Thermodynamic and kinetic characterization of hydroxyethylamine ß-secretase-1 inhibitors.

Alzheimer's disease (AD) is a devastating neurodegenerative disease affecting millions of people. ß-Secretase-1 (BACE-1), an enzyme involved in the processing of the amyloid precursor protein (APP) to form Aß, is a well validated target for AD. Herein, the authors characterize 10 randomly selected hydroxyethylamine (HEA) BACE-1 inhibitors in terms of their association and dissociation rate constants and thermodynamics of binding using surface plasmon resonance (SPR). Rate constants of association (ka) measured at 25 °C ranged from a low of 2.42×10(4) M(-1) s(-1) to the highest value of 8.3×10(5) M(-1) s(-1). Rate constants of dissociation (kd) ranged from 1.09×10(-4) s(-1) (corresponding to a residence time of close to three hours), to the fastest of 0.028 s(-1). Three compounds were selected for further thermodynamic analysis where it was shown that equilibrium binding was enthalpy driven while unfavorable entropy of binding was observed. Structural analysis revealed that upon ligand binding, the BACE-1flap folds down over the bound ligand causing an induced fit. The maximal difference between alpha carbon positions in the open and closed conformations of the flap was over 5 Å. Thus the negative entropy of binding determined using SPR analysis was consistent with an induced fit observed by structural analysis.

Pharmacogenomics in the preclinical development of vaccines: evaluation of efficacy and systemic toxicity in the mouse using array technology.

The development of vaccines, conventional protein based as well as nucleic acid based vaccines, and their delivery systems has been largely empirical and ineffective. This is partly due to a lack of methodology, since traditionally only a few markers are studied. By introducing gene expression analysis and bioinformatics into the design of vaccines and their delivery systems, vaccine development can be improved and accelerated considerably. Each vaccine antigen and delivery system combination is characterized by a unique genomic profile, a "fingerprint" that will give information of not only immunological and toxicological responses but also other related cellular responses e.g. cell cycle, apoptosis and carcinogenic effects. The resulting unique genomic fingerprint facilitates the establishment of molecular structure--pharmacological activity relationships and therefore leads to optimization of vaccine development.

Pharmacogenomics in the evaluation of efficacy and adverse events during clinical development of vaccines.

The understanding of vaccine-induced immune responses in adults and infants is limited. Current vaccination schedules for infants are frequently debated. Especially, the relationship among the timing, the frequency of the dosing, and the generation of an immunological memory are debated. Vaccine antigen-induced cytokine responses to vaccinations given in infancy are of particular interest because little is known about cellular responses in this age, and the information available is based on antibody responses. Pharmacogenomics is ideally suited to study cellular responses related to immune response; in addition, toxicity, inflammation, apoptosis, stress, and oncogenesis can be monitored, since the expression of thousands of genes can be measured in a single experiment.

Current Trends in Ligand Binding Real-Time Measurement Technologies.

Numerous advances in ligand binding assay (LBA) real-time measurement technologies have been made within the last several years, ranging from the development of novel platforms to drive technology expansion to the adaptation of existing platforms to optimize performance and throughput. In this review, we have chosen to focus on technologies that provide increased value to two distinct segments of the LBA community. First, experimentally, by measuring real-time binding events, these technologies provide data that can be used to interrogate receptor/ligand binding interactions. While overall the platforms are not new, they have made significant advances in throughput, multiplexing, and/or sensitivity. Second, clinically, these point-of-care (POC) technologies provide instantaneous information which facilitates rapid treatment decisions.

Pharmacogenomics and its potential impact on drug and formulation development.

Recent advances in genomic research have provided the basis for new insights into the importance of genetic and genomic markers during the different stages of drug development. A new field of research, pharmacogenomics, which studies the relationship between drug effects and the genome, has emerged. Structural pharmacogenomics maps the complete DNA sequences of whole genomes (genotypes) including individual variations, and functional pharmacogenomics assesses the expression levels of thousands of genes in one single experiment. Together, these two areas of pharmacogenomics have generated massive databases, which have become a challenge for the research field of informatics and have fostered a new branch of research, bioinformatics. If skillfully used, the databases generated by pharmacogenomics together with data mining on the Web promise to improve the drug development process in a variety of areas: identification of drug targets, evaluation of toxicity, classification of diseases, evaluation of formulations, assessment of drug response and treatment, post-marketing applications, and development of personalized medicines.

Targeting the intrinsically disordered structural ensemble of α-synuclein by small molecules as a potential therapeutic strategy for Parkinson's disease.

The misfolding of intrinsically disordered proteins such as α-synuclein, tau and the Aß peptide has been associated with many highly debilitating neurodegenerative syndromes including Parkinson's and Alzheimer's diseases. Therapeutic targeting of the monomeric state of such intrinsically disordered proteins by small molecules has, however, been a major challenge because of their heterogeneous conformational properties. We show here that a combination of computational and experimental techniques has led to the identification of a drug-like phenyl-sulfonamide compound (ELN484228), that targets α-synuclein, a key protein in Parkinson's disease. We found that this compound has substantial biological activity in cellular models of α-synuclein-mediated dysfunction, including rescue of α-synuclein-induced disruption of vesicle trafficking and dopaminergic neuronal loss and neurite retraction most likely by reducing the amount of α-synuclein targeted to sites of vesicle mobilization such as the synapse in neurons or the site of bead engulfment in microglial cells. These results indicate that targeting α-synuclein by small molecules represents a promising approach to the development of therapeutic treatments of Parkinson's disease and related conditions.

Development of a novel ß-secretase binding assay using the AlphaScreen platform.

Alzheimer's disease (AD) is a devastating neurodegenerative disease affecting millions of people. ß-secretase-1 (BACE1), an enzyme involved in the processing of the amyloid precursor protein (APP) to form Aß is a validated target for AD. Herein, the authors develop and validate a novel binding assay for BACE1 using the AlphaScreen platform that is amenable for high-throughput screening (HTS). Small-molecule BACE1 inhibitors of the hydroxyethylamine, hydantoin, and sulfamide classes were functionalized by biotin PEG linkers of varying lengths forming probes that were bound to streptavidin donor beads. BACE1 was coupled to nickel-chelate acceptor beads. Upon mixing, probes designed from all three classes registered high signal-to-background values in the AlphaScreen binding assay, where the interaction between probe and BACE1 was completely blocked by free parent compound. A probe from the hydantoin class was chosen for further optimization, where the final assay conditions of 50 nM BACE and 250 nM probe were used and Z(') values >0.75 were commonly observed. IC50 values determined by the AlphaScreen assay format exhibited ~10-fold greater sensitivity when compared with a fluorescence polarization-based activity assay. The assay was miniaturized to a 1536-well format for HTS, in which 525 000 compounds were screened.

Label free fragment screening using surface plasmon resonance as a tool for fragment finding - analyzing parkin, a difficult CNS target.

Surface Plasmon Resonance (SPR) is rarely used as a primary High-throughput Screening (HTS) tool in fragment-based approaches. With SPR instruments becoming increasingly high-throughput it is now possible to use SPR as a primary tool for fragment finding. SPR becomes, therefore, a valuable tool in the screening of difficult targets such as the ubiquitin E3 ligase Parkin. As a prerequisite for the screen, a large number of SPR tests were performed to characterize and validate the active form of Parkin. A set of compounds was designed and used to define optimal SPR assay conditions for this fragment screen. Using these conditions, more than 5000 pre-selected fragments from our in-house library were screened for binding to Parkin. Additionally, all fragments were simultaneously screened for binding to two off target proteins to exclude promiscuous binding compounds. A low hit rate was observed that is in line with hit rates usually obtained by other HTS screening assays. All hits were further tested in dose responses on the target protein by SPR for confirmation before channeling the hits into Nuclear Magnetic Resonance (NMR) and other hit-confirmation assays.

Laminin-411 is a vascular ligand for MCAM and facilitates TH17 cell entry into the CNS.

TH17 cells enter tissues to facilitate pathogenic autoimmune responses, including multiple sclerosis (MS). However, the adhesion molecules involved in the unique migratory capacity of TH17 cells, into both inflamed and uninflamed tissues remain unclear. Herein, we characterize MCAM (CD146) as an adhesion molecule that defines human TH17 cells in the circulation; following in vitro restimulation of human memory T cells, nearly all of the capacity to secrete IL-17 is contained within the population of cells expressing MCAM. Furthermore, we identify the MCAM ligand as laminin 411, an isoform of laminin expressed within the vascular endothelial basement membranes under inflammatory as well as homeotstatic conditions. Purified MCAM-Fc binds to laminin 411 with an affinity of 27 nM, and recognizes vascular basement membranes in mouse and human tissue. MCAM-Fc binding was undetectable in tissue from mice with targeted deletion of laminin 411, indicating that laminin 411 is a major tissue ligand for MCAM. An anti-MCAM monoclonal antibody, selected for inhibition of laminin binding, as well as soluble MCAM-Fc, inhibited T cell adhesion to laminin 411 in vitro. When administered in vivo, the antibody reduced TH17 cell infiltration into the CNS and ameliorated disease in an animal model of MS. Our data suggest that MCAM and laminin 411 interact to facilitate TH17 cell entry into tissues and promote inflammation.

Kinetics of asthma- and allergy-associated immune response gene expression in peripheral blood mononuclear cells from vaccinated infants after in vitro re-stimulation with vaccine antigen.

The global expression of immune response genes in infants after vaccination and their role in asthma and allergy is not clearly understood. Pharmacogenomics is ideally suited to study the involved cellular responses, since the expression of thousands of genes can be assessed simultaneously. Here, array technology was used to assess the expression kinetics of immune response genes with association to asthma and allergy in peripheral blood mononuclear cells (PBMC) of five healthy infants after vaccination with Infanrix-Polio+Hib. At 12h after in vitro re-stimulation of the PBMC with pertussis toxin (PT) antigen, 14 immune response pathways, 33 allergy-related and 66 asthma-related genes were found activated.

Gene expression profiles in mouse lung tissue after administration of two cationic polymers used for nonviral gene delivery.

PURPOSE: This study compared gene expression profiles in mouse lungs after administration of the cationic polymers polyethyleneimine (PEI) or chitosan alone or formulated with a luciferase reporter plasmid (PEI-pLuc, chitosan-pLuc). METHODS: The polymers and formulations were administered intratracheally to Balb/c mice at doses judged to be nontoxic according to intracellular dehydrogenase activity and tissue morphology. RNA was isolated from the lungs 24 or 72 h after administration, and a dedicated stress and toxicology cDNA array was used to monitor the in vivo response to the gene delivery system in the lung tissue. RESULTS: The gene expression profiles differed between the PEI and chitosan groups with regard to both the total number and the type of expressed genes. Chitosan-pLuc upregulated genes that protect the cell from oxidative stress and inflammation, such as heme oxygenase-1 and catalase, whereas PEI-pLuc upregulated genes involved in inflammatory processes, such as the cyclooxygenases 1 and 2, indicating possible involvement in the development of adverse reactions. However, both polymers activated genes involved in reaction to stress, such as DNA damage repair. Furthermore, in the PEI group, chaperone genes and members of the p38 mitogen-activated protein kinase pathway were also upregulated, suggesting a possible explanation for the better performance of PEI in gene delivery systems. CONCLUSIONS: The results indicate that gene expression profiling is a useful and sensitive tool for the evaluation of tissue responses after administration of polymers or gene delivery systems. The results also suggest a possible explanation for the differences in gene delivery performance between the two polymers in gene delivery systems.

Gene expression after vaccination of mice with formulations of diphtheria toxoid or tetanus toxoid and different adjuvants: identification of shared and vaccine-specific genes in spleen lymphocytes.

We immunized mice with four different combinations of diphtheria toxoid or tetanus toxoid with aluminum phosphate or Freund's adjuvant and studied the resulting gene expression profiles in spleen lymphocytes. Genes, which are unique for each combination or shared in several combinations, were found activated, with functions in immune response but also in other cellular processes like apoptosis or signal transduction. Using bioinformatic tools we show, that some of the genes may serve as indicators for adverse reactions, while other genes may be new immune response markers. The results also suggest that adjuvant participates in the formation of an immunological memory.

Enhancement by effectors and substrate nucleotides of R1-R2 interactions in Escherichia coli class Ia ribonucleotide reductase.

Ribonucleotide reductases are a family of essential enzymes that catalyze the reduction of ribonucleotides to their corresponding deoxyribonucleotides and provide cells with precursors for DNA synthesis. The different classes of ribonucleotide reductase are distinguished based on quaternary structures and enzyme activation mechanisms, but the components harboring the active site region in each class are evolutionarily related. With a few exceptions, ribonucleotide reductases are allosterically regulated by nucleoside triphosphates (ATP and dNTPs). We have used the surface plasmon resonance technique to study how allosteric effects govern the strength of quaternary interactions in the class Ia ribonucleotide reductase from Escherichia coli, which like all class I enzymes has a tetrameric alpha(2) beta(2) structure. The component alpha(2)called R1 harbors the active site and two types of binding sites for allosteric effector nucleotides, whereas the beta(2) component called R2 harbors the tyrosyl radical necessary for catalysis. Our results show that only the known allosteric effector nucleotides, but not non-interacting nucleotides, promote a specific interaction between R1 and R2. Interestingly, the presence of substrate together with allosteric effector nucleotide strengthens the complex 2-3 times with a similar free energy change as the mutual allosteric effects of substrate and effector nucleotide binding to protein R1 in solution experiments. The dual allosteric effects of dATP as positive allosteric effector at low concentrations and as negative allosteric effector at high concentrations coincided with an almost 100-fold stronger R1-R2 interaction. Based on the experimental setup, we propose that the inhibition of enzyme activity in the E. coli class Ia enzyme occurs in a tight 1:1 complex of R1 and R2. Most intriguingly, we also discovered that thioredoxin, one of the physiological reductants of ribonucleotide reductases, enhances the R1-R2 interaction 4-fold.