Dissection of the Recognition Properties of p38 MAP Kinase. Determination of the Binding Mode of a New Pyridinyl-Heterocycle Inhibitor Family Robert Soliva, ²,‡,§ Josep Lluis Gelpı ´, ²,‡, ⊥ Carmen Almansa, § Marina Virgili, § and Modesto Orozco* ,²,‡, ⊥,# Departament de Bioquı ´mica i Biologia Molecular, Facultat de Quı ´mica, UniVersitat de Barcelona, Martı ´ i Franque ` s 1, Barcelona 08028, Spain, Molecular Modeling and Bioinformatics Unit, Institut de Recerca Biome ´ dica, Parc Cientı ´fic de Barcelona, Josep Samitier 1-5, Barcelona 08028, Spain, Drug DiscoVery, J. Uriach y Cia, Camı ´ Reial 51-57, Poligon Industrial Riera de Caldes, Palau de Plegamans, Barcelona 08184, Spain, Computational Biology Program, Barcelona Supercomputing Center, Jordi Girona 31, Edifici Nexos II, Barcelona 08028, Spain, and Structural Bioinformatics Node, Instituto Nacional de Bioinforma ´ tica, Parc Cientı ´fic de Barcelona, Josep Samitier 1-5, Barcelona 08028, Spain ReceiVed September 12, 2006 The main recognition characteristics of the ATP binding site of p38 mitogen activated protein kinase alpha (p38R MAPK) have been explored by a combination of modeling and bioinformatics techniques, making special emphasis in the characteristics of the site that justifies binding specificity with respect to other MAP kinases. Particularly, we have analyzed the binding mode of a new family of p38 MAPK inhibitors based on the pyridinyl-heterocycle core. This family of compounds has a marked pseudosymmetry and can exist in different tautomeric forms, which makes the determination of the binding mode especially challenging. A combination of homology modeling, quantum mechanics, classical docking, and molecular dynamics calculations allowed us to determine the main characteristics defining the binding mode of this new scaffold in the ATP binding site of p38R. A set of free energy calculations allowed us to verify the binding mode proposed, giving an overall excellent agreement with the experimental values. Finally, the binding mode of this new family of compounds was compared to that of other members of the pyridinyl and pyrimidinyl heterocycle class. Introduction Tumor necrosis factor-R (TNFR) and interleukin-1 (IL-1) are two cytokines involved in many basic cellular processes such as response to infectious agents or cellular stress. 1 High levels of these cytokines are also associated with the develop- ment of a variety of inflammatory diseases such as Crohn’s disease, toxic shock syndrome, rheumatoid arthritis (RA), psoriasis, and inflammatory bowel disease (IBD). 2 Reduction in the levels of TNFR and IL-1 has been recognized as one of the most promising strategies to fight an acute or chronic inflammatory response. 3 The clinical success of Enbrel (Etan- ercept), 4 a soluble TNF-R receptor, and Remicade (Infliximab), 5 an antibody against TNF-R, in the treatment of RA and IBD has confirmed the utility of this approach. Unfortunately, and despite their success, both Enbrel and Remicade are facing the typical drawbacks associated with a protein therapeutic: high production costs and intravenous administration. This explains the great interest in developing new small molecules able to modulate the levels of TNFR and IL-1 without the problems inherent to the pharmaceutical use of proteins. A key step in the complex sequence of events leading to the production of TNF-R and IL-1 is the activation of p38 mitogen activated protein kinase (MAPK), 6 a serine/threonine kinase involved in major signal transduction pathways. Activation of p38 MAP kinase is due to upstream MAP kinases like MMK3 and MMK6 which phosphorylate residues Thr180 and Tyr182 located in the activation loop. 6 Once activated, p38 MAPK is able to phosphorylate other downstream kinases, transcription factors, and probably AU binding proteins leading to the production of TNF-R and IL-1. 7 This whole signal transduction pathway is activated when cells are exposed to a variety of extracellular stimuli ranging from bacterial lipopolysaccharide and UV light to proinflammatory cytokines or growth factors. 8 P38 MAPK has a large homology with other members of the MAPK superfamily like extracellular signal-regulated ki- nases (ERK) and c-Jun N-terminal kinases (JNK), with which p38 shares a high sequence identity. These three kinases share a common fold and a high level of sequence identity which handicapped the design of p38 MAPK inhibitors. 8,9 The issue of selectivity is further complicated by the existence of at least four splicing isoforms of p38 (R, , δ, and γ) which have a large degree of sequence identity, but different tissue distribu- tion, and probably also different functions. 7 Thus, inhibition of the R isoform of p38 MAPK by small molecules becomes an especially challenging task, since a drug must not only bind tightly to the target enzyme but also avoid interaction with other very closely related kinases, which might trigger unpredictable side effects. In this paper, we will try to clarify key differences between the binding site of p38 MAPK and those of other closely related enzymes. The three-dimensional structure of the complex between p38 MAPK and compound 1 (Figure 1) 10 showed the ATP binding site as the recognition region for the inhibitor. The complex was stabilized by two key interactions: (i) a hydrogen bond between pyridine nitrogen (that mimicking N1 of ATP) and the amide proton of Met109 and (ii) a hydrophobic interaction between the 4-fluorophenyl group and a buried apolar pocket in the protein. Additional contacts were a hydrogen bond between imidazole nitrogen and Lys53 and a π-stacking between the sulfonyl-phenyl group and Tyr35, both of which were partly exposed to solvent. This structural knowledge provided the basis * Corresponding author. E-mail: modesto@mmb.pcb.ub.es. Phone: 34 93 4037156. Fax: 34 93 4037157. ² Universitat de Barcelona. ‡ Institut de Recerca Biome ´dica. § J. Uriach y Cia. ⊥ Barcelona Supercomputing Center. # Instituto Nacional de Bioinforma ´tica. 283 J. Med. Chem. 2007, 50, 283-293 10.1021/jm061073h CCC: $37.00 © 2007 American Chemical Society Published on Web 12/22/2006