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Cerebral malaria (CM) is a life-threatening form of Plasmodium falciparum infection caused by brain inflammation. Brain endothelium dysfunction is a hallmark of CM pathology, which is also associated with the activation of the type I interferon (IFN) inflammatory pathway. The molecular triggers and sensors eliciting brain type I IFN cellular responses during CM remain largely unknown. We herein identified the stimulator of interferon response cGAMP interactor 1 (STING1) as the key innate immune sensor that induces Ifnβ1 transcription in the brain of mice infected with Plasmodium berghei ANKA ( Pba ). This STING1/IFNβ-mediated response increases brain CXCL10 governing the extent of brain leukocyte infiltration and blood–brain barrier (BBB) breakdown, and determining CM lethality. The critical role of brain endothelial cells (BECs) in fueling type I IFN–driven brain inflammation was demonstrated in brain endothelial–specific IFNβ-reporter and STING1-deficient Pba -infected mice, which were significantly protected from CM lethality. Moreover, extracellular particles (EPs) released from Pba -infected erythrocytes activated the STING1-dependent type I IFN response in BECs, a response requiring intracellular acidification. Fractionation of the EPs enabled us to identify a defined fraction carrying hemoglobin degradation remnants that activates STING1/IFNβ in the brain endothelium, a process correlated with heme content. Notably, stimulation of STING1-deficient BECs with heme, docking experiments, and in vitro binding assays unveiled that heme is a putative STING1 ligand. This work shows that heme resultant from the parasite heterotrophic activity operates as an alarmin, triggering brain endothelial inflammatory responses via the STING1/IFNβ/CXCL10 axis crucial to CM pathogenesis and lethality.

Urease is a metalloenzyme that catalyzes the hydrolysis of urea, and its modulation has an important role in both the agricultural and medical industry. Even though numerous molecules have been tested against ureases of different species, their clinical translation has been limited due to chemical and metabolic stability as well as side effects. Therefore, screening new compounds against urease would be of interest in part due to rising concerns regarding antibiotic resistance. In this work, we collected and curated a diverse set of 2640 publicly available small-molecule inhibitors of jack bean urease and developed a classifier using a random forest machine learning method with high predictive performance. In addition, the physicochemical features of compounds were paired with molecular docking and protein–ligand fingerprint analysis to gather insight into the current activity landscape. We observed that the docking score could not differentiate active from inactive compounds within each chemical family, but scores were correlated with compound activity when all compounds were considered. Additionally, a decision tree model was built based on 2D and 3D Morgan fingerprints to mine patterns of the known active-class compounds. The final machine learning model showed good prediction performance against the test set (81% and 77% precision for active and inactive compounds, respectively). Finally, this model was employed, as a proof-of-concept, on an in-house library to predict new hits that were then tested against urease and found to be active. This is, to date, the largest, most diverse dataset of compounds used to develop predictive in silico models. Overall, the results highlight the usefulness of using machine learning classifiers and molecular docking to predict novel urease inhibitors.

Nature has revealed to be a key source of innovative anticancer drugs. This study evaluated the antitumour potential of the marine bromoditerpene sphaerococcenol A on different cancer cellular models. Dose-response analyses (0.1–100 μM; 24 h) were accomplished in eight different tumour cell lines (A549, CACO-2, HCT-15, MCF-7, NCI-H226, PC-3, SH-SY5Y, SK-MEL-28). Deeper studies were conducted on MFC-7 cells, namely, determination of hydrogen peroxide (H2O2) levels and evaluation of apoptosis biomarkers (phosphatidylserine membrane translocation, mitochondrial dysfunction, Caspase-9 activity, and DNA changes). The ability of the compound to induce genotoxicity was verified in L929 fibroblasts. Sphaerococcenol A capacity to impact colorectal-cancer stem cells (CSCs) tumourspheres (HT29, HCT116, SW620) was evaluated by determining tumourspheres viability, number, and area, as well as the proteasome inhibitory activity. Sphaerococcenol A hepatoxicity was studied in AML12 hepatocytes. The compound exhibited cytotoxicity in all malignant cell lines (IC50 ranging from 4.5 to 16.6 μM). MCF-7 cells viability loss was accompanied by H2O2 generation, mitochondrial dysfunction, Caspase-9 activation and DNA nuclear morphology changes. Furthermore, the compound displayed the lowest IC50 on HT29-derived tumourspheres (0.70 μM), followed by HCT116 (1.77 μM) and SW620 (2.74 μM), impacting the HT29 tumoursphere formation by reducing their number and area. Finally, the compound displayed low cytotoxicity on AML12 hepatocytes without genotoxicity. Overall, sphaerococcenol A exhibits broad cytotoxic effects on different tumour cells, increasing H2O2 production and apoptosis. It also affects colorectal CSCs-enriched tumoursphere development. These data highlight the elevance to include sphaerococcenol A in further pharmacological studies aiming cancer treatments. info:eu-repo/semantics/publishedVersion

We report the synthesis of 3-hydroxyl-2-naphthoylhydrazones containing pyrrole (HL1), furane (HL2) and thiophene (HL3) moieties and their V(IV)O-, Cu(II)- and Zn(II)-complexes. All compounds are characterized by the usual analytical techniques and coordination of the ligands to the metal ions is discussed based on spectroscopic data (FTIR, UV-vis, EPR and NMR) as well as CAMB3LYP DFT/TDDFT calculations, indicating the formation of neutral ML2 type complexes. The photophysical properties of ligands and complexes are disclosed. The binding to Bovine Serum Albumin (BSA) is evaluated in detail using several spectroscopic tools. Circular dichroism shows that the compounds, and particularly the ligand precursors, stabilize BSA, increasing its a-helical content. Fluorescence studies indicate the formation of 1 : 1 protein-compound adducts, which is corroborated by molecular docking studies that show the interaction between Trp 213 of BSA and the naphthalene rings. The general toxicity is evaluated using the Artemia salina lethality assay, with all compounds showing general toxicity towards the brine shrimp model. The cytotoxicity on human cancer cells (H1299, MCF7, and HCT116) is assessed for all compounds and the half-maximal inhibitory concentration (IC50) values are in the range from 0.57 to 27.35 mu M. Compounds containing L-1 (pyrrole derivative) are the most cytotoxic, with the vanadium and zinc complexes performing better than the copper ones, and some of them depicting IC50 values lower than 1.1 mM. However, selectivity needs improvement as the compounds show toxicity towards Artemia salina and normal fibroblasts. Fundacao para a Ciencia e a Tecnologia (FCT)Portuguese Foundation for Science and Technology [UID/QUI/00100/2019, UID/MULTI/04349/2013, UID/BIO/04565/2013, RECI/QEQ-QIN/0189/2012, RECI/QEQ-MED/0330/2012, UID/DTP/04567/2016] Programa Operacional Regional de Lisboa [LISBOA-01-0145-FEDER-007317] [UID/04138/2019] [SAICTPAC/0019/2015] [PTDC/QUI-QAN/32242/2017] [PD/BD/128320/2017] [SFRH/BD/135797/2018] [SFRH/BPD/107834/2015] [UID/QUI/50006/2019] [RECI/BBB-BEP/0124/2012] [UID/DTP/04567/2019] [SFRH/BD/119144/2016] info:eu-repo/semantics/publishedVersion

A pharmacophore model for inhibitors of Escherichia coli’s DNA Gyrase B was developed, using computer-aided drug design. Subsequently, docking studies showed that 2,5(6)-substituted benzimidazole derivatives are promising molecules, as they possess key hydrogen bond donor/acceptor groups for an efficient interaction with this bacterial target. Furthermore, 5(6)-bromo-2-(2-nitrophenyl)-1H-benzimidazole, selected as a core molecule, was prepared on a multi-gram scale through condensation of 4-bromo-1,2-diaminobenzene with 2-nitrobenzaldehyde using a sustainable approach. The challenging functionalization of the 5(6)-position was carried out via palladium-catalyzed Suzuki–Miyaura and Buchwald-Hartwig amination cross-coupling reactions between N-protected-5-bromo-2-nitrophenyl-benzimidazole and aryl boronic acids or sulfonylanilines, with yields up to 81%. The final designed molecules (2-(aminophen-2-yl)-5(6)-substituted-1H-benzimidazoles), which encompass the appropriate functional groups in the 5(6)-position according to the pharmacophore model, were obtained in yields up to 91% after acid-mediated N-boc deprotection followed by Pd-catalyzed hydrogenation. These groups are predicted to favor interactions with DNA gyrase B residues Asn46, Asp73, and Asp173, aiming to promote an inhibitory effect.

Bio-contamination of water through biofouling, which involves the natural colonization of submerged surfaces by waterborne organisms, is a global socio-economic concern, allied to premature materials bio-corrosion and high human health risks. Most effective strategies release toxic and persistent disinfectant compounds into the aquatic medium, causing environmental problems and leading to more stringent legislation regarding their use. To minimize these side effects, a newly non-biocide-release coating strategy suitable for several polymeric matrices, namely polydimethylsiloxane and polyurethane (PU)-based coatings, was used to generate antimicrobial ceramic filters for water bio-decontamination. The best results, in terms of antimicrobial activity and biocide release, showed an expressed delay and a decrease of up to 66% in the population of methicillin-resistant Staphylococcus aureus bacteria on ceramic filters coated with polyurethane (PU)-based coatings containing grafted Econea biocide, and no evidence of biocide release after being submerged for 45 days in water. Biocidal PU-based surfaces were also less prone to Enterococcus faecalis biofilm formation under flow conditions with an average reduction of 60% after 48 h compared to a pristine PU-based surface. Biocidal coated filters show to be a potential eco-friendly alternative for minimizing the environmental risks associated with biofouling formation in water-based industrial systems., This research work was supported by national funds through FCT-Foundation for Science and Technology within the scope of research units grants to BioISI (UIDB/04046/2020 and UIDP/04046/2020), and also by Base Funding-UIDB/00511/2020 of the Laboratory for Process Engineering, Environment, Biotechnology, and Energy-LEPABE-funded by national funds through the FCT/MCTES (PIDDAC). O. Ferreira also acknowledges the Grant PD/BD/128370/2017. L.C.G. and E.R.S. thank the Portuguese Foundation for Science and Technology (FCT) for their work contracts through the Scientific Employment Stimulus-Individual Call (CEECIND/01700/2017 and CEECIND/03530/2018).

HIV-2 infection is frequently neglected in HIV/AIDS campaigns. However, a special emphasis must be given to HIV-2 as an untreated infection that also leads to AIDS and death, and for which the efficacy of most available drugs is limited against HIV-2. HIV envelope glycoproteins mediate binding to the receptor CD4 and co-receptors at the surface of the target cell, enabling fusion with the cell membrane and viral entry. Here, we developed and optimized a computer-assisted drug design approach of an important HIV-2 glycoprotein that allows us to explore and gain further insights at the molecular level into protein structures and interactions crucial for the inhibition of HIV-2 cell entry. The 3D structure of a key HIV-2ROD gp125 region was generated by a homology modeling campaign. To disclose the importance of the main structural features and compare them with experimental results, 3D-models of six mutants were also generated. These mutations revealed the selective impact on the behavior of the protein. Furthermore, molecular dynamics simulations were performed to optimize the models, and the dynamic behavior was tackled to account for structure flexibility and interactions network formation. Structurally, the mutations studied lead to a loss of aromatic features, which is very important for the establishment of π-π interactions and could induce a structural preference by a specific coreceptor. These new insights into the structure-function relationship of HIV-2 gp125 V3 and surrounding regions will help in the design of better models and the design of new small molecules capable to inhibit the attachment and binding of HIV with host cells.

Lipoaminoacids (LAA) are an important group of biosurfactants, formed by a polar hydrophilic part (amino acid) and a hydrophobic tail (lipid). The gemini LAA structures allow the formation of a supramolecular complex with bioactive molecules, like DNA, which provides them with good transfection efficiency. Since lipases are naturally involved in lipid and protein metabolism, they are an alternative to the chemical production of LAA, offering an eco-friendly biosynthetic process option. This work aimed to design the production of novel cystine derived gemini through a bioconversion system using immobilized lipases. Three lipases were used: porcine pancreatic lipase (PPL), lipase from Thermomyces lanuginosus (TLL), and lipase from Rizhomucor miehei (RML). PPL was immobilized in sol-gel lenses. L-cystine dihydrochloride and dodecylamine were used as substrates for the bioreaction. The production of LAA was evaluated by thin layer chromatography (TLC), and colorimetric reaction with eosin. The identification and quantification was carried out by High Performance Liquid Chromatographer-Mass Spectrometry (HPLC-MS/MS). The optimization of media design included co-solvent (methanol, dimethylsulfoxide), biphasic (n-hexane and 2-propanol) or solvent-free media, in order to improve the biocatalytic reaction rates and yields. Moreover, a new medium was tested where dodecylamine was melted and added to the cystine and to the biocatalyst, building a system of mainly undissolved substrates, leading to 5 mg/mL of LAA. Most of the volume turned into foam, which indicated the production of the biosurfactant. For the first time, the gemini derived cystine lipoaminoacid was produced, identified, and quantified in both co-solvent and solvent-free media, with the lipases PPL, RML, and TLL.

Introduction Verbascum betonicifolium (V. betonicifolium) is a plant used in traditional medicine for several ailments. The objective of this study was to determine the antioxidant activity and acetylcholinesterase inhibitory activity of the aqueous extract together with the metabolites responsible for these activities. This paper presents the first phytochemical characterization and bioactivities of aqueous extracts from aerial parts. Methods The compounds present in the aerial part aqueous extract were identified by high performance liquid chromatography, coupled to a diode-array detector (HPLC-DAD) and by high-resolution mass spectrometry (HRMS), using LC–MS/MS analyses. Antioxidant activity was measured as the capacity to scavenge the free radical DPPH and the AChE activity was determined using the Ellman test. The cytotoxicity was determined used HepG2 cell lines. Results Several types of metabolites were found, primary metabolites (malic, citric and gluconic acids), phenolic compounds (verbascoside, luteolin), terpenoids (iridoid glycoside, unedide), among others. The total phenol content of 28 μg of gallic acid equivalents/mg of extract was determined. The aqueous extract antioxidant activity had and EC50 of 70 μg/mL and the AChE inhibitory activity an IC50 of 750 μg/mL. No cytotoxicity towards HepG2 cells was detected, even using a concentration of 1 mg/mL. Conclusions The phenolic compounds present in the extract may be the main contributors to the bioactivities of V. betonicifolium. These results show for the first time the richness of phytochemicals and the strong bioactivities of V. betonicifolium and that the aqueous extract could be used as new natural sources of bioactive molecules.

Boronic acids (BAs) are a prominent functionality extensively used to design biologically active compounds and functional biomaterials. Boronic acids open shell can lead to unspecific reactivity of BAs with endogenous nucleophiles and to undesired off-target effects. Here, diazaborines are presented as a new class of boron-based warheads for serine proteases inhibition, in which the boron function is stabilized in the form of an aromatic boron-based heterocycle. In this study, diazaborines were readily synthesized in a single step in yields up to 96%, without any chromatographic operation and were shown to reversibly inhibit human neutrophil elastase (HNE) serine protease with IC50 values in the low μM range. Diazaborines were also selective toward HNE, as no inhibition was observed against a panel of five closely related serine proteases. Synthetic and theoretical studies performed on this system suggest that, similar to BAs, the reaction mechanism involves the formation of a reversible covalent bond between the diazaborine boron center and the catalytic serine oxygen. Finally, different from BAs, diazaborines were shown to be very stable in pH 7.4 phosphate buffer and human plasma.

Regulated necrosis or necroptosis, mediated by receptor-interacting kinase 1 (RIPK1), RIPK3 and pseudokinase mixed lineage kinase domain-like protein (MLKL), contributes to the pathogenesis of inflammatory, infectious and degenerative diseases. Recently identified necroptosis inhibitors display moderate specificity, suboptimal pharmacokinetics, off-target effects and toxicity, preventing these molecules from reaching the clinic. Here, we developed a cell-based high-throughput screening (HTS) cascade for the identification of small-molecule inhibitors of necroptosis. From the initial library of over 250,000 compounds, the primary screening phase identified 356 compounds that strongly inhibited TNF-α-induced necroptosis, but not apoptosis, in human and murine cell systems, with EC50 50 >100 μM, EC50 2.5–11.5 μM under long-term necroptosis execution in murine fibroblast L929 cells, and full protection from ATP depletion and membrane leakage in human and murine cells. As a proof of concept, compound SN-6109, with binding mode to RIPK1 similar to that of necrostatin-1, confirmed RIPK1 inhibitory activity and appropriate pharmacokinetic properties. SN-6109 was further tested in mice, showing efficacy against TNF-α-induced systemic inflammatory response syndrome. In conclusion, a phenotypic-driven HTS cascade promptly identified robust necroptosis inhibitors with in vivo activity, currently undergoing further medicinal chemistry optimization. Notably, the novel hits highlight the opportunity to identify new molecular mechanisms of action in necroptosis.

The role of metalloproteinases (MMPs) on the migration and invasion of cancer cells has been correlated with tumor aggressiveness, namely with the up-regulation of MMP-2 and 9. Herein, two pyridine-containing macrocyclic compounds, [15]pyN5 and [16]pyN5, were synthesized, chemically characterized and evaluated as potential MMP inhibitors for breast cancer therapy using 3D and 2D cellular models. [15]pyN5 and [16]pyN5 (5&ndash, 20 &micro, M) showed a marked inhibition of MMPs activity (100% at concentrations &ge, 7.5 &mu, M) when compared to ARP-100, a known MMP inhibitor. The inhibitory activity of [15]pyN5 and [16]pyN5 was further supported through in silico docking studies using Goldscore and ChemPLP scoring functions. Moreover, although no significant differences were observed in the invasion studies in the presence of all MMPs inhibitors, cell migration was significantly inhibited by both pyridine-containing macrocycles at concentrations above 5 &mu, M in 2D cells (p &lt, 0.05). In spheroids, the same effect was observed, but only with [16]pyN5 at 20 &mu, M and ARP-100 at 40 &mu, M. Overall, [15]pyN5 and [16]pyN5 led to impaired breast cancer cell migration and revealed to be potential inhibitors of MMPs 2 and 9.

Leucine-rich repeat kinase 2 (LRRK2) is a serine-threonine kinase involved in multiple cellular processes and signaling pathways. LRRK2 mutations are associated with autosomal-inherited Parkinson's disease (PD), and evidence suggests that LRRK2 pathogenic variants generally increase kinase activity. Therefore, inhibition of LRRK2 kinase function is a promising therapeutic strategy for PD treatment. The search for drug-like molecules capable of reducing LRRK2 kinase activity in PD led to the design of selective LRRK2 inhibitors predicted to be within the CNS drug-like space. This review highlights the journey that translates chemical tools for interrogating the role of LRRK2 in PD into promising drug candidates, addressing the challenges in discovering selective and brain-penetrant LRRK2 modulators and exploring the structure–activity relationship of distinct LRRK2 inhibitors.

Influenza NS1 protein is among the most promising novel druggable anti-influenza target, based on its structure; multiple interactions; and global function in influenza replication and pathogenesis. Notwithstanding, drug development guidance based on NS1 structural biology is lacking. Here, we design a promising strategy directed to highly conserved druggable regions as a result of an exhaustive large-scale sequence analysis and structure characterization of NS1 protein across human-infecting influenza A subtypes, over the past 100 years. We have identified 3 druggable pockets and 8 new potential hot spot residues in the NS1 protein, not described before, additionally to other 16 sites previously identified, which represent attractive targets for pharmacological modulation. This study provides a rationale towards structure-function studies of NS1 druggable sites, which have the potential to accelerate the NS1 target validation. This research also contributes to a deeper comprehension and insight into the evolutionary dynamics of influenza A NS1 protein. Highlights: Anti-influenza strategies based on highly conserved target structures are needed; Overall, the human NS1 protein is highly conserved in the RBD and ED domains; Three main consensus druggable pockets were found with high druggability score; 8 new potential hot spots were identified within the NS1-ED; The study discloses a new panel for NS1 structure-function studies. info:eu-repo/semantics/publishedVersion

From a screening study of various potential inhibitors for cholinesterases (ChEs), compound (rac)-1 (4-((3-hydroxy-2-oxo-3-phenylindolin-1-yl) methyl) piperidin-1-ium chloride) showed an IC50 of 18 μM for butyrylcholinesterase (BuChE). Herein we present a toxicological and pharmacological evaluation of (rac)-1 to determine its potential for use as an alternative ChE inhibitor for the treatment of Alzheimer’s disease. The strategy adopted included in vivo and ex vivo studies with mouse models, Molecular Modelling and Saturation Transfer Difference (STD) NMR studies. Preliminary molecular docking studies were conducted with both (R) and (S)-1 with acetylcholinesterase (AChE) and BuChE, prior to advancing to the mouse model, and indeed favorable interactions were observed, with (R)-1 showing the best binding with AChE and (S)-1 with BuChE. STD-NMR studies were used to successfully validate these results. Toxicological studies were also conducted using the Artemia salina model, with donepezil as reference. It was found that in the in vivo mouse studies that (rac)-1 presented a slightly better inhibition of AChE (0.096 µmol.min−1.mg−1) than donepezil (0.112 µmol.min−1.mg−1) and the same level of inhibition for BuChE as donepezil (0.014 µmol.min−1.mg−1).

Necroptosis is a regulated form of necrosis, which may be critical in the pathogenesis of neurodegenerative diseases. Neuroinflammation, characterized by the activation of glial cells such as microglia, is closely linked with neurodegenerative pathways and constitutes a major mechanism of neural damage and disease progression. Importantly, inhibition of necroptosis results in disease improvement, unveiling an alternative approach for therapeutic intervention. In the present study, we screened a small library of new molecules, potentially inhibitors of necroptosis, using two cellular models of necroptosis. A new oxazolone, Oxa12, reduced tumour necrosis factor α (TNF-α)-induced necroptosis in mouse L929 fibrosarcoma cells. Notably, Oxa12 strongly inhibited zVAD-fmk-induced necroptosis in murine BV2 microglial cells. Moreover, Oxa12 blocked phosphorylation of mixed-lineage kinase domain-like protein (MLKL), and interfered with necrosome complex formation, indicating that Oxa12 targets components upstream of MLKL. In fact, in silico molecular docking studies revealed that Oxa12 is occupying a region similar to the 1-aminoisoquinoline type II kinase inhibitor inside the receptor-interacting protein 1 (RIP1) kinase domain. Finally, in microglial cells, Oxa12 attenuated zVAD-fmk- and lipopolysaccharide (LPS)-induced inflammatory processes, as revealed by a marked decrease of TNF-α and/or IL-1β expression. More specifically, Oxa12 negatively targeted c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) pathways, as well as NF-κB activation. Overall, we identified a strong lead inhibitor of necroptosis that is also effective at reducing inflammation-associated events. Oxa12 is a promising candidate molecule for further development to target disease states dependent on RIP kinase activity.

Data mining approaches can uncover underlying patterns in chemical and pharmacological property space decisive for drug discovery and development. Two of the most common approaches are visualization and machine learning methods. Visualization methods use dimensionality reduction techniques in order to reduce multi-dimension data into 2D or 3D representations with a minimal loss of information. Machine learning attempts to find correlations between specific activities or classifications for a set of compounds and their features by means of recurring mathematical models. Both models take advantage of the different and deep relationships that can exist between features of compounds, and helpfully provide classification of compounds based on such features or in case of visualization methods uncover underlying patterns in the feature space. Drug-likeness has been studied from several viewpoints, but here we provide the first implementation in chemoinformatics of the t-Distributed Stochastic Neighbor Embedding (t-SNE) method for the visualization and the representation of chemical space, and the use of different machine learning methods separately and together to form a new ensemble learning method called AL Boost. The models obtained from AL Boost synergistically combine decision tree, random forests (RF), support vector machine (SVM), artificial neural network (ANN), k nearest neighbors (kNN), and logistic regression models. In this work, we show that together they form a predictive model that not only improves the predictive force but also decreases bias. This resulted in a corrected classification rate of over 0.81, as well as higher sensitivity and specificity rates for the models. In addition, separation and good models were also achieved for disease categories such as antineoplastic compounds and nervous system diseases, among others. Such models can be used to guide decision on the feature landscape of compounds and their likeness to either drugs or other characteristics, such as specific or multiple disease-category(ies) or organ(s) of action of a molecule.

A computer-aided campaign boosted the discovery of potent human neutrophil elastase (HNE) inhibitors. A pharmacophoric model was developed, validated and applied to filter an oxo-beta-lactam library previously generated by de novo design. This campaign led us to compound 1 which showed an inhibitory activity of 6.9 nM against HNE, more active than the only commercially available HNE inhibitor for therapeutic usage. Computer-aided methodologies proved again to be powerful tools to increase the rate of success for HNE inhibitor discovery either for therapeutic or activity-based probing development.

The Ubiquitin Proteasome Pathway (UPP) plays a pivotal role in intracellular protein degradation and turnover in eukaryotic cells.1 Therefore, modulation of the UPP emerged as a rational therapeutic approach in cancer, neurodegenerative diseases (Alzheimer, Parkinson), inflammatory pathologies (arthritis, psoriasis, asthma, colitis), organ transplant, infective diseases (malaria), among others.2 During the last two decades academia and pharmaceutical industry made huge efforts to develop natural and synthetic proteasome inhibitors (PI). In 2003 FDA approved the pioneering dipeptidyl boronic acid derivative PI bortezomib for the treatment of refractory multiple myeloma (MM) and subsequently frontline therapy for MM. However, despite the enormous potential of PI, their use is still limited to certain types of blood cancer and shows severe side effects, dose limiting toxicity, peripheral neuropathy, limited activity in solid tumour and innate or acquired drug resistance.3 In this work, we have used Molecular Dynamics (MD) simulations to perform the first conformational and structural characterization of the human native 20S proteasome structure4. We focused our analysis on the three catalytic subunits well known for their proteolytic activity (b1, b2 and b5) and we further extended our study to additional MD simulations of three different point mutations in the b5 catalytic subunit, with recognized importance in PI’s resistance: Ala49Thr, Ala50Val and Cys52Phe. Hopefully, our studies will be able to shed the light on the structural key determinants that regulate the observed PI’s resistance in the different mutations, and ultimately use the acquired knowledge in the development of new alternative and efficient proteasome inhibitors. Acknowledgements: We thank the Fundacao para a Ciencia e a Tecnologia for financial support PTDC/QEQ-MED/7042/2014, UID/DTP/04138/2013 and SAICTPAC/0019/2015.

Background: Gait parameters such as gait speed (GS) are important indicators of functional capacity. Frailty Syndrome is closely related to GS and is also capable of predicting adverse outcomes. The cognitive demand of gait control is usually explored with dual-task (DT) methodology. Objective: To investigate the effect of DT and frailty on the spatio-temporal parameters of gait in older people and identify which variables relate to GS. Method: The presence of frailty was verified by Fried's Frailty Criteria. Cognitive function was evaluated with the Mini-Mental State Exam (MMSE) and gait parameters were analyzed through the GAITRite(r) system in the single-task and DT conditions. The Kolmogorov-Smirnov, ANOVA, and Pearson's Correlation tests were administered. Results: The participants were assigned to the groups frail (FG), pre-frail (PFG), and non-frail (NFG). During the DT, the three groups showed a decrease in GS, cadence, and stride length and an increase in stride time (p

Thirteen pyrrolidine-based iminosugar derivatives have been synthesized and evaluated for inhibition of α-glucosidase from rat intestine. The compounds studied were the non-hydroxy, mono-hydroxy and dihydroxypyrrolidines. All the compounds were N -benzylated apart from one. Four of the compounds had a carbonyl group in the 2,5-position of the pyrrolidine ring. The most promising iminosugar was the trans -3,4-dihydroxypyrrolidine 5 giving an IC 50 of 2.97 ± 0.046 and a K I of 1.18 mM. Kinetic studies showed that the inhibition was of the mixed type, but predominantly competitive for all the compounds tested. Toxicological assay results showed that the compounds have low toxicity. Docking studies showed that all the compounds occupy the same region as the DNJ inhibitor on the enzyme binding site with the most active compounds establishing similar interactions with key residues. Our studies suggest that a rotation of ∼90° of some compounds inside the binding pocket is responsible for the complete loss of inhibitory activity. Despite the fact that activity was found only in the mM range, these compounds have served as simple molecular tools for probing the structural features of the enzyme, so that inhibition can be improved in further studies.

Herein we report the discovery of new modulators of human phenylalanine hydroxylase (hPAH) inspired by the structure of its substrate and regulator L-phenylalanine. These new hPAH modulators were simply prepared in good-to-excellent yields and excellent diastereoselectivities, based on a boron promoted assembly of L-phenylalanine, salicylaldehyde and aryl boronic acids. Iminoboronate 8, prepared with L-phenylalanine, para-methoxy-salicylaldehyde and phenyl boronic acid, was identified as the most efficient hPAH modulator, with an apparent binding affinity nearly identical to the natural allosteric activator L-phenylalanine.

Introduction: The acquired immunodeficiency syndrome (AIDS) is the culmination of the infection by the human immunodeficiency viruses upon destruction of CD4+ lymphocytes of the host [1]. The efficacy of the available antiretroviral drugs is very limited against HIV-2 and, most importantly, none of the current drugs effectively prevents entry into the cells. HIV envelope glycoproteins mediate binding to the receptor CD4 and co-receptors at the surface of the target cell, enabling fusion with the cell membrane and viral entry [2,3]. The discovery of multiple new hit compounds that can be used as useful starting points towards drug candidates for HIV-1 and HIV-2 therapy is the main goal of this work. The glycoproteins gp120 and gp125 are critical to the receptors recognition and internalization of viral material into the cell. The study aimed to assess if the modulation of the glycoproteins activity can lead to the disturbance of the entry viral mechanism. Materials and methods: Until now, it has been marked by the use of computational techniques to study the viral surface glycoproteins as potential drug targets against HIV-1 and HIV-2 infections. A 3D structure of HIV-2ROD gp125 was generated by homology modelling, using MOE2016 and MODELLER 9v19. Additionally, to disclose the importance of the main structural features and compare with experimental results, 3D-models of six V3 mutants were also generated using the C2V3C3 domain. Additionally, molecular dynamics is being performed, using Gromacs 2006.3, in order to better characterize this protein and disclose its the biological dynamic behaviour. The structures to use in molecular dynamic simulations were prepared using MOE 2016.0802 software and further exported as PDB files. Results: It is primordial to understand the structural behavior of these domains inserted in the main structure. These mutations revealed selectively impact in the behaviour of the protein. Structurally, the mutations studied leads to a loss of aromatic features, very important for the establishment of π-π interactions, which could induce a structural preference by a specific coreceptor. Discussion and conclusions: In the absence of a crystallographic structure of HIV-2 envelope gp125 comprising variable domains, computer aided modulation is crucial to identify structural features in the variable regions that correlate with HIV-2 tropism and susceptibility to neutralization, highly associated with the mutated residues. These new insights into the structure-function relationship will help in the design of better models and into the following of the design of new small molecules capable of use under the proposal presented inhibit the attachment and binding of HIV with host cells mediated by the envelope surface glycoproteins.

Increased resistance of Plasmodium falciparum to most available drugs challenges the control of malaria. Studies with protease inhibitors have suggested important roles for the falcipain family of cysteine proteases. These enzymes act in concert with other proteases to hydrolyze host erythrocyte hemoglobin in the parasite food vacuole. In order to find potential new antimalarial drugs, we screened in silico the ZINC database using two different protocols involving structure- and ligand-based methodologies. Our search identified 19 novel low micromolar inhibitors of cultured chloroquine resistant P. falciparum. The most active compound presented an IC50 value of 0.5 μM against cultured parasites and it also inhibited the cysteine protease falcipain-2 (IC50 = 25.5 μM). These results identify novel classes of antimalarials that are structurally different from those currently in use and which can be further derivatized to deliver leads suitable for optimisation.

© 2011 American Association of Pharmaceutical Scientists, The aim of this work is to prepare tretinoin/dimethyl-beta-cyclodextrin complexes and fully characterize them through various analytical techniques. According to the phase solubility studies performed, the equilibrium for maximum complexation is reached in about 8 days presenting an AL-type diagram (soluble complexes) corresponding mainly to 1:1 stoichiometry (Ks=13,600 M−1), although the possibility of the presence of 1:2 complexes was mathematically proven. Differential scanning calorimetry, X-ray diffraction and all the other analytical techniques have proven the presence of true complex formation in all the preparation methods tested. H-NMR and FTIR spectra allowed the selection of the best complexation method. The comparison between Raman spectra revealed that the more relevant feature is the band at 1,573 cm−1, which corresponds to the entire delocalization of the superconjugated system, and after inclusion is observed as a positive frequency shift. Based on these results and the data obtained by molecular modelling calculations, it is proposed that the structure of the drug included into the cyclodextrin corresponds to the side chain including the functional group COOH. The complex was also analysed by atomic force microscopy to determine its size distribution which was heterogeneous and polymodal. However, it could be observed that they all have the same phase constitution.

OBJECTIVES: To compare gait and functional performance parameters in elderly subjects who had or had not total hip arthroplasty (THA). METHODS: Our sample comprised 23 elderly subjects (72±6.5 years of age) with a mean of 2.6±1.3 years following THA, named the arthroplasty group (AG) and 23 asymptomatic elderly subjects (70.1±5.9 years of age), named the control group (CG). Case and control subjects were paired by gender, age, body mass index and level of physical activity. The GAITRite® system was used to analyze various gait parameters in four situations: usual speed (US), fast speed (FS), slow speed (SS) and dual task (DT); the Dynamic Gait Index (DGI) and Timed Up and Go test (TUG) methods were used to evaluate functional capacity. The statistical analyses were carried out using the Shapiro-Wilk test, Student's-t-tests for independent samples, chi-square tests, ANOVA for repeated measurements and paired Student's-t-tests. RESULTS: The AG had the worst results for gait speed (AG=1.18±0.13 and CG=1.39±0.09; p=0.012), symmetry index (SI) of step length (AG=3.60±1.01 and CG=1.12±0.59; p=0.000), SI of step time (AG=-2.65±0.92 and CG=0.99±0.74; p=0.000), SI of stance phase (AG=-2.55 and CG=-1.04±0.50; p=0.005), SI of single support phase (AG=-2.17±0.78 and CG=1.21±0.51; p=0.003), DGI (AG=20.04±1.91 and CG=21.69±1.45; p=0.001) and TUG (AG=14.67±1.94 and CG=10.08±1.49; p=0.001). CONCLUSION: Elderly subjects with a history of THA had changes in gait parameters and lower performance in TUG test even 2.6±1,3 years after surgery, which suggests functional impairment.OBJETIVOS: Comparar os parâmetros da marcha e o desempenho funcional de idosos com e sem artroplastia total de quadril (ATQ). MÉTODOS: Foram selecionados 23 idosos (72±6,5 anos) após média de 2,6±1,3 anos de ATQ e 23 idosos assintomáticos (70,1±5,9 anos), pareados por gênero, idade, índice de massa corpórea (IMC) e nível de atividade física. Utilizou-se o sistema GAITRite® em quatro situações distintas: velocidades habitual (VH), rápida (VR), lenta (VL) e tarefa dupla (TD). A capacidade funcional foi avaliada pelo Dynamic Gait Index (DGI) e Timed Up and Go (TUG). Na análise estatística, utilizaram-se os testes Shapiro-Wilk, t-Student para amostras independentes, Qui-quadrado, ANOVA com medidas repetidas e t-Student pareado. RESULTADOS: O grupo artroplastia (GA) apresentou piores resultados estatisticamente significantes no que se refere à velocidade de marcha (VM) (GA=1,18±0,13 e GC=1,39±0,09; p=0,012), índice de simetria (IS) do comprimento do passo (GA=3,60±1,01 e GC=1,12±0,59; p=0,000), IS do tempo do passo (GA=-2,65±0,92 e GC=0,99±0,74; p=0,000), IS da duração da fase de apoio (GA=-2,55 e GC=-1,04±0,50; p=0,005), IS da duração da subfase de apoio único (GA=-2,17±0,78 e GC=1,21±0,51; p=0,003), DGI (GA=20,04±1,91 e GC=21,69±1,45; p=0,001) e TUG (GA=14,67±1,94 e GC=10,08±1,49; p=0,001). CONCLUSÃO: Idosos com ATQ apresentaram alterações nos parâmetros da marcha, mesmo após 2,6±1,3 anos de cirurgia, e pior desempenho no teste TUG, indicando comprometimento funcional.

Phototoxic and radical-generating debromination reactions of monobrominated hypericin with bromine at one of four possible positions were investigated using density functional theory. The study was performed on two closely lying conformational isomers, differing in the relative orientations of the two anthracene units of the hypericin core. Calculated adiabatic electron affinities show that the molecules have the ability to, in aqueous solution, extract an electron from the surrounding. The electron might then be passed on to molecular oxygen, forming reactive superoxide radical anions. If electron extraction from the molecule does not occur in this step, the molecule might dissociate, generating a negatively charged bromine as a leaving group and a hypericin radical capable of forming direct binding to biological molecules. This reaction was found possible for those species substituted by Br at two of the four positions, with barriers of similar to 13 kcal/mol in aqueous solution. Debromination was not found energetically possible for neither the neutral ground state compounds nor the bay-deprotonated species. (c) 2008 Wiley Periodicals, Inc.

Carbamate and ester derivatives of the 1,1-dioxobenzo[b]thiophen-2-ylmethyloxycarbonyl (Bsmoc) scaffold react readily with thiols via a Michael addition at rates not significantly affected by the nature of the carboxylic or carbamic acid leaving group. These Michael acceptors are irreversible inhibitors of the cysteine proteases papain and human liver cathepsin B, displaying first-order kinetics with respect to inhibitor concentration. In contrast, none of the Bsmoc derivatives inhibited porcine pancreatic elastase, a serine protease. (C) 2006 Elsevier Ltd. All rights reserved.

Hypericin (hyp) displays photodynamic activity under the influence of visible light, which has opened up new possibilities of utilizing hypericin derivatives as potential photodynamic therapy agents. In the current work, the effects of halogen substitution were considered through a detailed computational study of bromine, chlorine, and fluorine substitution on the geometrical structure, and electronic and phototoxic properties of the hypericin molecule by the use of density functional theory, integral equation formalism polarized continuum models, and a time-dependent formalism (TD-DFT) to the study of singlet and triplet excited states. It is concluded that the effect of halogen substitution is primarily an increase of the electron affinities and thereby increased quantum efficiency of superoxide formation of importance in photodynamic therapy.