Synergistic Use of Protein Crystallography and Solution‐phase NMR Spectroscopy in Structure‐based Drug Design: Strategies and Tactics

Abstract

Structural biology is an integral part of the drug discovery process in the pharmaceutical industry. Two complementary methods dominate among the experimental techniques capable of providing detailed three-dimensional (3D) structural information of therapeutically relevant targets, most often with bound ligands: protein crystallography and solution-phase NMR spectroscopy. The structural information these techniques provide continues to grow in depth and breadth, impacting every step of drug discovery from lead identification to preclinical testing ( Fig. 12.1). Used together, protein crystallography and solution-phase NMR can significantly increase both the pace of discovery and the quality of the resulting compounds that are moved forward into development.The benefits to drug discovery of rapidly available, high-resolution 3D structural information provided by protein crystallography are well established [1,2]. While the technique's success depends on diffraction-quality protein crystals, significant advances in protein biochemistry and protein crystallization technologies have dramatically increased the number of targets amenable to structure determination by X-ray diffraction. Driven in large part by a number of worldwide structural genomics efforts underway in both academia and industry [3], these advances include robotic systems for cloning, expression, protein purification and crystallization [4]. In addition, continued hardware and software developments, both at synchrotron and in-house X-ray sources, have decreased the time of de novo structure solution from months to days, while co-crystal structures are routinely obtained within hours. Thus, once diffraction-quality crystals are obtained, structure determination is not a bottleneck in the discovery process. In fact, a number of drugs on the market were discovered in large part by routine use of structural information provided by protein crystallography [2, 5], a clear indication that this technique has become a driver of the design process and not a retrospective analysis tool.In academia, solution-phase NMR spectroscopy continues to develop as a technique for obtaining 3D structural information of macromolecules including target-ligand complexes. Recent advances have made NMR structural work possible 249 Fragment-based Approaches in Drug Discovery. Edited

Fragment-Based Drug Discovery: What has it Achieved so Far?

“…Due to the characteristics of its active site, evolved to recognise the substrate phosphotyrosine, PTP1B has proven to be a challenging target for drug discovery. Extensive HTS screening of PTP1B using conventional methods did not provide any viable leads [18].…”

“…Each of the 62 examples is listed in Table 1 with the structure of the initial fragment hit, the resulting lead structure together with their potencies and ligand efficiencies. The individual targets are c-Src [67], adenosine kinase [68], t-RNA-guanine transglycosylase [69], PTP1B [18,70,71,72], DNA gyrase [73], thrombin [25,74,75], FKBP [33], U1061A RNA [76], MMP3 [77,78], human papillomavirus E2 protein (HPV E2) [79], Bcl-xL [80], Bcl-2 [81], LFA-1/ICAM-1 [82], glycogenphosphorylase [83] urokinase [84,85,86], gelatinase B [87], Phosphodiesterase-4 (PDE4) [88], HPV E1 helicase [89], thymidylate synthase [90], ErmAM [91], P38 [92,93,94], carbonic anhydrase [95,96,97], neuraminidase [98], NS3 protease [99], cyclin dependent kinase-2 (CDK2) [100,101], caspase-3 [102], Dipeptidylpeptidase IV (DPP IV) [103,104], Fab I [105], crucain [106], CXC-chemokine receptor 2 ...…”

“…The technologies that have played a major role in driving the development of fragment discovery are NMR spectroscopy [4][5][6], X-ray crystallography [7][8][9][10][11], mass spectrometry [12] as well as in silico approaches like virtual screening [13][14][15][16], and more recently ITC experiments [17]. These methods are also sometimes used in a synergistic approach, for example combining NMR-based screening with X-ray structure determination *Address correspondence to this author at the Pfizer Global Research and Development, Sandwich Laboratories, Ramsgate Road, Sandwich, Kent CT13 9NJ, United Kingdom; E-mail: alexander.alex@pfizer.com [18]. Fragment-based drug discovery has been a very active field of research in the last ten years and there are a number of recent reviews on the fundamental principles, current appro-aches and new developments in the field of fragment screening [13,19] and fragment-based discovery ("Fragonomics") [20].…”