We report the fabrication of enthalpy arrays and their use to detect molecular interactions, including protein-ligand binding, enzymatic turnover, and mitochondrial respiration. Enthalpy arrays provide a universal assay methodology with no need for specific assay development such as fluorescent labeling or immobilization of reagents, which can adversely affect the interaction. Microscale technology enables the fabrication of 96-detector enthalpy arrays on large substrates. The reduction in scale results in large decreases in both the sample quantity and the measurement time compared with conventional microcalorimetry. We demonstrate the utility of the enthalpy arrays by showing measurements for two proteinligand binding interactions (RNase A ؉ cytidine 2 -monophosphate and streptavidin ؉ biotin), phosphorylation of glucose by hexokinase, and respiration of mitochondria in the presence of 2,4-dinitrophenol uncoupler.U nderstanding the thermodynamics of molecular interactions is central to biology and chemistry. Although a number of methods are available, calorimetry is the only universal assay for the complete thermodynamic characterization of these interactions. Under favorable circumstances, the enthalpy, entropy, free energy, and stoichiometry of a reaction can be determined (1, 2). In addition, calorimetry does not require any labeling or immobilization of the reactants and hence offers a completely generic method for characterizing the interactions. Indeed, titration calorimetry is widely used in both drug discovery and basic science, but its use is severely constrained to a small number of very high-value measurements by the large sample requirements and long measurement times. No currently available methods for calorimetric measurements lend themselves to modern approaches in which large libraries of compounds, ranging from small molecules in combinatorial libraries to proteins and other macromolecules, are studied.Here we report a low-cost nanocalorimetry detector that can be used as a high-throughput assay tool to detect enthalpies of binding interactions, enzymatic turnover, and other chemical reactions. The detectors are made by using microscale fabrication technology, resulting in a nearly 3 orders of magnitude reduction in both the sample quantity and the measurement time over conventional microcalorimetry. The fabrication technology is low-cost and enables fabrication of 96-detector arrays, which we call enthalpy arrays, on large substrates. Accordingly, the technology will scale to high-volume production of disposable arrays. This increase in performance and reduction in cost promises to enable calorimetry to be used to investigate a substantial number of samples. Nanocalorimetry in the enthalpy array format has valuable applications in proteomics for protein interaction and protein chemistry research and in high-throughput screening and lead optimization for drug discovery. Materials and MethodsDevice Fabrication. The schematic cross section of a nanocalorimeter detector is shown in Fig. 1a. The d...

Higher throughput thermodynamic measurements can provide value in structure-based drug discovery during fragment screening, hit validation, and lead optimization. Enthalpy can be used to detect and characterize ligand binding, and changes that affect the interaction of protein and ligand can sometimes be detected more readily from changes in the enthalpy of binding than from the corresponding free-energy changes or from protein-ligand structures. Newer, higher throughput calorimeters are being incorporated into the drug discovery process. Improvements in titration calorimeters come from extensions of a mature technology and face limitations in scaling. Conversely, array calorimetry, an emerging technology, shows promise for substantial improvements in throughput and material utilization, but improved sensitivity is needed.

A metodologia de modelos mistos (REML/BLUP) tem sido empregada para estudar os efeitos da interação genótipo x ambiente (G x E) em várias culturas, como: arroz, feijão, cana-de-açúcar, cajueiro e eucalipto, porém ainda não foi aplicada em feijão-caupi. Assim, o objetivo deste trabalho foi selecionar simultaneamente genótipos de feijão-caupi semiprostrado cultivados no Estado do Mato Grosso do Sul, via modelos mistos, que reúnam alta adaptabilidade, estabilidade e produtividade de grãos. Foram conduzidos quatro ensaios de valor de cultivo e uso com genótipos de genótipos de feijão-caupi nos anos de 2005 e 2006 em Aquidauana, Chapadão do Sul e Dourados. O delineamento experimental utilizado foi o de blocos completos casualizados, com 20 genótipos e 4 repetições. Os parâmetros genéticos foram estimados pela metodologia REML/BLUP, e a seleção baseou-se no método da média harmônica do desempenho relativo dos valores genéticos (MHPRVG), em três estratégias: seleção com base no valor genético predito, tendo-se considerado o desempenho médio dos genótipos em todos os ambientes (sem efeito de interação) ou o desempenho em cada ambiente (com efeito da interação); e seleção simultânea quanto à produtividade de grãos, estabilidade e adaptabilidade. Os genótipos BRS Paraguaçu, MNC99-542F-5 e MNC99-508G-1 podem ser cultivados em vários ambientes, pois reúnem alta produtividade de grãos, adaptabilidade e estabilidade. A herdabilidade da média dos genótipos apresentou magnitude variando de moderada a alta, fato que indica excelentes possibilidades para a seleção, permitindo acurácia seletiva de 82%.Palavras-chave: BLUP/REML, parâmetros genéticos, Vigna unguiculata L. Genotype x environment interaction in semiprostrade cowpea genotypes via mixed models AbstractThe mixed model methodology (REML / BLUP) has been used to study the effects of genotype x environment interaction (G x E) in various crops, such as: rice, common bean, cane sugar, cashew and eucalyptus, but still was not applied in cowpea. The aim of this work was to select simultaneously semiprostrade cowpea genotypes grown in the state of Mato Grosso do Sul, via mixed models, bringing together high adaptability, stability and yield grain. It were conducted four growing amount of trials and use of genotypes of cowpea genotypes in 2005 and 2006 in Aquidauana, Chapadão do Sul and Dourados. The experimental design was a randomized complete blocks with four replications and 20 genotypes. The genetic parameters were estimated by REML/BLUP methodology and, the selection was based on the MHPRVG method (harmonic mean of the relative performance of genetic values) in three strategies: selection based on predicted breeding value, having considered the performance mean of genotypes in all environments (no interaction effect) or performance in each environment (with interaction effect); and simultaneous selection for grain yield, stability and adaptability. BRS Paraguaçu, MNC99-542F-5 and MNC99-508G-1 genotypes can be grown in various environments, as they bring together high grain ...

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We demonstrate rapid mixing of sub-microliter droplets (250nl) using miniaturized magnetic stir bars (400 μm by 200 μm by 15 μm). The stir bars are fabricated using laser micromachining and placed on the substrate on which the drops are manipulated. They are activated by an externally applied magnetic field and used in combination with on-demand drop merging in enthalpy arrays. This technique results in a 10-fold increase in mixing rate, and a mixing time constant of about 2 seconds. Drop mixing times are measured by Förster resonance energy transfer (FRET) and verified by thermodynamic measurements of binding and enzymatic reactions.

Fragment-based lead discovery (FBLD) is a technique in which, small, low-complexity chemical fragments of 6 to 15 heavy atoms are screened for binding to or inhibiting activity of the target. Hits are then linked and/ or elaborated into tightly binding ligands, ideally yielding early lead compounds for drug discovery. Calorimetry provides a label-free method to assay binding and enzymatic activity that is unaffected by the spectroscopic properties of the sample. Conventional microcalorimetry is hampered by requiring large quantities of reagents and long measurement times. Nanocalorimeters can overcome these limitations of conventional isothermal titration calorimetry. Here we use enthalpy arrays, which are arrays of nanocalorimeters, to perform an enzyme activity-based fragment screen for competitive inhibitors of phosphodiesterase 10A (PDE10A). Two dozen fragments with KI <2 mM were identified and moved to crystal soaking trials. All soak experiments yielded high resolution diffraction with two-thirds of the fragments yielding high-resolution co-crystal structures with PDE10A. The structural information was used to elaborate fragment hits, yielding leads with KI <1 µM. This study shows how array calorimetry can be used as a prescreening method for fragment-based lead discovery with enzyme targets and paired successfully with an x-ray crystallography secondary screen.