Mercury ions impact the kinetic and thermal stabilities of human lens γ-crystallins via direct metal-protein interactions.

Loss of metal homeostasis may be involved in several age-related diseases, such as cataracts. Cataracts are caused by the aggregation of lens proteins into light-scattering high molecular weight complexes that impair vision. Environmental exposure to heavy metals, such as mercury, is a risk factor for cataract development. Indeed, mercury ions induce the non-amyloid aggregation of human γC- and γS crystallins, while human γD-crystallin is not sensitive to this metal. Using Differential Scanning Calorimetry (DSC), we evaluate the impact of mercury ions on the kinetic stability of the three most abundant human γ-crystallins. The metal/crystallin interactions were characterized using Isothermal Titration Calorimetry (ITC). Human γD-crystallins exhibited kinetic stabilization due to the presence of mercury ions, despite its thermal stability being decreased. In contrast, human γC- and γS-crystallins are both, thermally and kinetically destabilized by this metal, consistent with their sensitivity to mercury-induced aggregation. The interaction of human γ-crystallins with mercury ions is highly exothermic and complex, since the protein interacts with the metal at more than three sites. The isolated domains of human γ-D and its variant with the H22Q mutation were also studied, revealing the importance of these regions in the mercury-induced stabilization by a direct metal-protein interaction.

PEDOT-Polyamine-Based Organic Electrochemical Transistors for Monitoring Protein Binding.

The fabrication of efficient organic electrochemical transistors (OECTs)-based biosensors requires the design of biocompatible interfaces for the immobilization of biorecognition elements, as well as the development of robust channel materials to enable the transduction of the biochemical event into a reliable electrical signal. In this work, PEDOT-polyamine blends are shown as versatile organic films that can act as both highly conducting channels of the transistors and non-denaturing platforms for the construction of the biomolecular architectures that operate as sensing surfaces. To achieve this goal, we synthesized and characterized films of PEDOT and polyallylamine hydrochloride (PAH) and employed them as conducting channels in the construction of OECTs. Next, we studied the response of the obtained devices to protein adsorption, using glucose oxidase (GOx) as a model system, through two different strategies: The direct electrostatic adsorption of GOx on the PEDOT-PAH film and the specific recognition of the protein by a lectin attached to the surface. Firstly, we used surface plasmon resonance to monitor the adsorption of the proteins and the stability of the assemblies on PEDOT-PAH films. Then, we monitored the same processes with the OECT showing the capability of the device to perform the detection of the protein binding process in real time. In addition, the sensing mechanisms enabling the monitoring of the adsorption process with the OECTs for the two strategies are discussed.

Human variation in the protein receptor ACE2 affects its binding affinity to SARS-CoV-2 in a variant-dependent manner.

SARS-CoV-2 infection depend on the binding of the viral Spike glycoprotein (S) to the human receptor Angiotensin Converting Enzyme 2 (ACE2) to induce virus-cell membrane fusion. S protein evolved diverse amino acid changes that are possibly linked to more efficient binding to human ACE2, which might explain part of the increase in frequency of SARS-CoV-2 Variants Of Concern (VOCs). In this work, we investigated the role of ACE2 protein variations that are naturally found in human populations and its binding affinity with S protein from SARS-CoV-2 representative genotypes, based on a series of in silico approaches involving molecular modelling, docking and molecular dynamics simulations. Our results indicate that SARS-CoV-2 VOCs bind more efficiently to the human receptor ACE2 than the ancestral Wuhan genotype. Additionally, variations in the ACE2 protein can affect SARS-CoV-2 binding and protein-protein stability, mostly making the interaction weaker and unstable in some cases. We show that some VOCs, such as B.1.1.7 and P.1 are much less sensitive to ACE2 variants, while others like B.1.351 appear to be specifically optimized to bind to the widespread wild-type ACE2 protein.Communicated by Ramaswamy H. Sarma.

Identification of VEGFR2 as the Histatin-1 receptor in endothelial cells.

Histatin-1 is a salivary peptide with antimicrobial and wound healing promoting activities, which was previously shown to stimulate angiogenesis in vitro and in vivo via inducing endothelial cell migration. The mechanisms underlying the proangiogenic effects of Histatin-1 remain poorly understood and specifically, the endothelial receptor for this peptide, is unknown. Based on the similarities between Histatin-1-dependent responses and those induced by the prototypical angiogenic receptor, vascular endothelial growth factor receptor 2 (VEGFR2), we hypothesized that VEGFR2 is the Histatin-1 receptor in endothelial cells. First, we observed that VEGFR2 is necessary for Histatin-1-induced endothelial cell migration, as shown by both pharmacological inhibition studies and siRNA-mediated ablation of VEGFR2. Moreover, Histatin-1 co-immunoprecipitated and co-localized with VEGFR2, associating spatial proximity between these proteins with receptor activation. Indeed, pulldown assays with pure, tagged and non-tagged proteins showed that Histatin-1 and VEGFR2 directly interact in vitro. Optical tweezers experiments permitted estimating kinetic parameters and rupture forces, indicating that the Histatin-1-VEGFR2 interaction is transient, but specific and direct. Sequence alignment and molecular modeling identified residues Phe26, Tyr30 and Tyr34 within the C-terminal domain of Histatin-1 as relevant for VEGFR2 binding and activation. This was corroborated by mutation and molecular dynamics analyses, as well as in direct binding assays. Importantly, these residues were required for Histatin-1 to induce endothelial cell migration and angiogenesis in vitro. Taken together, our findings reveal that VEGFR2 is the endothelial cell receptor of Histatin-1 and provide insights to the mechanism by which this peptide promotes endothelial cell migration and angiogenesis.

New Insights into Hemopexin-Binding to Hemin and Hemoglobin.

Hemopexin (Hx) is a plasma glycoprotein that scavenges heme (Fe(III) protoporphyrin IX). Hx has important implications in hemolytic disorders and hemorrhagic conditions because releasing hemoglobin increases the labile heme, which is potentially toxic, thus producing oxidative stress. Therefore, Hx has been considered for therapeutic use and diagnostics. In this work, we analyzed and mapped the interaction sequences of Hx with hemin and hemoglobin. The spot-synthesis technique was used to map human hemopexin (P02790) binding to hemin and human hemoglobin. A library of 15 amino acid peptides with a 10-amino acid overlap was designed to represent the entire coding region (aa 1-462) of hemopexin and synthesized onto cellulose membranes. An in silico approach was taken to analyze the amino acid frequency in the identified interaction regions, and molecular docking was applied to assess the protein-protein interaction. Seven linear peptide sequences in Hx were identified to bind hemin (H1-H7), and five were described for Hb (Hb1-Hb5) interaction, with just two sequences shared between hemin and Hb. The amino acid composition of the identified sequences demonstrated that histidine residues are relevant for heme binding. H105, H293, H373, H400, H429, and H462 were distributed in the H1-H7 peptide sequences, but other residues may also play an important role. Molecular docking analysis demonstrated Hx's association with the ß-chain of Hb, with several hotspot amino acids that coordinated the interaction. This study provides new insights into Hx-hemin binding motifs and protein-protein interactions with Hb. The identified binding sequences and specific peptides can be used for therapeutic purposes and diagnostics as hemopexin is under investigation to treat different diseases and there is an urgent need for diagnostics using labile heme when monitoring hemolysis.

Crosslinked zwitterionic polymeric ionic liquid-functionalized nitinol wires for fiber-in-tube solid-phase microextraction and UHPLC-MS/MS as an amyloid beta peptide binding protein assay in biological fluids.

Alzheimer disease (AD) is a neurodegenerative disorder characterized by extracellular accumulation of amyloid-ß peptide (Aß) in the brain interstitium. Human serum albumin (HSA) highly binds to Aß in blood plasma and is thought to inhibit plaque formation in peripheral tissue. Thus, the evaluation of albumin binding to Aß is an important key to understand the dynamics of these molecules in the biological system of patients with AD. In this work, a fiber-in-tube solid-phase microextraction (fiber-in-tube SPME) and ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed to estimate Aß fraction binding to HSA in cerebrospinal fluid (CSF) and plasma samples. Crosslinked zwitterionic polymeric ionic liquid (zwitterionic PIL)-coated nitinol wires were developed and packed into a polyether ether ketone (PEEK) capillary for a fiber-in-tube SPME and UHPLC-MS/MS method. Zwitterionic PIL sorbent was synthetized from 1-vinyl-3-(butanesulfonate)imidazolium ([VIm+C4SO3-]) and 1,12-di(3-vinylimidazolium)dodecane dibromide ([(VIm)2C12]2[Br]) monomers by in-situ thermally-initiated polymerization. Morphological characterization by scanning electron microscopy (SEM) and atomic force microscopy (AFM) revealed a decrease in the surface roughness of the nitinol wires from ∼17 nm to 1 nm after the in-situ polymerization. The zwitterionic PIL sorbent selectively preconcentrates Aß through a two-pronged interaction mechanism. The fiber-in-tube SPME and UHPLC-MS/MS method presented lower limits of quantification (LLOQ) of 0.4 ng mL-1 for Aß38 and 0.3 ng mL-1 for Aß40 and Aß42, a linear range from LLOQ values to 15 ng mL-1 with coefficients of determination higher than 0.99, precision with coefficient of variation (CV) values ranging from 2.1 to 7.3% and accuracy with relative standard deviation (RSD) values from -0.3 to 7.4. This method was successfully applied to evaluate the binding of HSA to Aß in cerebrospinal fluid (CSF) and plasma samples.

SARS-CoV-2 Proteins Bind to Hemoglobin and Its Metabolites.

(1) Background: coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been linked to hematological dysfunctions, but there are little experimental data that explain this. Spike (S) and Nucleoprotein (N) proteins have been putatively associated with these dysfunctions. In this work, we analyzed the recruitment of hemoglobin (Hb) and other metabolites (hemin and protoporphyrin IX-PpIX) by SARS-Cov2 proteins using different approaches. (2) Methods: shotgun proteomics (LC-MS/MS) after affinity column adsorption identified hemin-binding SARS-CoV-2 proteins. The parallel synthesis of the peptides technique was used to study the interaction of the receptor bind domain (RBD) and N-terminal domain (NTD) of the S protein with Hb and in silico analysis to identify the binding motifs of the N protein. The plaque assay was used to investigate the inhibitory effect of Hb and the metabolites hemin and PpIX on virus adsorption and replication in Vero cells. (3) Results: the proteomic analysis by LC-MS/MS identified the S, N, M, Nsp3, and Nsp7 as putative hemin-binding proteins. Six short sequences in the RBD and 11 in the NTD of the spike were identified by microarray of peptides to interact with Hb and tree motifs in the N protein by in silico analysis to bind with heme. An inhibitory effect in vitro of Hb, hemin, and PpIX at different levels was observed. Strikingly, free Hb at 1mM suppressed viral replication (99%), and its interaction with SARS-CoV-2 was localized into the RBD region of the spike protein. (4) Conclusions: in this study, we identified that (at least) five proteins (S, N, M, Nsp3, and Nsp7) of SARS-CoV-2 recruit Hb/metabolites. The motifs of the RDB of SARS-CoV-2 spike, which binds Hb, and the sites of the heme bind-N protein were disclosed. In addition, these compounds and PpIX block the virus's adsorption and replication. Furthermore, we also identified heme-binding motifs and interaction with hemin in N protein and other structural (S and M) and non-structural (Nsp3 and Nsp7) proteins.

Release, transfer and partition of fluorescent dyes from polymeric nanocarriers to serum proteins monitored by asymmetric flow field-flow fractionation.

Fluorescent probes are used in drug nanocarrier pre-clinical studies or as active compounds in theranostics and photodynamic therapy. In the biological medium, nanoparticles interact with proteins, which can result in the off-target release of their cargo. The present study used asymmetric flow field-flow fractionation with online multi-angle laser light scattering and fluorescence detection (AF4-MALLS-FLD) to study the release, transfer, and partition of fluorescent dyes from polymeric nanoparticles (NP). NP formulations containing the dyes Rose Bengal, Rhodamine B, DiI, 3-(α-azidoacetyl)coumarin and its polymer conjugate, Nile Red, and IR780 and its polymer conjugate were prepared. NP suspensions were incubated in a medium with serum proteins and then analyzed by AF4. AF4 allowed efficient separation of proteins (< 10 nm) from fluorescently labeled NP (range of 54 - 180 nm in diameters). The AF4 analyses showed that some dyes, such as Rose Bengal, IR780, and Coumarin were transferred to a high extent (68-77%) from NP to proteins. By contrast, for DiI and dye-polymer conjugates, transfer occured to a lower extent. The studies of dye release kinetics showed that the transfer of IR780 from NP to proteins occurs at a high extent (~50%) and rate, while Nile Red was slowly released from the NP over time with reduced association with proteins (~20%). This experiment assesses the stability of fluorescence labeling of nanocarriers and probes the transfer of fluorescent dyes from NP to proteins, which is otherwise not accessible with commonly used techniques of separation, such as dialysis and ultrafiltration/centrifugation employed in drug encapsulation and release studies of nanocarriers. Determining the interaction and transfer of dyes to proteins is of utmost importance in the pre-clinical evaluation of drug nanocarriers for improved correlation between in vitro and in vivo studies.

Helical water-soluble NiII complexes with pyridoxal ligand derivatives: Structural evaluation and interaction with biomacromolecules.

This article deals with the synthesis of Schiff-based bis-azomethine-based ligands derived from pyridoxal and aliphatic dihydrazides and the synthesis of nickel(II) complexes C1-C4. The synthesized complexes had their structures elucidated by monocrystal X-ray diffraction and were characterized by vibrational and absorption spectroscopy. The synthesized ligands have characteristics that allow the formation of self-assembly processes, thus, the flexibility or rigidity of the coordination of organic molecules added to the orbitals of the NiII cation leads to the formation of helical complexes with double helix and a dinucler nickel(II) complex. Moreover, compounds was their interactions with CT-DNA and HSA absorption and emission analysis and molecular docking calculations.

The structure of unliganded sterol carrier protein 2 from Yarrowia lipolytica unveils a mechanism for binding site occlusion.

Isolated or as a part of multidomain proteins, Sterol Carrier Protein 2 (SCP2) exhibits high affinity and broad specificity for different lipidic and hydrophobic compounds. A wealth of structural information on SCP2 domains in all forms of life is currently available; however, many aspects of its ligand binding activity are poorly understood. ylSCP2 is a well-characterized single domain SCP2 from the yeast Yarrowia lipolytica. Herein, we report the X-ray structure of unliganded ylSCP2 refined to 2.0 Å resolution. Comparison with the previously solved liganded ylSCP2 structure unveiled a novel mechanism for binding site occlusion. The liganded ylSCP2 binding site is a large cavity with a volume of more than 800 Å3. In unliganded ylSCP2 the binding site is reduced to about 140 Å3. The obliteration is caused by a swing movement of the C-terminal α helix 5 and a subtle compaction of helices 2-4. Previous pairwise comparisons were between homologous SCP2 domains with a uncertain binding status. The reported unliganded ylSCP2 structure allows for the first time a fully controlled comparative analysis of the conformational effects of ligand occupation dispelling several doubts regarding the architecture of SCP2 binding site.

Identification of key genes for type 1 diabetes mellitus by network-based guilt by association

SUMMARY OBJECTIVE This study aimed to propose a co-expression-network (CEN) based gene functional inference by extending the "Guilt by Association" (GBA) principle to predict candidate gene functions for type 1 diabetes mellitus (T1DM). METHODS Firstly, transcriptome data of T1DM were retrieved from the genomics data repository for differentially expressed gene (DEGs) analysis, and a weighted differential CEN was generated. The area under the receiver operating characteristics curve (AUC) was chosen to determine the performance metric for each Gene Ontology (GO) term. Differential expression analysis identified 325 DEGs in T1DM, and co-expression analysis generated a differential CEN of edge weight > 0.8. RESULTS A total of 282 GO annotations with DEGs > 20 remained for functional inference. By calculating the multifunctionality score of genes, gene function inference was performed to identify the optimal gene functions for T1DM based on the optimal ranking gene list. Considering an AUC > 0.7, six optimal gene functions for T1DM were identified, such as regulation of immune system process and receptor activity. CONCLUSIONS CEN-based gene functional inference by extending the GBA principle predicted 6 optimal gene functions for T1DM. The results may be potential paths for therapeutic or preventive treatments of T1DM.

RESUMO OBJETIVO O objetivo deste estudo é realizar uma inferência funcional genética baseada na rede de coexpressão (CEN), expandindo o escopo do princípio de "Culpa por Associação" (GBA - Guilt by Association) para prever as funções genéticas do diabetes mellitus tipo 1 (T1DM). MÉTODOS Primeiro, os dados transcritos do T1DM foram recuperados do repositório de dados genômicos para a análise dos genes diferenciais (DEGs), e foi gerada uma CEN diferencial ponderada. A área sob a curva ROC (AUC) foi escolhida para determinar a métrica de desempenho para cada termo de Ontologia Genética (GO). A análise da expressão diferencial identificou 325 DEGs no T1DM, e a análise de coexpressão gerou uma CEN diferencial com aresta de peso >0,8. RESULTADOS Um total de 282 anotações de GO com DEGs >20 foram mantidas para inferência funcional. Ao calcular a pontuação de multifuncionalidade dos genes, a inferência da função genética foi realizada para identificar as funções genéticas ideais para T1DM com base na lista de classificação genética ideal. Considerando um valor de AUC >0,7, foram identificadas seis funções genéticas ideais para a T1DM, tais como a regulação do processo imunológico e da atividade dos receptores. CONCLUSÕES A inferência funcional genética baseada em CEN, ao expandir o princípio de GBA, previu seis funções genéticas ideais para o T1DM. Os resultados podem ser caminhos potenciais para tratamentos terapêuticos ou preventivos do T1DM.

Hydroethanolic extract from Endopleura uchi (Huber) Cuatrecasas and its marker bergenin: Toxicological and pharmacokinetic studies in silico and in vivo on zebrafish.

Endopleura uchi, is used for the treatment of inflammatory disease and related to the female reproductive tract. The aim of this study was to evaluate the acute toxicity of the Endopleura uchi stem bark hydroethanolic extract (EEu) in zebrafish, emphasizing the histopathological and biochemical parameters, as well as evaluating the in silico pharmacokinetic and toxicological parameters of the phytochemical/pharmacological marker, bergenin, as their metabolites. The animals were orally treated with EEu at a single dose of 75 mg/kg, 500 mg/kg, 1000 mg/kg and 3000 mg/kg. the oral LD50 of the EEu higher to the dose of 3000 mg/kg. Behavioral, biochemical and histopathological changes were dose dependent. In silico pharmacokinetic predictions for bergenin and its metabolites showed moderate absorption in high human intestinal absorption (HIA) and Caco-2 models, reduced plasma protein binding, by low brain tissue binding and no P-glycoprotein (P-Gp) inhibition. Their metabolism is defined by the CYP450 enzyme, in addition to bergenin inhibition of CYP2C9, CYP3A4 and CYP2C19. In the bergenin and its metabolites in silico toxicity test it have been shown to cause carcinogenicity and a greater involvement of the bergenin with the CYP enzymes in the I and II hepatic and renal metabolism's phases was observed. It is possible to suggest that the histopathological damages are involved with the interaction of this major compound and its metabolites at the level of the cellular-biochemical mechanisms which involve the absorption, metabolization and excretion of these possible prodrug and drug.

Interaction with Blood Proteins of a Ruthenium(II) Nitrofuryl Semicarbazone Complex: Effect on the Antitumoral Activity.

The steady rise in the cancer burden and grim statistics set a vital need for new therapeutic solutions. Given their high efficiency, metallodrugs are quite appealing in cancer chemotherapy. This work examined the anticancer activity of an anti-trypanosomal ruthenium-based compound bearing the 5-nitrofuryl pharmacophore, [RuII(dmso)2(5-nitro-2-furaldehyde semicarbazone)] (abbreviated as RuNTF; dmso is the dimethyl sulfoxide ligand). The cytotoxicity of RuNTF was evaluated in vitro against ovarian adenocarcinoma, hormone-dependent breast adenocarcinoma, prostate carcinoma (grade IV) and V79 lung fibroblasts human cells. The activity of RuNTF was similar to the benchmark metallodrug cisplatin for the breast line and inactive against the prostate line and lung fibroblasts. Given the known role of serum protein binding in drug bioavailability and the distribution via blood plasma, this study assessed the interaction of RuNTF with human serum albumin (HSA) by circular dichroism (CD) and fluorescence spectroscopy. The fluorescence emission quenching from the HSA-Trp214 residue and the lifetime data upon RuNTF binding evidenced the formation of a 1:1 {RuNTF-albumin} adduct with log Ksv = (4.58 ± 0.01) and log KB = (4.55 ± 0.01). This is supported by CD data with an induced CD broad band observed at ~450 nm even after short incubation times. Importantly, the binding to either HSA or human apo-transferrin is beneficial to the cytotoxicity of the complex towards human cancer cells by enhancing the cytotoxic activity of RuNTF.

Label-free evaluation of small-molecule-protein interaction using magnetic capture and electrochemical detection.

The evaluation of interaction between small molecules and protein is an important step in the discovery of new drugs and to study complex biological systems. In this work, an alternative method was presented to evaluate small-molecule-protein interaction by using ligand capture by protein-coated magnetic particles (MPs) and disposable electrochemical cells. The interaction study was conducted using [10]-gingerol from ginger rhizome and a transmembrane protein αVß3 integrin. Initially, the electrochemical behavior of the natural compound [10]-gingerol was evaluated with the disposable carbon-based electrodes and presented an irreversible oxidation process controlled by diffusion. The analytical curve for [10]-gingerol was obtained in the range of 1.0 to 20.0 µmol L-1, with limit of detection of 0.26 µmol L-1. Then MPs coated with αVß3 integrin were incubated with standard solutions and extracts of ginger rhizome for [10]-gingerol capture and separation. The bioconjugate obtained was dropped to the disposable electrochemical cells, keeping a permanent magnet behind the working electrode, and the binding process was evaluated by the electrochemical detection of [10]-gingerol. The assay method proposed was also employed to calculate the [10]-gingerol-αVß3 integrin association constant, which was calculated as 4.3 × 107 M-1. The method proposed proved to be a good label-free alternative to ligand-protein interaction studies. Graphical abstract ᅟ.

Pharmacokinetics and tissue distribution of N-(2-hydroxyphenyl)-2-propylpentanamide in Wistar Rats and its binding properties to human serum albumin.

N-(2-hydroxyphenyl)-2-propylpentanamide (HO-AAVPA) is a novel valproic acid derivative that has shown anti-proliferative activity against epitheloid cervix carcinoma (HeLa), rhabdomyosarcoma (A204), and several breast cancer cell lines. The aim of this research was to evaluate the pharmacokinetic profile and tissue distribution of HO-AAVPA in Wistar rats, as well as its human serum albumin binding potential by experimental and in silico methods. A single dose of HO-AAVPA was given to male rats by intravenous, intragastric or intraperitoneal routes at doses of 25, 100, and 100 mg/kg, respectively. Then, blood samples were drawn at predetermined intervals of time, and the HO-AAVPA concentration in the plasma was quantified with a validated HPLC method. The elimination half-life (t1/2) was approximately 222 min, and the systemic clearance (CL) and apparent volume of distribution (Vd) were 2.20 mL/min/kg and 0.70 L/kg, respectively. The absolute oral bioavailability of HO-AAVPA was 33.8%, and the binding rate of HO-AAVPA with rat plasma proteins was between 66.2% and 83.0%. Additionally, in silico, UV and Raman spectroscopy data showed weak interactions between the test compound and human serum albumin. Thus, the results that were obtained demonstrated that despite its low oral bioavailability, the potential anticancer agent HO-AAVPA exhibits acceptable pharmacokinetic properties that would allow it to reach its site of action and exert its pharmacological effect in Wistar Rats, and it has a convenient profile for future assays to evaluate its human applications.

Binding properties of sterol carrier protein 2 (SCP2) characterized using Laurdan.

Sterol carrier protein 2 (SCP2) binds lipids with high affinity and broad specificity. The overall hydrophobicity, fluidity, and dipolar dynamics of the binding site of SCP2 from Yarrowia lipolytica were characterized using the environmentally-sensitive fluorescent probe Laurdan. The study revealed a binding site with an overall polarity similar to that of dichloromethane and an internal phase comparable to that of phospholipid membranes with coexisting solid-ordered and liquid-crystalline states. The fluorescence properties of bound Laurdan also revealed that the binding site of SCP2 can accommodate competitively more than one ligand, with micro and nanomolar dissociation constants. The much higher affinity for the second than for the first ligand implies that the most prominent SCP2 species in the cellular context are those occupied by two ligands. Thus SCP2 may carry a highly populated lipid in the background and a second one, specific for the functional purpose of SCP2. Our findings are important for the characterization of SCP2 biological functions and the design of specific inhibitors.

Neurospora crassa developmental control mediated by the FLB-3 transcription factor.

Here, we report that the Neurospora crassa FLB-3 protein, the ortholog of the Aspergillus nidulans FlbC transcription factor, is required for developmental control. Deletion of flb-3 leads to changes in hyphae morphology and affects sexual and asexual development. We identified, as putative FLB-3 targets, the N. crassa aba-1, wet-1 and vos-1 genes, orthologs of the ones involved in A. nidulans asexual development and that work downstream of FlbC (abaA, wetA and vosA). In N. crassa, these three genes require FLB-3 for proper expression; however, they appear not to be required for normal development, as demonstrated by gene expression analyses during vegetative growth and asexual development. Moreover, mutant strains in the three genes conidiate well and produce viable conidia. We also determined FLB-3 DNA-binding preferences via protein-binding microarrays (PBMs) and demonstrated by chromatin immunoprecipitation (ChIP) that FLB-3 binds the aba-1, wet-1 and vos-1 promoters. Our data support an important role for FLB-3 in N. crassa development and highlight differences between the regulatory pathways controlled by this transcription factor in different fungal species.

Stability and plasmatic protein binding of novel zidovudine prodrugs: Targeting site ii of human serum albumin.

Despite its vastly demonstrated clinical efficacy, zidovudine (AZT) exhibits several suboptimal pharmacokinetic properties. In particular, its short plasmatic half-life (t1/2 ~ 1 h) is related to its low bound fraction to whole plasmatic proteins and in particular to human serum albumin (HSA). The design of prodrugs constitutes a promising strategy to enhance AZT pharmacokinetic properties, including its affinity for HSA. Recently, we reported the synthesis and chemical stability evaluation of three novel prodrugs of AZT obtained by derivatization with dicarboxylic acids (1-3). In this work, we present the design, synthesis and evaluation of chemical and enzymatic stabilities of a novel series of double prodrugs of AZT obtained by derivatization of 1-3 with a methylated l-phenylalanine moiety (4-6). In addition, the plasmatic protein binding properties were studied both by experimental and theoretical techniques. Prodrugs 4-6 were found to be relatively stable at pH 7.4 (t1/2 between 4.1 and 57.8 h), while also demonstrated adequate stabilities in human plasma at 37 °C (t1/2 between 1.0 and 2.1 h). Also, prodrugs 4-6 were able to regenerate AZT at a rate that depended on the length of the alkyl chain in 1-3. Additionally, 4-6 exhibited a significantly increased binding to plasmatic proteins (between 52.1 and 72.5%) with respect to AZT (12%) and 1-3 (between 26 and 34%). It is noteworthy that the displacement experiments with HSA site I and II markers, demonstrated that 4-6 bound to a different site than that of AZT and 1-3. Molecular modeling studies (i.e. molecular docking and free energy of binding analysis) were applied to shed light at an atomistic level on the pharmacodynamic properties driving the interaction of 4-6 with HSA. Overall, the present work provides a state of the art contribution to the design and development of novel prodrugs of AZT with optimized pharmacokinetic properties.

Application of a pharmacokinetics-pharmacodynamics approach to the free propofol plasma levels during coronary artery bypass grafting surgery with hypothermic cardiopulmonary bypass

OBJECTIVES: The objective of this study was to apply a pharmacokinetics-pharmacodynamics approach to investigate the free propofol plasma levels in patients undergoing coronary artery bypass grafting under hypothermic conditions compared with the off-pump procedure. METHODS: Nineteen patients scheduled for on-pump coronary artery bypass grafting under hypothermic conditions (n=10) or the equivalent off-pump surgery (n=9) were anesthetized with sufentanil and propofol target-controlled infusion (2 μg/mL) during surgery. The propofol concentration was then reduced to 1 μg/mL, and a pharmacokinetics-pharmacodynamics analysis using the maximum-effect-sigmoid model obtained by plotting the bispectral index values against the free propofol plasma levels was performed. RESULTS: Significant increases (two- to five-fold) in the free propofol plasma levels were observed in the patients subjected to coronary artery bypass grafting under hypothermic conditions. The pharmacokinetics of propofol varied according to the free drug levels in the hypothermic on-pump group versus the off-pump group. After hypothermic coronary artery bypass was initiated, the distribution volume increased, and the distribution half-life was prolonged. Propofol target-controlled infusion was discontinued when orotracheal extubation was indicated, and the time to patient extubation was significantly higher in the hypothermic on-pump group than in the off-pump group (459 versus 273 min, p=0.0048). CONCLUSIONS: The orotracheal intubation time was significantly longer in the hypothermic on-pump group than in the off-pump group. Additionally, residual hypnosis was identified through the pharmacokinetics-pharmacodynamics approach based on decreases in drug plasma protein binding in the hypothermic on-pump group, which could explain the increased hypnosis observed with this drug in this group of patients.