Fragment-based lead discovery using X-ray crystallography.

Fragment screening offers an alternative to traditional screening for discovering new leads in drug discovery programs. This paper describes a fragment screening methodology based on high throughput X-ray crystallography. The method is illustrated against five proteins (p38 MAP kinase, CDK2, thrombin, ribonuclease A, and PTP1B). The fragments identified have weak potency (>100 microM) but are efficient binders relative to their size and may therefore represent suitable starting points for evolution to good quality lead compounds. The examples illustrate that a range of molecular interactions (i.e., lipophilic, charge-charge, neutral hydrogen bonds) can drive fragment binding and also that fragments can induce protein movement. We believe that the method has great potential for the discovery of novel lead compounds against a range of targets, and the companion paper illustrates how lead compounds have been identified for p38 MAP kinase starting from fragments such as those described in this paper.

[1]  Paul Greengard,et al.  Pharmacological inhibitors of cyclin-dependent kinases. , 2002, Trends in pharmacological sciences.

[2]  L Meijer,et al.  Cyclin-dependent kinases: initial approaches to exploit a novel therapeutic target. , 1999, Pharmacology & therapeutics.

[3]  Youwei Yan,et al.  Small, low nanomolar, noncovalent thrombin inhibitors lacking a group to fill the 'distal binding pocket'. , 2003, Bioorganic & medicinal chemistry letters.

[4]  J. Pflugrath,et al.  The finer things in X-ray diffraction data collection. , 1999, Acta crystallographica. Section D, Biological crystallography.

[5]  G. Bemis,et al.  Kinase inhibitors and the case for CH…O hydrogen bonds in protein–ligand binding , 2002, Proteins.

[6]  T. N. Bhat,et al.  The Protein Data Bank , 2000, Nucleic Acids Res..

[7]  E. Mandine,et al.  Requirements for specific binding of low affinity inhibitor fragments to the SH2 domain of (pp60)Src are identical to those for high affinity binding of full length inhibitors. , 2003, Journal of medicinal chemistry.

[8]  S F Campbell,et al.  Science, art and drug discovery: a personal perspective. , 2000, Clinical science.

[9]  Johan Schultz,et al.  Structure-based screening and design in drug discovery. , 2002, Drug discovery today.

[10]  Christopher W Murray,et al.  Identification of novel p38alpha MAP kinase inhibitors using fragment-based lead generation. , 2005, Journal of medicinal chemistry.

[11]  Jean M. Severin,et al.  Discovery of potent inhibitors of dihydroneopterin aldolase using CrystaLEAD high-throughput X-ray crystallographic screening and structure-directed lead optimization. , 2004, Journal of medicinal chemistry.

[12]  Andrew R. Leach,et al.  Molecular Complexity and Its Impact on the Probability of Finding Leads for Drug Discovery , 2001, J. Chem. Inf. Comput. Sci..

[13]  Jean-Pierre Marquette,et al.  SAR and X-ray. A new approach combining fragment-based screening and rational drug design: application to the discovery of nanomolar inhibitors of Src SH2. , 2002, Journal of medicinal chemistry.

[14]  Andrew J. Sharff.  HIGH THROUGHPUT CRYSTALLOGRAPHY ON AN IN-HOUSE SOURCE, USING ACTOR , 2003 .

[15]  G. Bemis,et al.  Inhibitors of p38 MAP kinase: therapeutic intervention in cytokine-mediated diseases. , 1999, Current medicinal chemistry.

[16]  S. Kim,et al.  High-resolution crystal structures of human cyclin-dependent kinase 2 with and without ATP: bound waters and natural ligand as guides for inhibitor design. , 1996, Journal of medicinal chemistry.

[17]  P J Thomas,et al.  Crystal structures of thrombin complexed to a novel series of synthetic inhibitors containing a 5,5-trans-lactone template. , 1999, Biochemistry.

[18]  S. Brady,et al.  Design and synthesis of a series of potent and orally bioavailable noncovalent thrombin inhibitors that utilize nonbasic groups in the P1 position. , 1998, Journal of medicinal chemistry.

[19]  K. Acharya,et al.  Crystal structures of ribonuclease A complexes with 5'-diphosphoadenosine 3'-phosphate and 5'-diphosphoadenosine 2'-phosphate at 1.7 A resolution. , 1997, Biochemistry.

[20]  D. Kostrewa,et al.  Novel inhibitors of DNA gyrase: 3D structure based biased needle screening, hit validation by biophysical methods, and 3D guided optimization. A promising alternative to random screening. , 2000, Journal of medicinal chemistry.

[21]  M. Congreve,et al.  A 'rule of three' for fragment-based lead discovery? , 2003, Drug discovery today.

[22]  Vicki L. Nienaber,et al.  Discovering novel ligands for macromolecules using X-ray crystallographic screening , 2000, Nature Biotechnology.

[23]  T R Burke,et al.  Structural basis for inhibition of the protein tyrosine phosphatase 1B by phosphotyrosine peptide mimetics. , 1998, Biochemistry.

[24]  Karl A. Walter,et al.  ChemInform Abstract: Discovery of Potent Nonpeptide Inhibitors of Stromelysin Using SAR by NMR. , 1997 .

[25]  J. Maraganore,et al.  Thrombin structure and function: why thrombin is the primary target for antithrombotics. , 1991, Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis.

[26]  Protein-Ligand Interactions,et al.  Knowledge-based Scoring Function to Predict , 2000 .

[27]  R. Murray.  Molecular recognition. , 2009, Analytical Chemistry.

[28]  D. Zaller,et al.  Structural basis for p38alpha MAP kinase quinazolinone and pyridol-pyrimidine inhibitor specificity. , 2003 .

[29]  J. Gasteiger,et al.  Automatic generation of 3D-atomic coordinates for organic molecules , 1990 .

[30]  D. E. Clark,et al.  Flexible docking using tabu search and an empirical estimate of binding affinity , 1998, Proteins.

[31]  Ajay,et al.  The SHAPES strategy: an NMR-based approach for lead generation in drug discovery. , 1999, Chemistry & biology.

[32]  J. Navaza,et al.  AMoRe: an automated package for molecular replacement , 1994 .

[33]  Richard D. Taylor,et al.  Improved protein–ligand docking using GOLD , 2003, Proteins.

[34]  Marcel L. Verdonk,et al.  The consequences of translational and rotational entropy lost by small molecules on binding to proteins , 2002, J. Comput. Aided Mol. Des..

[35]  R. Stroud,et al.  Site-directed ligand discovery. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[36]  A. Bitonti,et al.  Crystal structure of human cyclin-dependent kinase 2 in complex with the adenine-derived inhibitor H717. , 2001, Journal of medicinal chemistry.

[37]  Paul Watson,et al.  A web-based platform for virtual screening. , 2003, Journal of molecular graphics & modelling.

[38]  E. Goldsmith,et al.  The structure of mitogen-activated protein kinase p38 at 2.1-A resolution. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[39]  J. Greer,et al.  Automated crystal mounting and data collection for protein crystallography. , 2000, Structure.

[40]  R. Glen,et al.  Molecular recognition of receptor sites using a genetic algorithm with a description of desolvation. , 1995, Journal of molecular biology.

[41]  A. Hopkins,et al.  Ligand efficiency: a useful metric for lead selection. , 2004, Drug discovery today.

[42]  D. Lane,et al.  Inhibitors of cyclin-dependent kinases as anti-cancer therapeutics. , 2000, Current medicinal chemistry.

[43]  Tudor I. Oprea,et al.  The Design of Leadlike Combinatorial Libraries. , 1999, Angewandte Chemie.

[44]  Bin Ye,et al.  Crystal structures of two potent nonamidine inhibitors bound to factor Xa. , 2002, Biochemistry.

[45]  Dustin J Maly,et al.  Combinatorial target-guided ligand assembly: identification of potent subtype-selective c-Src inhibitors. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[46]  Elizabeth J. Goldsmith,et al.  Acquisition of Sensitivity of Stress-activated Protein Kinases to the p38 Inhibitor, SB 203580, by Alteration of One or More Amino Acids within the ATP Binding Pocket* , 1998, The Journal of Biological Chemistry.

[47]  G. Bemis,et al.  Properties of known drugs. 2. Side chains. , 1999, Journal of medicinal chemistry.

[48]  P Willett,et al.  Development and validation of a genetic algorithm for flexible docking. , 1997, Journal of molecular biology.

[49]  L. Iversen,et al.  2-(Oxalylamino)-Benzoic Acid Is a General, Competitive Inhibitor of Protein-tyrosine Phosphatases* , 2000, The Journal of Biological Chemistry.

[50]  T. J. R. Harris.  High throughput X-ray crystallography for Drug Discovery , 2000 .

[51]  B. Kennedy,et al.  Structure of protein tyrosine phosphatase 1B in complex with inhibitors bearing two phosphotyrosine mimetics. , 2001, Journal of medicinal chemistry.

[52]  Johan Schultz,et al.  Structure-based screening as applied to human FABP4: a highly efficient alternative to HTS for hit generation. , 2002, Journal of the American Chemical Society.

[53]  N. Tonks,et al.  Protein tyrosine phosphatases: a diverse family of intracellular and transmembrane enzymes. , 1991, Science.

[54]  Tudor I. Oprea,et al.  Is There a Difference Between Leads and Drugs? A Historical Perspective. , 2001 .

[55]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.

[56]  L. Vitagliano,et al.  Productive and nonproductive binding to ribonuclease A: X‐ray structure of two complexes with uridylyl(2′,5′)guanosine , 2000, Protein science : a publication of the Protein Society.

[57]  G. Bemis,et al.  The properties of known drugs. 1. Molecular frameworks. , 1996, Journal of medicinal chemistry.

[58]  A V Finkelstein,et al.  The price of lost freedom: entropy of bimolecular complex formation. , 1989, Protein engineering.

[59]  Michael J Hartshorn,et al.  AstexViewer: a visualisation aid for structure-based drug design. , 2002, Journal of computer-aided molecular design.

[60]  H. Jhoti,et al.  Structure-based screening of low-affinity compounds. , 2002, Drug discovery today.

[61]  G. Murshudov,et al.  Refinement of macromolecular structures by the maximum-likelihood method. , 1997, Acta crystallographica. Section D, Biological crystallography.

[62]  F. Lombardo,et al.  Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. , 2001, Advanced drug delivery reviews.