Fragment Based Drug Design, Volume 493: Tools, Practical Approaches, and Examples (Methods in Enzymology)

Fragment Based Drug Design, Volume 493: Tools, Practical Approaches, and Examples (Methods in Enzymology)

Language: English

Pages: 645

ISBN: 0123812747

Format: PDF / Kindle (mobi) / ePub


There are numerous excellent reviews on fragment-based drug discovery (FBDD), but there are to date no hand-holding guides or protocols with which one can embark on this orthogonal approach to complement traditional high throughput screening methodologies. This Methods in Enzymology volume offers the tools, practical approaches, and hit-to-lead examples on how to conduct FBDD screens. The chapters in this volume cover methods that have proven to be successful in generating leads from fragments, including chapters on how to apply computational techniques, nuclear magnetic resonance, surface plasma resonance, thermal shift and binding assays, protein crystallography, and medicinal chemistry in FBDD. Also elaborated by experienced researchers in FBDD are sample preparations of fragments, proteins, and GPCR as well as examples of how to generate leads from hits.

Offers the tools, practical approaches, and hit-to-lead examples on how to conduct FBDD screens The chapters in this volume cover methods that have proven to be successful in generating leads from fragments, including chapters on how to apply computational techniques, nuclear magnetic resonance, surface plasma resonance, thermal shift and binding assays, protein crystallography, and medicinal chemistry in FBDD

 

 

 

 

 

 

 

 

 

 

canulae, 100% oxygen is distributed to each well at a flow rate of $3.5 cfm. We have found that the dual-functioning canulae, providing both oxygenation and lift (large bubble size), necessitate a high percentage of oxygen addition for the greatest yield. This is potentially dangerous, and strong system ventilation must be used for safety. When OD600 reaches 5–6 units, each tube receives IPTG (1 mM final concentration) and Antifoam Y-30 Emulsion through ports in the Airlift Fermentation System

times until each well has received 1000 mL of the appropriate buffer. A candidate-purified protein is then transferred ($70 mL) to each well to sample all the 12 conditions. Buffer-exchanged samples are collected following centrifugation for 2 min at 1000Âg-force. The eluate containing the bufferexchanged protein is then loaded into 1.7-mm NMR microprobe tubes using a Gilson 215 Liquid Handler. Each tube is stored at room temperature and scored by visual inspection for precipitation after 10

times until each well has received 1000 mL of the appropriate buffer. A candidate-purified protein is then transferred ($70 mL) to each well to sample all the 12 conditions. Buffer-exchanged samples are collected following centrifugation for 2 min at 1000Âg-force. The eluate containing the bufferexchanged protein is then loaded into 1.7-mm NMR microprobe tubes using a Gilson 215 Liquid Handler. Each tube is stored at room temperature and scored by visual inspection for precipitation after 10

Therefore, a very conservative hit selection was made using a T/R ratio of about 0.5, which resulted in 79 fragments being defined as hits. We assessed whether the decay in the amount of functional b1AR affected our ability to detect ligand binding by investigating a potential relationship between the hits and the cycle in which they were analyzed. While a decline in the number of hits found per cycle was not found, it is entirely possible that the selection became more stringent as the screen

literature offers few examples of purely computational FBDD campaigns, there are reports of hits found computationally that can be developed into lead molecules of sufficient interest to spur a lead optimization campaign (e.g., Bo¨hm et al., 2000 and Teotico et al., 2009). In addition, computational methods can play a key role in selecting a set of fragments and in the structurebased elaboration of a fragment hit identified by experimental screening whatever the screening method may be. As our

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