We propose to develop general and robust HTS methods that enable the discovery of small molecules that switch, for example, disease states to healthy states without requiring knowledge of the relevant cellular target(s) in advance of the screen. These methods require the ability to perform multiple measurements per well that together define signatures of the relevant states. Small-molecule, high-throughput screens often entail a single measurement per well, for example, the activity of a singular, purified enzyme. In principle and in practice, the ability to perform multiple measurements per well provides novel scientific insights, as demonstrated through high-content expression- and image-based screening. To illustrate: 1) cells treated with small molecules have been probed with Luminex beads to determine the relative amounts of RNAs (mRNA, miRNA)13'43, 2) multiple cellular features have been measured and selected as state classifiers using image-analysis software developed by researchers at the Broad Institute38'39, and 3) protein microarray and Luminex bead-based methods have been adapted to the analyses of phosphoproteins in cells65'66. Such capabilities underlie signature-based state-switching screens, which permit the probing of biological and disease circuitries in order to discover small molecules able to switch one state to the other. (Methods for making multiple measurements can also permit extremely efficient screens where each of the individual measurements is of particular, but not necessarily related, importance, e.g., where the many RNAs being measured each represent a singular biological or therapeutic probe.)
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