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The Assay Guidance Manual (AGM) is an eBook of best practices for the design, development, and implementation of robust assays for early drug discovery. Initiated by pharmaceutical company scientists, the manual provides guidance for designing a "testing funnel" of assays to identify genuine hits using high-throughput screening (HTS) and advancing them through preclinical development. Combined with a workshop/tutorial component, the overall goal of the AGM is to provide a valuable resource for training translational scientists.

Before insulin can stimulate myocytes to take up glucose, it must first move from the circulation to the interstitial space. The continuous endothelium of skeletal muscle (SkM) capillaries restricts insulin's access to myocytes. The mechanism by which insulin crosses this continuous endothelium is critical to understand insulin action and insulin resistance; however, methodological obstacles have limited understanding of endothelial insulin transport in vivo. Here, we present an intravital microscopy technique to measure the rate of insulin efflux across the endothelium of SkM capillaries. This method involves development of a fully bioactive, fluorescent insulin probe, a gastrocnemius preparation for intravital microscopy, an automated vascular segmentation algorithm, and the use of mathematical models to estimate endothelial transport parameters. We combined direct visualization of insulin efflux from SkM capillaries with modeling of insulin efflux kinetics to identify fluid-phase transport as the major mode of transendothelial insulin efflux in mice. Model-independent experiments demonstrating that insulin movement is neither saturable nor affected by insulin receptor antagonism supported this result. Our finding that insulin enters the SkM interstitium by fluid-phase transport may have implications in the pathophysiology of SkM insulin resistance as well as in the treatment of diabetes with various insulin analogs., Introduction Interorgan communication is essential for maintaining physiological homeostasis. One way organs communicate with each other is by sending molecular signals through the circulation. Before these signals can stimulate biological [...]

Those suffering from Duchenne muscular dystrophy share with mdx mice a lack of dystrophin, which induces a loss of dystrophin-associated proteins (DAPs) in the sarcolemma. This loss could be mitigated by the dystrophin-related protein (DRP) utrophin, which is found in the sarcolemma in cardiac and skeletal muscles of adult mdx mice. Because DRP and DAP colocalize to the neuromuscular junction, DRP could help retain DAP in the sarcolemma.

Duchenne muscular dystrophy (DMD) is a disease of the muscles caused by a defective gene on the X chromosome; it affects only boys and usually results in death by age 20. Study of this defective DMD gene has revealed coding for dystrophin, an integral protein in the membrane of normal skeletal muscle fiber, but people with DMD are missing this protein. The study of other genetic diseases has revealed that when one protein is affected, others associated with it are affected also. In DMD patients, one of the four glycoproteins (carbohydrate and protein complexes) that make up the structure of this protein is deficient. It is hypothesized that the reduction of this dystrophin-associated glycoprotein may hold a clue to discovering the cause of this type of muscular dystrophy. Reduction of this glycoprotein can lead to a destabilization of the plasma membrane around the skeletal muscle cell, and the proteins associated with this membrane. This membrane destabilization can in turn lead to a deregulation of the intracellular calcium, which could cause abnormal activation of calcium-dependent protease (enzymes that breakdown proteins) activity. In DMD the muscle protein gradually degenerates, which could be explained by this proposed abnormal enzymatic activity. (Consumer Summary produced by Reliance Medical Information, Inc.

Abstract: The Assay Guidance Manual (AGM) is an eBook of best practices for the design, development, and implementation of robust assays for early drug discovery. Initiated by pharmaceutical company scientists, the manual provides guidance for designing a “testing funnel” of assays to identify genuine hits using high‐throughput screening (HTS) and advancing them through preclinical development. Combined with a workshop/tutorial component, the overall goal of the AGM is to provide a valuable resource for training translational scientists. [ABSTRACT FROM AUTHOR]

High Throughput Screening (HTS) assays are used to rapidly identify promising drug candidates or leads from hundreds of thousands of compounds. Hence, it is important that the assay should be designed to discriminate responses from the active and inactive compounds and the background noise. We introduce the idea of a signal window which provides a degree of separation between signals. This allows one to correctly identify new molecular entities with desired level of activity (called "hits") in the presence of variability. The statistical criteria for setting and calculating signal windows are presented along with illustrative examples. Results show that the ideal signal window should be 2 standard deviations (SD) of the largest signal in screening assays, although a 1 SD window size is the minimum acceptable limit. When signal windows are set below 2 SD, the probability of missing "hits" increases significantly. [ABSTRACT FROM PUBLISHER]

Membranes prepared from stable transfected cells expressing the human gene encoding a functional 5-hydroxytryptaminelE (5HT1E) receptor were used to investigate high-affinity [3H]serotonin ([3H]5-HT) binding with scintillation proximity assay (SPA) technology. In this nonseparation format, membranes are captured by WGA-coated SPA fluoromicrospheres for detection of receptor-bound radioligand. Total binding observed was approximately 2000 cpm compared to a nonspecific signal of 100 cpm determined in the presence of 10,uM unlabeled 5-HT. Non-proximity effects (NPE) for the radiolabel and SPA beads averaged less than 100 cpm. Saturation binding analysis yielded an equilibrium dissociation constant (Kd) of 5.38 ± 0.43 nM and a maximum binding capacity (Bmax) of 6.42 + 0.15 pmol/mg protein. Competition with unlabeled serotonergic compounds demonstrated a specificity of the assay with rank potencies (5-HT > a-Me-5-HT > 2-Me-5-HT > 5-CT) similar to those observed using traditional fitration techniques. The variability of the assay and the stability of all reagents were investigated to validate the assay for extended use throughout a typical high throughput screen operation lasting several months. [ABSTRACT FROM PUBLISHER]