Surface charge modulation of rifampicin-loaded PLA nanoparticles to improve antibiotic delivery in Staphylococcus aureus biofilms.

BACKGROUND: After the golden age of antibiotic discovery, bacterial infections still represent a major challenge for public health worldwide. The biofilm mode of growth is mostly responsible for chronic infections that current therapeutics fail to cure and it is well-established that novel strategies must be investigated. Particulate drug delivery systems are considered as a promising strategy to face issues related to antibiotic treatments in a biofilm context. Particularly, poly-lactic acid (PLA) nanoparticles present a great interest due to their ability to migrate into biofilms thanks to their submicronic size. However, questions still remain unresolved about their mode of action in biofilms depending on their surface properties. In the current study, we have investigated the impact of their surface charge, firstly on their behavior within a bacterial biofilm, and secondly on the antibiotic delivery and the treatment efficacy. RESULTS: Rifampicin-loaded PLA nanoparticles were synthetized by nanoprecipitation and characterized. A high and superficial loading of rifampicin, confirmed by an in silico simulation, enabled to deliver effective antibiotic doses with a two-phase release, appropriate for biofilm-associated treatments. These nanoparticles were functionalized with poly-L-lysine, a cationic peptide, by surface coating inducing charge reversal without altering the other physicochemical properties of these particles. Positively charged nanoparticles were able to interact stronger than negative ones with Staphylococcus aureus, under planktonic and biofilm modes of growth, leading to a slowed particle migration in the biofilm thickness and to an improved retention of these cationic particles in biofilms. While rifampicin was totally ineffective in biofilms after washing, the increased retention capacity of poly-L-lysine-coated rifampicin-loaded PLA nanoparticles has been associated with a better antibiotic efficacy than uncoated negatively charged ones. CONCLUSIONS: Correlating the carrier retention capacity in biofilms with the treatment efficacy, positively charged rifampicin-loaded PLA nanoparticles are therefore proposed as an adapted and promising approach to improve antibiotic delivery in S. aureus biofilms.

Degradation of High Energy Materials Using Biological Reduction: A Rational Way to Reach Bioremediation.

Explosives molecules have been widely used since World War II, leading to considerable contamination of soil and groundwater. Recently, bioremediation has emerged as an environmentally friendly approach to solve such contamination issues. However, the 1,3,5,7-tetranitro-1,3,5,7-tetrazocane (HMX) explosive, which has very low solubility in water, does not provide satisfying results with this approach. In this study, we used a rational design strategy for improving the specificity of the nitroreductase from E. Cloacae (PDB ID 5J8G) toward HMX. We used the Coupled Moves algorithm from Rosetta to redesign the active site around HMX. Molecular Dynamics (MD) simulations and affinity calculations allowed us to study the newly designed protein. Five mutations were performed. The designed nitroreductase has a better fit with HMX. We observed more H-bonds, which productively stabilized the HMX molecule for the mutant than for the wild type enzyme. Thus, HMX's nitro groups are close enough to the reductive cofactor to enable a hydride transfer. Also, the HMX affinity for the designed enzyme is better than for the wild type. These results are encouraging. However, the total reduction reaction implies numerous HMX derivatives, and each of them has to be tested to check how far the reaction can' go.

In Silico, in Vitro, and in Vivo Evaluation of New Candidates for α-Synuclein PET Imaging.

Accumulation of α-synuclein (α-syn) is a neuropathological hallmark of synucleinopathies. To date, no selective α-syn positron emission tomography (PET) radiotracer has been identified. Our objective was to develop the first original, selective, and specific α-syn PET radiotracer. Chemical design inspired from three structural families that demonstrated interesting α-syn binding characteristics was used as a starting point. Bioinformatics modeling of α-syn fibrils was then employed to select the best molecular candidates before their syntheses. An in vitro binding assay was performed to evaluate the affinity of the compounds. Radiotracer specificity and selectivity were assessed by in vitro autoradiography and in vivo PET studies in animal (rodents) models. Finally, gold standard in vitro autoradiography with patients' postmortem tissues was performed to confirm/infirm the α-syn binding characteristics. Two compounds exhibited a good brain availability and bound to α-syn and Aß fibrils in a rat model. In contrast, no signal was observed in a mouse model of synucleinopathy. Experiments in human tissues confirmed these negative results.

Deciphering the molecular mechanisms underlying the binding of the TWIST1/E12 complex to regulatory E-box sequences.

The TWIST1 bHLH transcription factor controls embryonic development and cancer processes. Although molecular and genetic analyses have provided a wealth of data on the role of bHLH transcription factors, very little is known on the molecular mechanisms underlying their binding affinity to the E-box sequence of the promoter. Here, we used an in silico model of the TWIST1/E12 (TE) heterocomplex and performed molecular dynamics (MD) simulations of its binding to specific (TE-box) and modified E-box sequences. We focused on (i) active E-box and inactive E-box sequences, on (ii) modified active E-box sequences, as well as on (iii) two box sequences with modified adjacent bases the AT- and TA-boxes. Our in silico models were supported by functional in vitro binding assays. This exploration highlighted the predominant role of protein side-chain residues, close to the heart of the complex, at anchoring the dimer to DNA sequences, and unveiled a shift towards adjacent ((-1) and (-1*)) bases and conserved bases of modified E-box sequences. In conclusion, our study provides proof of the predictive value of these MD simulations, which may contribute to the characterization of specific inhibitors by docking approaches, and their use in pharmacological therapies by blocking the tumoral TWIST1/E12 function in cancers.

Destabilization of the TWIST1/E12 complex dimerization following the R154P point-mutation of TWIST1: an in silico approach.

BACKGROUND: The bHLH transcription factor TWIST1 plays a key role in the embryonic development and in tumorigenesis. Some loss-of-function mutations of the TWIST1 gene have been shown to cause an autosomal dominant craniosynostosis, known as the Saethre-Chotzen syndrome (SCS). Although the functional impacts of many TWIST1 mutations have been experimentally reported, little is known on the molecular mechanisms underlying their loss-of-function. In a previous study, we highlighted the predictive value of in silico molecular dynamics (MD) simulations in deciphering the molecular function of TWIST1 residues. RESULTS: Here, since the substitution of the arginine 154 amino acid by a glycine residue (R154G) is responsible for the SCS phenotype and the substitution of arginine 154 by a proline experimentally decreases the dimerizing ability of TWIST1, we investigated the molecular impact of this point mutation using MD approaches. Consistently, MD simulations highlighted a clear decrease in the stability of the α-helix during the dimerization of the mutated R154P TWIST1/E12 dimer compared to the wild-type TE complex, which was further confirmed in vitro using immunoassays. CONCLUSIONS: Our study demonstrates that MD simulations provide a structural explanation for the loss-of-function associated with the SCS TWIST1 mutation and provides a proof of concept of the predictive value of these MD simulations. This in silico methodology could be used to determine reliable pharmacophore sites, leading to the application of docking approaches in order to identify specific inhibitors of TWIST1 complexes.

Bioinspired Design and Oriented Synthesis of Immunogenic Site-Specifically Penicilloylated Peptides.

ß-Lactam antibiotics allergy is recognized as a public health concern. By covalently binding to serum proteins, penicillins are known to form immunogenic complexes. The latter are recognized and digested by antigen-presenting cells into drug-hapten peptides leading to the immunization of treated persons and IgE-mediated hypersensitivity reactions encompassing anaphylaxis. If type I allergic reactions to drugs are often unpredictable, they are known to be dependent on CD4+ T-cells. This fundamental study revisits the chemical basis of the benzylpenicillin (BP) allergy with the aim of identifying immunologically relevant biomimetic benzylpenicilloylated peptides through the analysis of BP-conjugated human serum albumin (BP-HSA) profile and the evaluation of the naïve CD4+ T-cell responses to candidate BP-HSA-derived peptides. The chemical structures of BP-HSA bioconjugates synthesized in vitro at both physiological and basic pH were investigated by mass spectrometry. From the ten most representative lysine residues grafted by BP-hapten, HSA-bioinspired 15-mer peptide sequences were designed and the potential T-cell epitope profile of each peptide was predicted using two complementary in silico approaches, i.e., HLA class II binding prediction tools from the Immune Epitope Database and Analysis Resource (IEDB) and computational alanine scanning mutagenesis. Twelve structurally diversified benzylpenicilloylated peptides (BP-Ps) were selected and synthesized with the aid of a flexible synthesis pathway using an original benzylpenicilloylated lysine monomer as common precursor. In order to corroborate their predicted "epitope" profile, the naïve CD4+ T-cell response specific to BP was evaluated through a coculture approach. To our knowledge, this study showed for the first time the ability of bioinspired peptides structurally stemming from BP-HSA to be recognized by naïve CD4+ T-cells thus identifying a pre-existing T-cell repertoire for penicillin molecules bound to proteins. It also established a promising model approach expandable to other most frequently used penicillin classes of antibiotics to reveal biomimetic drug-modified antigenic peptides relevant for qualitative and quantitative drug allergy studies.

Daclatasvir-like inhibitors of NS5A block early biogenesis of hepatitis C virus-induced membranous replication factories, independent of RNA replication.

BACKGROUND & AIMS: Direct-acting antivirals that target nonstructural protein 5A (NS5A), such as daclatasvir, have high potency against the hepatitis C virus (HCV). They are promising clinical candidates, yet little is known about their antiviral mechanisms. We investigated the mechanisms of daclatasvir derivatives. METHODS: We used a combination of biochemical assays, in silico docking models, and high-resolution imaging to investigate inhibitor-induced changes in properties of NS5A, including its interaction with phosphatidylinositol-4 kinase IIIα and induction of the membranous web, which is the site of HCV replication. Analyses were conducted with replicons, infectious virus, and human hepatoma cells that express a HCV polyprotein. Studies included a set of daclatasvir derivatives and HCV variants with the NS5A inhibitor class-defining resistance mutation Y93H. RESULTS: NS5A inhibitors did not affect NS5A stability or dimerization. A daclatasvir derivative interacted with NS5A and molecular docking studies revealed a plausible mode by which the inhibitor bound to NS5A dimers. This interaction was impaired in mutant forms of NS5A that are resistant to daclatavir, providing a possible explanation for the reduced sensitivity of the HCV variants to this drug. Potent NS5A inhibitors were found to block HCV replication by preventing formation of the membranous web, which was not linked to an inhibition of phosphatidylinositol-4 kinase IIIα. Correlative light-electron microscopy revealed unequivocally that NS5A inhibitors had no overall effect on the subcellular distribution of NS5A, but completely prevented biogenesis of the membranous web. CONCLUSIONS: Highly potent inhibitors of NS5A, such as daclatasvir, block replication of HCV RNA at the stage of membranous web biogenesis-a new paradigm in antiviral therapy.

Quantification and kinetic study in plasma and tissues of (E)-1,1,4,4-tetramethyl-2-tetrazene, a liquid propellant and a transformation product of 1,1-dimethyl hydrazine.

(E)-1,1,4,4-tetramethyl-2-tetrazene (TMTZ) is formed from the oxidation of the unsymmetrical 1,1-dimethylhydrazine (UDMH) and is used as a storable liquid fuel which can be considered as a new potential propellant for space rocket propulsion. To better understand the toxicological behavior of the compound, an intraperitoneal administration of TMTZ was performed in mice to define its toxicokinetics and tissue distribution. A fully validated liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) assay was developed to determine TMTZ levels in biological samples. Determination of TMTZ was achieved using 50 µL of plasma or tissue solution. Precipitation with ammonium sulfate and acetonitrile was used for sample preparation. Liquid chromatography was performed on an Atlantis HILIC Silica column (Waters; 3 µm, 150 mm × 2.1 mm i.d.). Isocratic elution with a mixture of ammonium acetate buffer (pH 5, 100 mM)/water/acetonitrile (3:2:95, v/v/v) was used. The detection was conducted using an electrospray source in positive ion mode. TMTZ and (15)N2-TMTZ (internal standard) were quantitated in selected reaction monitoring mode using the transition m/z 117→72 and 119→74, respectively. Standard curves exhibited excellent linearity in the range of 10-500 ng/mL for plasma and 50-2000 ng/mL for all tissues (heart, liver, brain, kidney, and lung) analyzed, and acceptable precision and accuracy (<10 %) were obtained. The elimination rate constant strongly suggests that TMTZ was very quickly eliminated from the body. The results of tissue distribution experiments indicated that TMTZ underwent a rapid distribution into limited organs such as the liver, kidney, and brain.

Calcineurin A versus NS5A-TP2/HD domain containing 2: a case study of site-directed low-frequency random mutagenesis for dissecting target specificity of peptide aptamers.

We previously identified a peptide aptamer (named R5G42) via functional selection for its capacity to slow cell proliferation. A yeast two-hybrid screen of human cDNA libraries, using R5G42 as "bait," allowed the identification of two binding proteins with very different functions: calcineurin A (CnA) (PP2B/PPP3CA), a protein phosphatase well characterized for its role in the immune response, and NS5A-TP2/HD domain containing 2, a much less studied protein induced subsequent to hepatitis C virus non-structural protein 5A expression in HepG2 hepatocellular carcinoma cells, with no known activity. Our objective in the present study was to dissect the dual target specificity of R5G42 in order to have tools with which to better characterize the actions of the peptide aptamers toward their individual targets. This was achieved through the selection of random mutants of the variable loop, derived from R5G42, evaluating their specificity toward CnA and NS5A-TP2 and analyzing their sequence. An interdisciplinary approach involving biomolecular computer simulations with integration of the sequence data and yeast two-hybrid binding phenotypes of these mutants yielded two structurally distinct conformers affording the potential molecular basis of the binding diversity of R5G42. Evaluation of the biological impact of CnA- versus NS5A-TP2-specific peptide aptamers indicated that although both contributed to the anti-proliferative effect of R5G42, CnA-binding was essential to stimulate the nuclear translocation of nuclear factor of activated T cells, indicative of the activation of endogenous CnA. By dissecting the target specificity of R5G42, we have generated novel tools with which to study each target individually. Apta-C8 is capable of directly activating CnA independent of binding to NS5A-TP2 and will be an important tool in studying the role of CnA activation in the regulation of different signaling pathways, whereas Apta-E1 will allow dissection of the function of NS5A-TP2, serving as an example of the usefulness of peptide aptamer technology for investigating signaling pathways.

Localization of putative binding sites for cyclic guanosine monophosphate and the anti-cancer drug 5-fluoro-2'-deoxyuridine-5'-monophosphate on ABCC11 in silico models.

BACKGROUND: The Multidrug Resistance Protein ABCC11/MRP8 is expressed in physiological barriers and tumor breast tissues in which it secretes various substrates including cGMP (cyclic guanosine monophosphate) and 5FdUMP (5-fluoro-2'-deoxyuridine-5'-monophosphate), the active metabolite of the anticancer drug 5-FluoroUracil (frequently included to anticancer therapy).Previously, we described that ABCC11 high levels are associated to the estrogen receptor (ER) expression level in breast tumors and in cell lines resistant to tamoxifen. Consequently, by lowering the intracellular concentration of anticancer drugs, ABCC11 likely promotes a multidrug resistance (MDR) phenotype and decreases efficiency of anticancer therapy of 5FdUMP. Since no experimental data about binding sites of ABCC11 substrate are available, we decided to in silico localize putative substrate interaction sites of the nucleotide derivatives. Taking advantage of molecular dynamics simulation, we also analysed their evolution under computational physiological conditions and during the time. RESULTS: Since ABCC11 crystal structure is not resolved yet, we used the X-ray structures of the mouse mdr3 (homologous to human ABCB1) and of the bacterial homolog Sav1866 to generate two independent ABCC11 homology models in inward- and outward-facing conformations. Based on docking analyses, two putative binding pockets, for cGMP and 5FdUMP, were localized in both inward- and outward-facing conformations. Furthermore, based on our 3D models, and available biochemical data from homologous transporters, we identified several residues, potentially critical in ABCC11 transport function. Additionally, molecular dynamics simulation on our inward-facing model revealed for the first time conformation changes assumed to occur during transport process. CONCLUSIONS: ABCC11 would present two binding sites for cGMP and for 5FdUMP. Substrates likely first bind at the intracellular side of the transmembrane segment while ABCC11 is open forward the cytoplasm (inward-facing conformation). Then, along with conformational changes, it would pass through ABCC11 and fix the second site (close to the extracellular side), until the protein open itself to the extracellular space and allow substrate release.

Induction of a strong and long-lasting neutralizing immune response by dPreS1-TLR2 agonist nanovaccine against hepatitis B virus.

Hepatitis B virus remains a major medical burden with more than 250 million chronically infected patients worldwide and 900,000 deaths each year, due to the disease progression towards severe complications (cirrhosis, hepatocellular carcinoma). Despite the availability of a prophylactic vaccine, this infection is still pandemic in Western Pacific and African regions, where around 6% of the adult population is infected. Among novel anti-HBV strategies, innovative drug delivery systems, such as nanoparticle platforms to deliver vaccine antigens or therapeutic molecules have been investigated. Here, we developed polylactic acid-based biodegradable nanoparticles as an innovative and efficient vaccine. They are twice functionalized by (i) the entrapment of Pam3CSK4, an immunomodulator and ligand to Toll-Like-Receptor 1/2, and by (ii) the adsorption/coating of myristoylated (2-48) derived PreS1 from the HBV surface antigen, identified as the major viral attachment site on hepatocytes. We demonstrate that such formulations mimic HBV virion with an efficient peptide recognition by the immune system, and elicit potent and durable antibody responses in naive mice during at least one year. We also show that the most efficient in vitro viral neutralization was observed with NP-Pam3CSK4-dPreS1 sera. The immunogenicity of the derived HBV antigen is modulated by the likely synergistic action of both the dPreS1 coated nanovector and the adjuvant moiety. This formulation represents a promising vaccine alternative to fight HBV infection.

Bacterial tyrosine-kinases: structure-function analysis and therapeutic potential.

Since the characterization of genes encoding Ser/Thr-kinases and Tyr-kinases in bacteria, in 1991 and 1997, respectively, a growing body of evidence has been reported showing the important role of these enzymes in the regulation of bacterial physiology. While most Ser/Thr-kinases share structural similarity with their eukaryotic counterparts, it seems that bacteria have developed their own Tyr-kinases to catalyze protein phosphorylation on tyrosine. Different types of Tyr-kinases have been identified in bacteria and a large number of them are similar to ATP-binding proteins with Walker motifs. These enzymes have been grouped in the same family (BY-kinases) and the crystal structures of two of them have been recently characterized. Phosphoproteome analysis suggest that BY-kinases are involved in several cellular processes and to date, the best-characterized role of BY-kinases concerns the control of extracellular polysaccharide synthesis. Knowing the role of these compounds in the virulence of bacterial pathogens, BY-kinases can be considered as promising targets to combat some diseases. Here, we review the current knowledge on BY-kinases and discuss their potential for the development of new antibiotics.

Identification of structural and molecular determinants of the tyrosine-kinase Wzc and implications in capsular polysaccharide export.

Capsular polysaccharides are well-established virulence factors of pathogenic bacteria. Their biosynthesis and export are regulated within the transmembrane polysaccharide assembly machinery by the autophosphorylation of atypical tyrosine-kinases, named BY-kinases. However, the accurate functioning of these tyrosine-kinases remains unknown. Here, we report the crystal structure of the non-phosphorylated cytoplasmic domain of the tyrosine-kinase Wzc from Escherichia coli in complex with ADP showing that it forms a ring-shaped octamer. Mutational analysis demonstrates that a conserved EX(2) RX(2) R motif involved in subunit interactions is essential for polysaccharide export. We also elucidate the role of a putative internal regulatory tyrosine and we show that BY-kinases from proteobacteria autophosphorylate on their C-terminal tyrosine cluster via a single-step intermolecular mechanism. This structure-function analysis also allows us to demonstrate that two different parts of a conserved basic region called the RK-cluster are essential for polysaccharide export and for kinase activity respectively. Based on these data, we revisit the dichotomy made between BY-kinases from proteobacteria and firmicutes and we propose a unique process of oligomerization and phosphorylation. We also reassess the function of BY-kinases in the capsular polysaccharide assembly machinery.

C(16)-methyl corticosteroids are far less allergenic than the non-methylated molecules.

BACKGROUND: It has been confirmed that binding to amino acids in skin proteins takes place at C(21) after oxidation of the corticosteroid molecule, which gives to the constituents of the D-ring an essential role in cross-reactivity patterns. In 2000, Matura et al. subdivided the corticosteroid esters of the D-group into two subgroups: D1, the 'stable' esters; and D2, the 'labile' esters. Recent data have indicated that non-methylated corticosteroids selectively react with arginine to form stable cyclic adducts, which are probably implicated in sensitization to corticosteroids. OBJECTIVES: To compare the patch test results and reactivity of C(16)-methylated and non-methylated corticosteroids. METHODS: Three hundred and fifteen subjects with a proven corticosteroid contact allergy underwent patch testing with an extended corticosteroid series. Statistical analysis was performed with the Wilcoxon signed rank test to compare the number of reactions to molecules with and without C(16)-methyl substitution. RESULTS: Positive patch test reactions to corticosteroid molecules without C(16)-methyl substitution groups A and D2, were, with statistical significance, much more frequently observed than to those with a C(16)-methyl group, groups D1 and C. CONCLUSIONS: C(16)-methyl substitution, interfering with protein binding, and halogenation, seem to reduce the allergenicity of corticosteroid molecules. Hence, when indicated, C(16)-methylated corticosteroids should be preferentially prescribed.

Latent TGF-ß Activation Is a Hallmark of the Tenascin Family.

Transforming growth factor-ß (TGF-ß) isoforms are secreted as inactive complexes formed through non-covalent interactions between bioactive TGF-ß entities and their N-terminal pro-domains called latency-associated peptides (LAP). Extracellular activation of latent TGF-ß within this complex is a crucial step in the regulation of TGF-ß activity for tissue homeostasis and immune cell function. We previously showed that the matrix glycoprotein Tenascin-X (TN-X) interacted with the small latent TGF-ß complex and triggered the activation of the latent cytokine into a bioactive TGF-ß. This activation most likely occurs through a conformational change within the latent TGF-ß complex and requires the C-terminal fibrinogen-like (FBG) domain of the glycoprotein. As the FBG-like domain is highly conserved among the Tenascin family members, we hypothesized that Tenascin-C (TN-C), Tenascin-R (TN-R) and Tenascin-W (TN-W) might share with TN-X the ability to regulate TGF-ß bioavailability through their C-terminal domain. Here, we demonstrate that purified recombinant full-length Tenascins associate with the small latent TGF-ß complex through their FBG-like domains. This association promotes activation of the latent cytokine and subsequent TGF-ß cell responses in mammary epithelial cells, such as cytostasis and epithelial-to-mesenchymal transition (EMT). Considering the pleiotropic role of TGF-ß in numerous physiological and pathological contexts, our data indicate a novel common function for the Tenascin family in the regulation of tissue homeostasis under healthy and pathological conditions.

Discovery of a Novel Non-Narcotic Analgesic Derived from the CL-20 Explosive: Synthesis, Pharmacology, and Target Identification of Thiowurtzine, a Potent Inhibitor of the Opioid Receptors and the Ion Channels.

The number of candidate molecules for new non-narcotic analgesics is extremely limited. Here, we report the identification of thiowurtzine, a new potent analgesic molecule with promising application in chronic pain treatment. We describe the chemical synthesis of this unique compound derived from the hexaazaisowurtzitane (CL-20) explosive molecule. Then, we use animal experiments to assess its analgesic activity in vivo upon chemical, thermal, and mechanical exposures, compared to the effect of several reference drugs. Finally, we investigate the potential receptors of thiowurtzine in order to better understand its complex mechanism of action. We use docking, molecular modeling, and molecular dynamics simulations to identify and characterize the potential targets of the drug and confirm the results of the animal experiments. Our findings finally indicate that thiowurtzine may have a complex mechanism of action by essentially targeting the mu opioid receptor, the TRPA1 ion channel, and the Cav voltage-gated calcium channel.

Uncompetitive nanomolar dimeric indenoindole inhibitors of the human breast cancer resistance pump ABCG2.

Multidrug resistance membrane pumps reduce the efficacy of chemotherapies by exporting a wide panel of structurally-divergent drugs. Here, to take advantage of the polyspecificity of the human Breast Cancer Resistance Protein (BCRP/ABCG2) and the dimeric nature of this pump, new dimeric indenoindole-based inhibitors from the monomeric α,ß-unsaturated ketone 4b and phenolic derivative 5a were designed. A library of 18 homo/hetero-dimers was synthesised. Homo-dimerization shifted the inhibition efficacy from sub-micromolar to nanomolar range, correlated with the presence of 5a, linked by a 2-6 methylene-long linker. Non-toxic, the best dimers displayed a therapeutic ratio as high as 70,000. It has been found that the high potency of the best compound 7b that displays a KI of 17 nM is due to an uncompetitive behavior toward mitoxantrone efflux and specific for that drug, compared to Hoechst 33342 efflux. Such property may be useful to target such anticancer drug efflux mediated by ABCG2. Finally, at a molecular level, an uncompetitive mechanism by which substrate promotes inhibitor binding implies that at least 2 ligands should bind simultaneously to the drug-binding pocket of ABCG2.

Construction of atomic models of full hepatitis B vaccine particles at different stages of maturation.

Hepatitis B, one of the world's most common liver infections, is caused by the Hepatitis B Virus (HBV). Via the infected cells, this virus generates non pathogen particles with similar surface structures as those found in the full virus. These particles are used in a recombinant form (HBsAg) to produce efficient vaccines. The atomic structure of the HBsAg particles is currently unsolved, and the only existing structural data for the full particle were obtained by electronic microscopy with a maximum resolution of 12 Å. As many vaccines, HBsAg is a complex bio-system. This complexity results from numerous sources of heterogeneity, and traditional bio-immuno-chemistry analytic tools are often limited in their ability to fully describe the molecular surface or the particle. For the Hepatitis B vaccine particle (HBsAg), no atomic data are available so far. In this study, we used the principal well-known elements of HBsAg structure to reconstitute and model the full HBsAg particle assembly at a molecular level (protein assembly, particle formation and maturation). Full HBsAg particle atomic models were built based on an exhaustive experimental data review, amino acid sequence analysis, iterative threading modeling, and molecular dynamic approaches.

Molecular Dynamics Studies of Poly(Lactic Acid) Nanoparticles and Their Interactions with Vitamin E and TLR Agonists Pam1CSK4 and Pam3CSK4.

Poly(lactic acid) (PLA) nanoparticles (NPs) are widely investigated due to their bioresorbable, biocompatible and low immunogen properties. Interestingly, many recent studies show that they can be efficiently used as drug delivery systems or as adjuvants to enhance vaccine efficacy. Our work focuses on the molecular mechanisms involved during the nanoprecipitation of PLA NPs from concentrated solutions of lactic acid polymeric chains, and their specific interactions with biologically relevant molecules. In this study, we evaluated the ability of a PLA-based nanoparticle drug carrier to vectorize either vitamin E or the Toll-like receptor (TLR) agonists Pam1CSK4 and Pam3CSK4, which are potent activators of the proinflammatory transcription factor NF-κB. We used dissipative particle dynamics (DPD) to simulate large systems mimicking the nanoprecipitation process for a complete NP. Our results evidenced that after the NP formation, Pam1CSK4 and Pam3CSK4 molecules end up located on the surface of the particle, interacting with the PLA chains via their fatty acid chains, whereas vitamin E molecules are buried deeper in the core of the particle. Our results allow for a better understanding of the molecular mechanisms responsible for the formation of the PLA NPs and their interactions with biological molecules located either on their surfaces or encapsulated within them. This work should allow for a rapid development of better biodegradable and safe vectorization systems with new drugs in the near future.

Delayed hypersensitivity to corticosteroids in a series of 315 patients: clinical data and patch test results.

BACKGROUND: Corticosteroids may cause immediate or delayed hypersensitivity. In 1989, based on structural and clinical characteristics, we put forward a classification of corticosteroids into four cross-reacting groups, namely group A, B, C, and D, the latter later subdivided into two subgroups, i.e. D1 and D2. The constituents on the D-ring of the corticosteroid-molecule are considered to have a central role for binding to skin proteins and for cross-reactions patterns; however, halogenation of the molecules is also interfering. OBJECTIVE: To study the clinical data and analyse simultaneous positive reactions obtained in a large group of corticosteroid-allergic patients. METHODS: Patch tests were performed with the baseline series, to which hydrocortisone butyrate and prednisolone caproate were added, as well as with the corticosteroids to which the patients had been exposed. Three hundred and forty subjects with a presumed or proven corticosteroid allergy were further investigated with an extended series containing 72 molecules. RESULTS: Out of 11 596 patients investigated, 315 subjects reacted positively to at least 1 corticosteroid-molecule, with most of them presenting with multiple positive reactions. CONCLUSION: A prevalence of corticosteroid allergy of 2.7% was found. Despite validity of the ABCD (sub)classification in many cases, possible adjustments may have to be considered.