How oxidized EGCG remodels α-synuclein fibrils into non-toxic aggregates: insights from computational simulations.

The misfolding and aggregation of the presynaptic protein α-synuclein (α-syn) is a pathological hallmark of Parkinson's disease (PD). Targeting α-syn has emerged as a promising therapeutic strategy for PD. Emerging in vitro evidence supports a dual action of epigallocatechin-3-gallate (EGCG) against amyloid neurotoxicity. EGCG can halt the formation of toxic aggregates by redirecting the amyloid fibril aggregation pathway toward non-toxic aggregates and remodeling the existing toxic fibrils into non-toxic aggregates. Moreover, EGCG oxidation can enhance fibril's remodeling by forming Schiff bases, leading to crosslinking of the fibril. However, this covalent modification is not required for amyloid remodeling, and establishing non-specific hydrophobic interactions with sidechains seems to be the main driver of amyloid remodeling by EGCG. Thioflavin (ThT) is a gold standard probe to detect amyloid fibrils in vitro, and oxidized EGCG competes with ThT for amyloid fibrils' binding sites. In this work, we performed docking and molecular dynamics (MD) simulations to gain insights into the intermolecular interactions of oxidized EGCG and ThT with a mature α-syn fibril. We find that oxidized EGCG moves within lysine-rich sites within the hydrophobic core of the α-syn fibril, forming aromatic and hydrogen-bonding (H-bond) interactions with different residues during the whole MD simulation time. In contrast, ThT, which does not remodel amyloid fibrils, was docked to the same sites but only via aromatic interactions. Our findings suggest that non-covalent interactions play a role in oxidized EGCG binding into the hydrophobic core, including H-bond and aromatic interactions with some residues in the amyloid remodeling processes. These interactions would ultimately lead to a disturbance of structural features as determinants for stabilizing this fibril into a compact and pathogenic Greek key topology.

Subtilisin of Leishmania amazonensis as Potential Druggable Target: Subcellular Localization, In Vitro Leishmanicidal Activity and Molecular Docking of PF-429242, a Subtilisin Inhibitor.

Subtilisin proteases, found in all organisms, are enzymes important in the post-translational steps of protein processing. In Leishmania major and L. donovani, this enzyme has been described as essential to their survival; however, few compounds that target subtilisin have been investigated for their potential as an antileishmanial drug. In this study, we first show, by electron microscopy and flow cytometry, that subtilisin has broad localization throughout the cytoplasm and membrane of the parasite in the promastigote form with foci in the flagellar pocket. Through in silico analysis, the similarity between subtilisin of different Leishmania species and that of humans were determined, and based on molecular docking, we evaluated the interaction capacity of a serine protease inhibitor against both life cycle forms of Leishmania. The selected inhibitor, known as PF-429242, has already been used against the dengue virus, arenaviruses, and the hepatitis C virus. Moreover, it proved to have antilipogenic activity in a mouse model and caused hypolipidemia in human cells in vitro. Here, PF-429242 significantly inhibited the growth of L. amazonensis promastigotes of four different strains (IC50 values = 3.07 ± 0.20; 0.83 ± 0.12; 2.02 ± 0.27 and 5.83 ± 1.2 µM against LTB0016, PH8, Josefa and LV78 strains) whilst having low toxicity in the host macrophages (CC50 = 170.30 µM). We detected by flow cytometry that there is a greater expression of subtilisin in the amastigote form; however, PF-429242 had a low effect against this intracellular form with an IC50 of >100 µM for intracellular amastigotes, as well as against axenic amastigotes (94.12 ± 2.8 µM for the LV78 strain). In conclusion, even though PF-429242 does not affect the intracellular forms, this drug will serve as a tool to explore pharmacological and potentially leishmanicidal targets.

Diterpenes isolated from Canistrocarpus cervicornis with virucidal activity against HIV-1: an in silico evaluation.

HIV is a public health problem, which makes necessary the development of new drugs. Natural products are known for their anti-HIV potential and a good strategy to suggest its mechanism of action is using in silico tools. Herein, diterpenes 1-3 had the binding mode evaluated in the HIV-1 glycoprotein; and properties ADMET in silico performed. In molecular docking important interactions between the hydrophobic cavity, and 1 and 2 were observed. In the molecular dynamics, 1 remained stable covering the entire hydrophobic cavity and performed hydrogen bond during all simulation. ADMET evaluation showed good properties for the diterpenes. Based on these findings, it was possible to suggest the potential from natural products as entry inhibitor and HIV-1 treatment.

Small Angle X-ray Scattering, Molecular Modeling, and Chemometric Studies from a Thrombin-Like (Lmr-47) Enzyme of Lachesis m. rhombeata Venom.

INTRODUCTION: Snakebite envenomation is considered a neglected tropical disease, and SVTLEs critical elements are involved in serious coagulopathies that occur on envenoming. Although some enzymes of this group have been structurally investigated, it is essential to characterize other proteins to better understand their unique properties such as the Lachesis muta rhombeata 47 kDa (Lmr-47) venom serine protease. METHODS: The structure of Lmr-47 was studied in solution, using SAXS, DLS, CD, and in silico by homology modeling. Molecular docking experiments simulated 21 competitive inhibitors. RESULTS: At pH 8.0, Lmr-47 has an Rg of 34.5 ± 0.6 Å, Dmax of 130 Å, and SR of 50 Å, according to DLS data. Kratky plot analysis indicates a rigid shape at pH 8.0. Conversely, the pH variation does not change the center of mass's intrinsic fluorescence, possibly indicating the absence of fluorescent amino acids in the regions affected by pH variation. CD experiments show a substantially random coiled secondary structure not affected by pH. The low-resolution model of Lmr-47 presented a prolate elongated shape at pH 8.0. Using the 3D structure obtained by molecular modeling, docking experiments identified five good and three suitable competitive inhibitors. CONCLUSION: Together, our work provided insights into the structure of the Lmr-47 and identified inhibitors that may enhance our understanding of thrombin-like family proteins.

Green Tea Epigallocatechin-3-gallate (EGCG) Targeting Protein Misfolding in Drug Discovery for Neurodegenerative Diseases.

The potential to treat neurodegenerative diseases (NDs) of the major bioactive compound of green tea, epigallocatechin-3-gallate (EGCG), is well documented. Numerous findings now suggest that EGCG targets protein misfolding and aggregation, a common cause and pathological mechanism in many NDs. Several studies have shown that EGCG interacts with misfolded proteins such as amyloid beta-peptide (Aß), linked to Alzheimer's disease (AD), and α-synuclein, linked to Parkinson's disease (PD). To date, NDs constitute a serious public health problem, causing a financial burden for health care systems worldwide. Although current treatments provide symptomatic relief, they do not stop or even slow the progression of these devastating disorders. Therefore, there is an urgent need to develop effective drugs for these incurable ailments. It is expected that targeting protein misfolding can serve as a therapeutic strategy for many NDs since protein misfolding is a common cause of neurodegeneration. In this context, EGCG may offer great potential opportunities in drug discovery for NDs. Therefore, this review critically discusses the role of EGCG in NDs drug discovery and provides updated information on the scientific evidence that EGCG can potentially be used to treat many of these fatal brain disorders.

Leishmanicidal therapy targeted to parasite proteases.

Leishmaniasis is considered a serious public health problem and the current available therapy has several disadvantages, which makes the search for new therapeutic targets and alternative treatments extremely necessary. In this context, this review focuses on the importance of parasite proteases as target drugs against Leishmania parasites, as a chemotherapy approach. Initially, we discuss about the current scenario for the treatment of leishmaniasis, highlighting the main drugs used and the problems related to their use. Subsequently, we describe the inhibitors of major proteases of Leishmania already discovered, such as Compound s9 (aziridine-2,3-dicarboxylate), Compound 1c (benzophenone derivative), Au2Phen (gold complex), AubipyC (gold complex), MDL 28170 (dipeptidyl aldehyde), K11777, Hirudin, diazo-acetyl norleucine methyl ester, Nelfinavir, Saquinavir, Nelfinavir, Saquinavir, Indinavir, Saquinavir, GNF5343 (azabenzoxazole), GNF6702 (azabenzoxazole), Benzamidine and TPCK. Next, we discuss the importance of the protease gene to parasite survival and the aspects of the validation of proteases as target drugs, with emphasis on gene disruption. Then, we describe novel important strategies that can be used to support the research of new antiparasitic drugs, such as molecular modeling and nanotechnology, whose main targets are parasitic proteases. And finally, we discuss possible perspectives to improve drug development. Based on all findings, proteases could be considered potential targets against leishmaniasis.

Insights into cytochrome bc1 complex binding mode of antimalarial 2-hydroxy-1, 4-naphthoquinones through molecular modelling

BACKGROUND Malaria persists as a major public health problem. Atovaquone is a drug that inhibits the respiratory chain of Plasmodium falciparum, but with serious limitations like known resistance, low bioavailability and high plasma protein binding. OBJECTIVES The aim of this work was to perform molecular modelling studies of 2-hydroxy-1,4-naphthoquinones analogues of atovaquone on the Qo site of P. falciparum cytochrome bc1 complex (Pfbc1) to suggest structural modifications that could improve their antimalarial activity. METHODS We have built the homology model of the cytochrome b (CYB) and Rieske iron-sulfur protein (ISP) subunits from Pfbc1 and performed the molecular docking of 41 2-hydroxy-1,4-naphthoquinones with known in vitro antimalarial activity and predicted to act on this target. FINDINGS Results suggest that large hydrophobic R2 substituents may be important for filling the deep hydrophobic Qo site pocket. Moreover, our analysis indicates that the H-donor 2-hydroxyl group may not be crucial for efficient binding and inhibition of Pfbc1 by these atovaquone analogues. The C1 carbonyl group (H-acceptor) is more frequently involved in the important hydrogen bonding interaction with His152 of the Rieske ISP subunit. MAIN CONCLUSIONS Additional interactions involving residues such as Ile258 and residues required for efficient catalysis (e.g., Glu261) could be explored in drug design to avoid development of drug resistance by the parasite.

Insights into cytochrome bc1 complex binding mode of antimalarial 2-hydroxy-1,4-naphthoquinones through molecular modelling.

BACKGROUND: Malaria persists as a major public health problem. Atovaquone is a drug that inhibits the respiratory chain of Plasmodium falciparum, but with serious limitations like known resistance, low bioavailability and high plasma protein binding. OBJECTIVES: The aim of this work was to perform molecular modelling studies of 2-hydroxy-1,4-naphthoquinones analogues of atovaquone on the Qo site of P. falciparum cytochrome bc1 complex (Pfbc1) to suggest structural modifications that could improve their antimalarial activity. METHODS: We have built the homology model of the cytochrome b (CYB) and Rieske iron-sulfur protein (ISP) subunits from Pfbc1 and performed the molecular docking of 41 2-hydroxy-1,4-naphthoquinones with known in vitro antimalarial activity and predicted to act on this target. FINDINGS: Results suggest that large hydrophobic R2 substituents may be important for filling the deep hydrophobic Qo site pocket. Moreover, our analysis indicates that the H-donor 2-hydroxyl group may not be crucial for efficient binding and inhibition of Pfbc1 by these atovaquone analogues. The C1 carbonyl group (H-acceptor) is more frequently involved in the important hydrogen bonding interaction with His152 of the Rieske ISP subunit. MAIN CONCLUSIONS: Additional interactions involving residues such as Ile258 and residues required for efficient catalysis (e.g., Glu261) could be explored in drug design to avoid development of drug resistance by the parasite.

Hologram QSAR models of 4-[(diethylamino)methyl]-phenol inhibitors of acetyl/butyrylcholinesterase enzymes as potential anti-Alzheimer agents.

Hologram QSAR models were developed for a series of 36 inhibitors (29 training set and seven test set compounds) of acetyl/butyrylcholinesterase (AChE/BChE) enzymes, an attractive molecular target for Alzheimer's disease (AD) treatment. The HQSAR models (N = 29) exhibited significant cross-validated (AChE, q2 = 0.787; BChE, q2 = 0. 904) and non-cross-validated (AChE, r2 = 0.965; BChE, r2= 0.952) correlation coefficients. The models were used to predict the inhibitory potencies of the test set compounds, and agreement between the experimental and predicted values was verified, exhibiting a powerful predictive capability. Contribution maps show that structural fragments containing aromatic moieties and long side chains increase potency. Both the HQSAR models and the contribution maps should be useful for the further design of novel, structurally related cholinesterase inhibitors.

Application of 4D-QSAR studies to a series of raloxifene analogs and design of potential selective estrogen receptor modulators.

Four-dimensional quantitative structure-activity relationship (4D-QSAR) analysis was applied on a series of 54 2-arylbenzothiophene derivatives, synthesized by Grese and coworkers, based on raloxifene (an estrogen receptor-alpha antagonist), and evaluated as ERa ligands and as inhibitors of estrogen-stimulated proliferation of MCF-7 breast cancer cells. The conformations of each analogue, sampled from a molecular dynamics simulation, were placed in a grid cell lattice according to three trial alignments, considering two grid cell sizes (1.0 and 2.0 Å). The QSAR equations, generated by a combined scheme of genetic algorithms (GA) and partial least squares (PLS) regression, were evaluated by "leave-one-out" cross-validation, using a training set of 41 compounds. External validation was performed using a test set of 13 compounds. The obtained 4D-QSAR models are in agreement with the proposed mechanism of action for raloxifene. This study allowed a quantitative prediction of compounds' potency and supported the design of new raloxifene analogs.

Investigating the differential activation of vascular endothelial growth factor (VEGF) receptors.

The vascular endothelial growth factors are key mediators of angiogenesis and are also related to several physiological processes such as monocyte chemotaxis, dendritic cell development, hematopoietic stem cell survival, and many others. PlGF, VEGF, VEGFB, VEGFC and VEGFD were identified as members of the vascular endothelial growth factor family. They act by differential activation of three receptors: Flt-1, KDR and Flt-4. PlGF and VEGFB only activate Flt-1. VEGF activates both Flt-1 and KDR. VEGFC and VEGFD activate KDR and Flt-4. The available three dimensional structures of VEGF and PlGF, in complex with the domain-2 of Flt-1, show that both proteins bind in a very similar way to Flt-1 receptor. Here we construct the three dimensional model of the domain-2 of KDR receptor using the same domain of Flt-1 as template. We also construct the model complexes VEGF/KDR, VEGFB/Flt-1, VEGFB/KDR and PlGF/KDR. Molecular dynamics simulations with explicit solvent are carried out on eleven molecular systems: unbound VEGF, VEGF/Flt-1(D2), VEGF/KDR(D2), unbound PlGF, PlGF/Flt-1(D2), PlGF/KDR(D2), unbound VEGFB, VEGFB/Flt-1(D2), VEGFB/KDR(D2), unbound Flt-1(D2) and unbound KDR(D2). We analyze protein-protein interactions, shape complementarity, charge complementarity and hydrogen bonds. As a coarse estimation of the desolvation penalties, we assume a correlation to the number of hydrogen bonds with solvent molecules that are lost upon complex formation. The results herein are consistent with the experimental selectivity profile (VEGF being able to activate both Flt-1 and KDR receptors while VEGFB and PlGF being only able to activate Flt-1), and provide a collection of evidences sustaining the complementarity of polar interactions as the main responsible for protein recognition and selectivity.