Promiscuous compound filter

Experiment Serum Dilution Positive Ion Negative ion Unique Cpds MurF Hits/ Serum Hits Serum Hits / Total Library 

There are approximately 2700 compounds per primary screening mixture, and the readout is in essence multiplexed the ligands are individually ionized and identified in the mass spectrometer according to their exact mass positions. The readout, however, does not unambiguously identify compounds, as multiple compounds in a single mixture may have the same mass, i.e., a particular peak may correspond to as many as 31 compounds with closely related masses. The protein excess over individual compounds coupled with the rarity of potent ligands within a randomly assembled library minimizes competition between ligands for 

174 I 4 Library Screening Using Ultrafiltration and Mass Spectrometry 

Both round 0 data (before affinity selection) and round 3 data (after three rounds of affinity selection) are shown, where round 0 represents a sampling prior to any ultrafiltration. Round 0 and round 3 samples undergo identical denaturation/solvent extraction procedures. Data were generated 

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Researchers from BMS have detailed the results of their efforts to enhance their HTS process [34]. Their papers discuss the subjectivity of the typical chemical structure triage process as it relates to HTS analysis, a topic worth some investigation by anyone involved in this type of selection process. With the aim of reducing subjectivity in this area, the authors systematically review their filters for physicochemical properties (this topic discussed in more detail below) and unwanted chemical moieties. Much of the work is aimed at understanding and detecting promiscuous compounds - those that initially appear to be hits in many screens (this topic discussed in more detail below). Importantly, the authors have made some of the related tools available for public use. 

However, these compounds and the fragments are not without their intrinsic problems and should not be used as is. Some examples of potentially problematic compounds include those with chemically reactive groups, dyes, and fluorescent compounds which interfere with assays, frequent hitters/promiscuous binders, and inorganic complexes (55). It is important, then, to a priori filter out such compounds or reagents which are practically useless from a drug discovery point of view. 

The compounds with final high rank orders are then tested experimentally using inhibition assays. The number of compounds to test is generally limited by the monetary and time cost of the experimental assays. The assays can then be repeated in the presence of detergent, e.g., Triton X-100, as a first test to filter out promiscuous, aggregate-based inhibitors (46). The inhibitory effect of the active compounds can also be tested on similar enzymes to investigate the specificity of the compounds for the enzyme of interest (see Note 16). 

The first HTS yielded approximately 50,000 hits capable of imparting >95% inhibition of S. aureus growth at 40 pM. Since cherry-picking capacities were limited at this time, a strategy was designed to select the most promising candidates from this hit list. The hypothesis was that the application of strict in silico filters could achieve an enrichment of novel compounds with favorable drug-like properties, and the least potential for promiscuity (24, 25). 

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