Hit confirmation

At the completion of a primary screening of a compound library, a collection of hits will be identified that meet or exceed the inhibition percentage cutoff for hit declaration (as described above). The next step is to ensure the validity of these primary screening results through a series of experimental procedures aimed at addressing two aspects of hit validation hit confirmation and hit verification. 

The term hit confirmation, as we define it, involves three components reproducibility, confirmation of chemical structure, and confirmation of chemical purity. Confirmation of hit reproducibility requires that the subset of library compounds designated as hits in the primary screen be identified, that samples of each of these be obtained from the library bank (a process often referred to as cherry picking ), and that these samples be retested, at least once but preferably multiple times, to determine if they reproducibly confer an inhibition percentage of the target enzyme... 

Dataset Primary (%) Confirmation (%) Compounds Hits" Confirmation rate (%)... 

Our experience in QSAR model development and validation has led us to establishing a complex strategy that is summarized in Fig. 6.2. It describes the predictive QSAR modeling work-flow, which focuses on delivering validated models and ultimately, computational hits confirmed by the experimental validation. We... 

Subsequently, a 1 pL per well substrate solution is dispensed to the assay plates in another Multidrop-Combi equipped with plate stackers. After 30-min incubation at room temperature, the operator manually moves the assay plates to a plate detector with stackers and executes the measurement of assay plates in a specific detection mode (fluorescence intensity in this case). The data file from the plate reader and the tracer file will be copied to a computer for data analysis after the experiment. This screening platform is very useful for laboratories that screen small compound collections, assay validation with the LOPAC collection, and follow-up screens for hit confirmation and lead optimization. 

Fig. 1 The Screening Paradigm. After selection of a target, a project has to pass through a variety of stages including assay development, optimization of the assay, adaptation of the screen to HTS, validation, primary screening of the compound collection, and hit confirmation with dose response determination, finally resulting in the identification of lead compounds.

The overall process and critical activities for robotic implementation of automated HTS from intake of the researcher s benchtop assay through to the identification of a validated, optimized lead series of compounds around a bona fide chemical scaffold are shown in Fig. 2. The downstream steps of hit confirmation/ validation and structure-activity relationship (SAR) elucidation and hit to lead (HTL) have been included to emphasize that the same robotic assay used for production HTS can and should be used by the HTS team to perform high-volume hit confirmation and first-line automated dose-response analysis of confirmed hits to obtain quantitative potency (IC50) rankings to elucidate the nascent SAR of emergent hit series... 

Overall, we have to accept that a perfect match of experimental conditions is not feasible and that a lack of hit confirmation may not result from an issue with a particular biophysical method. Further we need to accept that some valid hits will not be confirmed and, consequently, not considered for follow up work. 

D fingerprint Tanimoto similarity of the hits confirmed that the field-based approaches were better at scaffold hopping and delivered more diverse hits. 

Principle activities Target assessment Early target validation Early assay development Start preparing TPP Screening Hit confirmation Hit-to-lead chemistry Optimization for activity, selectivity, ADMET In vivo proof-of-ooncept Process development Animal pharmacology Toxicology Proposed clinical protocols... 

Zhang, J.H. et al. Probing the primaiy screening efficiency by multiple replicate testing A quantitative analysis of hit confirmation and false screening results of a biochemical assay. J. Biomol. Screen. 2005,10, 695-704. 

DHFR) inhibitor classes (e.g., methotrexate and methylprednisolone), they induced the AMLl/ETO abrogation signature at low nanomolar concentration. Hit confirmation was done by cell-based assays, where the compounds induced myeloid differentiation, dramatically reduced cell viability, and ultimately induced apoptosis. 

Target identification Screening Hit confirmation => lead Lead optimization Based on medical symptoms Using laboratory animals or cell cultures Use of second animal species Classical synthesis of 10 to 20 similar compounds, testing on laboratory animals Based on receptor hindii Using receptor hindii assays with st detection techniques Use of second assay type High throughput synthesis of 1000 to 100,000 compounds tested with HTS receptor hindii assays... 

Cell number can be increased to ensure proper differentiation, and the number should be re-titrated when developing this assay, as counting can vary from counter to counter. If viability is lower than 90 % it is possible to increase cell number per well, but this is only recommended when running small-scale hit confirmation experiments. 

---