Hit validation

The following paragraphs will report on basic NMR techniques that are highly relevant for hit validation and for FBDD applications. 

A simple NMR technique, and arguably the most widely used and effective for hit validation, is the chemical shift perturbation method. In this approach, a reference spectrum of isotopically labeled target is recorded in absence and presence of a given test ligand (or a mixture of test ligands). Commonly, differences in chemical shift between free and bound protein target are observed in 2D [15N, 1H and/or 2D [13C, H] correlation spectra of a protein (or nucleic acid) upon titration of a ligand... 

HTS data and secondary screening for hit validation and determination of kinetic on/off rates. These data have been successfully incorporated into hit triage by enhancing the understanding of SAR differences between potential scaffolds [24]. In this example, those differences allowed informed decision making in the choice of which series to pursue and which to deprioritize. 

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. 

For the application of biomolecular NMR in the process of hit identification and hit validation, four important components have to be optimized (i) supply of the target molecule, (ii) selection of ligands, (iii) hardware for automated sample preparation and high-sensitivity measurements, and (iv) software tools for automated data evaluation. 

Boehm, H.J., Boehringer, M., Bur, D., Gmuender, H., Huber, W., Klaus, W., Kostrewa, D., Kuehne, H., Luebbers, T., Meunier-Keller, N., and Mueller, F. Novel inhibitors of DNAgyrase 3D structure based biased needle screening, hit validation by biophysical methods,... 

Epigenetic Targets in Drug Discovery Cell-Based Assays for HDAC Inhibitor Hit Validation... 

A major advantage is seen upon hit validation. Subsequent hit-lead transitions may be dramatically shorter as relatively simple and fast protocols are already in place. 

The 48-channel MCFB reactor and the catalyst synthesis workflow components were similarly validated in experiments where bulk samples were prepared in library format, screened in the array format, and the data compared with known examples. This part of the workflow was used for initial hit validation and to opti-... 

Kuehne, T. Luebbers, N. Meunler-Keller, and F. Mueller,/. Med. Chem., 43, 2664 (2000). Novel Inhibitors of DNA Gyrase 3D Structure-Based Needle Screening, Hit Validation by Biophysical Methods, and 3D Guided Optimization. A Promising Alternative to Random Screening. 

Figure 2 The 384-microtube plate developed for high-throughput retrieval of compound subsets from large compound collections. At Roche, the 384-microtube plate is used for high-throughput cherry picking of compound samples from the HTS library for hit validation and secondary screening...

The storage of the compound collection, the assembly of the HTS library, and the retrieval of the compound subsets required for hit validation, secondary screening. 

Cherry picking of compound samples for hit validation and secondary screening High-throughput... 

Table 2 Sample aliquoting tree for the supply of the vast amount of samples needed for high-throughput screening. The Liquid Master Store assembles editions of master solutions for preparation of the compound aliquots needed for multiple HTS projects. The Smart Compound Depository delivers the compound subsets from the HTS library needed for hit validation and secondary screening...

NMR can assist the above process at different stages, namely, in hit finding, hit validation, and lead optimization. These three areas of application will be discussed in detail in the following sections. The drug discovery process is summarized in Figure 1, in which shaded boxes indicate where incorporation of NMR techniques is possible. 

In the following detailed discussion of the application of NMR techniques to drug discovery, a short description for hit finding, hit validation, and hit (lead) optimization is given. In addition, the corresponding requirements for the NMR experiments and the resulting implementations are described. Only typical experiments are given explicitly, but other techniques are sunamarized in Table 2. 

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