of antidepressants. Molecular docking and molecular dynamics simulations were carried out within the ion-channel of the α7 nAChR, revealing that the antidepressants adopt poses along the receptor channel, with slightly different binding-free energy values. Furthermore, the inhibition of ICh and free energy values for each antidepressant-receptor complex were highly correlated. Thus, the α7 nAChR is negatively modulated by a variety of antidepressants interacting in the ion-channel.

Computer-Aided Drug Design for the Identification of Novel Antischistosomal Compounds.

Schistosomiasis is a chronic neglected tropical disease, highlighted by the presence of Schistosoma worms, which presents in advanced cases in approximately 80 countries, affecting almost 300 million people. The treatment is based on only one drug, praziquantel, a drug discovered in the 1970s that shows moderate efficacy against the adult parasite, but low efficacy against the larval stages of the parasite. Therefore, the use of only one drug has brought concerns and losses on drug-resistance cases, necessitating the development of new effective chemotherapeutic agents against Schistosoma species. One of the strategies that have been implemented in drug development is the computer-aided drug design (CADD), investigating the structural characteristics of the compounds and targets in order to understand their actions and biological activities through 3D virtual manipulation, as the QSAR applied to ligands and molecular docking applied to a respective biological target. These studies help to extract information and characteristics relevant to the activity, as well as to predict potential applications and activity. Therefore, this chapter will present the main validated biological targets of the genus Schistosoma, as thioredoxin glutathione reductase (TGR), histone deacetylases (HDAC 1, HDAC 8), dihydroorotate dehydrogenase, sirtuin protein and cathepsin L1, as well as reports of CADD in literature applied to the development of drugs against schistosomiasis, providing compounds with high pharmacological potential and high specificity.

Inhibition of key enzymes in the inflammatory pathway by hybrid molecules of terpenes and synthetic drugs: In vitro and in silico studies.

The aim of this work was to compare the anti-inflammatory activity of compounds prepared from terpenes and the synthetic drugs ibuprofen and naproxen. The anti-inflammatory activity of the hybrid compounds was compared with the activity of the parent compounds. This was accomplished using in vitro inhibition of lipoxygenases (LOX) and COX-2, and in silico docking studies in 15-LOX and COX-2. The synthesized hybrids showed an inhibition of COX-2 and LOX between 9.8%-57.4% and 0.0%-97.7%, respectively. None of the hybrids showed an improvement in the inhibitory effect toward these pro-inflammatory enzymes, compared to the parent terpenes and non-steroidal anti-inflammatory drugs. The docking studies allowed us to predict the potential binding modes of hybrids 6-15 within COX-2 and 15-LOX active sites. The relative affinity of the compounds inside the binding sites could be explained by forming non-covalent interactions with most important and known amino acids reported for those enzymes. A good correlation (r2  = 0.745) between docking energies and inhibition percentages against COX-2 was found. The high inhibition obtained for compound 10 against COX-2 was explained by hydrogen bond interactions at the enzyme binding site. New synthetic possibilities could be obtained from our in silico models, improving the potency of these hybrid compounds.

Combined In Vitro Studies and in Silico Target Fishing for the Evaluation of the Biological Activities of Diphylleia cymosa and Podophyllum hexandrum.

This paper reports the in silico prediction of biological activities of lignans from Diphylleia cymosa and Podophyllum hexandrum combined with an in vitro bioassays. The extracts from the leaves, roots and rhizomes of both species were evaluated for their antibacterial, anticholinesterasic, antioxidant and cytotoxic activities. A group of 27 lignans was selected for biological activities prediction using the Active-IT system with 1987 ligand-based bioactivity models. The in silico approach was properly validated and several ethnopharmacological uses and known biological activities were confirmed, whilst others should be investigated for new drugs with potential clinical use. The extracts from roots of D. cymosa and from rhizomes and roots of P. hexandrum were very effective against Bacillus cereus and Staphylococcus aureus, while podophyllotoxin inhibited the growth of Staphylococcus aureus and Escherichia coli. D. cymosa leaves and roots showed anticholinesterasic and antioxidant activities, respectively. The evaluated extracts showed to be moderately toxic to THP-1 cells. The chromatographic characterization indicated that podophyllotoxin was the major constituent of P. hexandrum extract while kaempferol and its hexoside were the main constituents of D. cymosa leaves and roots, respectively. These results suggest that the podophyllotoxin could be the major antibacterial lignan, while flavonoids could be responsible for the antioxidant activity.

Recombinant production and biochemical and in silico characterization of lactate dehydrogenase from Geobacillus thermodenitrificans DSM-465

Background: Lactate dehydrogenase (LDH) is an enzyme of glycolytic pathway, ubiquitously found in living organisms. Increased glycolysis and LDH activity are associated with many pathologic conditions including inflammation and cancer, thereby making the enzyme a suitable drug target. Studies on conserved structural and functional domains of LDH from various species reveal novel inhibitory molecules. Our study describes Escherichia coli production and characterization of a moderately thermostable LDH (LDH-GT) from Geobacillus thermodenitrificans DSM-465. An in silico 3D model of recombinant enzyme and molecular docking with a set of potential inhibitors are also described. Results: The recombinant enzyme was overexpressed in E. coli and purified to electrophoretic homogeneity. The molecular weight of the enzyme determined by MALDI-TOF was 34,798.96 Da. It exhibited maximum activity at 65°C and pH 7.5 with a KM value for pyruvate as 45 µM. LDH-GT and human LDH-A have only 35.6% identity in the amino acid sequence. On the contrary, comparison by in silico structural alignment reveals that LDH-GT monomer has approximately 80% identity to that of truncated LDH-A. The amino acids "GEHGD" as well as His179 and His193 in the active site are conserved. Docking studies have shown the binding free energy changes of potential inhibitors with LDH-A and LDH-GT ranging from −407.11 to −127.31 kJ mol−1 . Conclusions: By highlighting the conserved structural and functional domains of LDH from two entirely different species, this study has graded potential inhibitory molecules on the basis of their binding affinities so that they can be applied for in vivo anticancer studies

In silico design, chemical synthesis and toxicological evaluation of 1,3-thiazolidine-2,4-dione derivatives as PPARγ agonists.

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors involved in the metabolism of lipids and carbohydrates. The exogenous ligands of these receptors are thiazolidinediones (TZDs), which are used to treat type 2 diabetes mellitus (DM2). However, drugs from this group produce adverse effects such as hepatic steatosis. Hence, the aim of this work was to design a set of small molecules that can activate the γ isoform of PPARs while minimizing the adverse effects. The derivatives were designed containing the polar head of TZD and an aromatic body, serving simultaneously as the body and tail. Two ligands were selected out of 130 tested. These compounds were synthesized in a solvent-free reaction and their physicochemical properties and toxicity were examined. Acute oral toxicity was determined by administering these compounds to female Wistar rats in increasing doses (as per the OECD protocol 425). The median lethal dose (LD50) of the compound substituted with a hydroxyl heteroatom was above 2000 mg/kg, and that of the compound substituted with halogens was 700-1400 mg/kg. The results suggest that the compounds can interact with PPARγ and elicit biological responses similar to other TZDs, but without showing adverse effects. The compounds will be subsequently evaluated in a DM2 animal model.

Oleanolic acid (OA) as an antileishmanial agent: Biological evaluation and in silico mechanistic insights.

Although a worldwide health problem, leishmaniasis is considered a highly neglected disease, lacking efficient and low toxic treatment. The efforts for new drug development are based on alternatives such as new uses for well-known drugs, in silico and synthetic studies and naturally derived compounds. Oleanolic acid (OA) is a pentacyclic triterpenoid widely distributed throughout the Plantae kingdom that displays several pharmacological activities. OA showed potent leishmancidal effects in different Leishmania species, both against promastigotes (IC(50 L. braziliensis) 30.47 ± 6.35 µM; IC(50 L. amazonensis) 40.46 ± 14.21 µM; IC(50 L. infantum) 65.93 ± 15.12 µM) and amastigotes (IC(50 L. braziliensis) 68.75 ± 16.55 µM; IC(50 L. amazonensis) 38.45 ± 12.05 µM; IC(50 L. infantum) 64.08 ± 23.52 µM), with low cytotoxicity against mouse peritoneal macrophages (CC(50) 235.80 ± 36.95 µM). Moreover, in silico studies performed to evaluate OA molecular properties and to elucidate the possible mechanism of action over the Leishmania enzyme sterol 14α-demethylase (CYP51) suggested that OA interacts efficiently with CYP51 and could inhibit the ergosterol synthesis pathway. Collectively, these data indicate that OA is a good candidate as leading compound for the development of a new leishmaniasis treatment.