Molecular basis of cyclooxygenase enzymes (COXs) selective inhibition Vittorio Limongellia,b,1, Massimiliano Bonomia, Luciana Marinellib, Francesco Luigi Gervasioc, Andrea Cavallid,e, Ettore Novellinob, and Michele Parrinelloa,1 aComputational Science, Department of Chemistry and Applied Biosciences, Eidgenössiche Technische Hochschule (ETH) Zürich, Università della Svizzera Italiana Campus, Via Giuseppe Buffi 13, CH-6900 Lugano, Switzerland; bDipartimento di Chimica Farmaceutica e Tossicologica, Università degli Studi di Napoli “Federico II”, Via D. Montesano, 49, I-80131 Naples, Italy; cComputational Biophysics Group, Spanish National Cancer Research Center (CNIO), calle Melchor Fernández Almagro, 3, E-28029 Madrid, Spain; dDepartment of Pharmaceutical Science, University of Bologna, Via Belmerolo 6, I-40126, Bologna, Italy; and eDepartment of Drug Discovery and Development, Italian Institute of Technology, Via Morego 30, I-16163, Genoa, Italy Edited by Michael L. Klein, University of Pennsylvania, Philadelphia, PA, and approved January 25, 2010 (received for review November 20, 2009) The widely used nonsteroidal anti-inflammatory drugs block the and in recent years, much effort has been paid in elucidating the cyclooxygenase enzymes (COXs) and are clinically used for the dynamic binding mechanism of COX inhibitors (7–9). Lanzo et treatment of inflammation, pain, and cancers. A selective inhibition al. (8) have found, by means of the fluorescence quenching tech- of the different isoforms, particularly COX-2, is desirable, and nique, that while the association of the COX-2 selective inhibitor consequently a deeper understanding of the molecular basis of SC-299 with COX-1 and COX-2 occurs at similar rates, the dis- selective inhibition is of great demand. Using an advanced compu- sociation of SC-299 from COX-2 takes hours, which is thousands- tational technique we have simulated the full dissociation process fold slower than in COX-1 (≈30 s). This finding clearly shows that of a highly potent and selective inhibitor, SC-558, in both COX-1 there is a correlation between the relative rates of dissociation and COX-2. We have found a previously unreported alternative and the selectivity of the isoenzyme inhibition, and this correla- binding mode in COX-2 explaining the time-dependent inhibition tion is confirmed also in additional experiments on other COX-2 exhibited by this class of inhibitors and consequently their long selective inhibitors (9). These phenomena have been so far as- residence time inside this isoform. Our metadynamics-based cribed to a more stable binding mode of selective inhibitors into approach allows us to illuminate the highly dynamical character the COX-2 isoform due to the presence of diverse residues such BIOPHYSICS AND of the ligand/protein recognition process, thus explaining a wealth as Val434 in COX-2 instead of Isoleucine in COX-1. However, COMPUTATIONAL BIOLOGY of experimental data and paving the way to an innovative strategy this represents a very simplistic way to address the different rates for designing new COX inhibitors with tuned selectivity. of dissociation of an inhibitor from an enzyme, and very likely other molecular mechanisms might be involved (8–11). nonsteroidal anti-inflammatory drugs ∣ COX selectivity ∣ coxibs ∣ On the other hand, it has to be pointed out that processes that path collective variables ∣ metadynamics take from microseconds to hundreds of seconds, such as binding and unbinding of ligands into protein, are impossible to simu- onsteroidal anti-inflammatory drugs (NSAIDs) are widely late with standard computational techniques such as molecular Nused as therapeutic agents for the treatment of pain and in- dynamics (MD), whose typical timescale is hundreds of nanose- flammation, and in addition several evidences have been very re- conds. Thus, computer simulation of the binding and especially of cently reported on their chemopreventive effect in colorectal the unbinding, which for COX-2 selective inhibitors is responsible cancer (1). Their mechanism of action is based on the blockage of the differences in the rates of dissociation, is an ongoing of the cyclooxygenase enzymes (COXs) (2) by sterically hindering challenge we decided to deal with. the entrance of the physiological binder arachidonic acid. The Thus, to reveal at an atomic level what might happen during classical NSAIDs such as aspirin, ibuprofen, or flurbiprofen the complex formation and dissociation and with the aim to over- are nonselective and inhibit indifferently all the COXs isoforms. come the large free-energy barriers toward the inhibitor-enzyme In the last few decades, the interest of scientists has been mostly undocking process in an affordable computational time, we used focused on a selective inhibition between COX-1/COX-2. In fact, the recently developed well-tempered metadynamics (12), which the inhibition of COX-1, particularly in the gastrointestinal sys- is an evolution of standard metadynamics (13). For this purpose, tem, may lead to dangerous side effects such as ulcers. As a one of the most potent and selective COX-2 inhibitors, SC-558 consequence, a selective inhibition of COX-2 has been sought (IC50 ¼ 9.3 nM) (6), was selected. for decades, and recently a new generation of NSAIDs, namely We have found a previously unreported alternative binding coxibs, was found. However, some of them, such as rofecoxib mode of SC-558 into COX-2, which is very similar to that experi- (Vioxx®), have been withdrawn from the market due to their car- mentally found for some nonselective COX inhibitors (14, 15). diotoxicity (3–5). This was not the case of other COX-2 selective This has important consequences on the classical way of perform- drugs such as celecoxib and nimesulide, with the latter one largely ing rational drug design, which is mainly based on understanding used as an anti-inflammatory agent in many diseases. As a con- only the main binding mode. Here we show that in so doing one sequence, today, in the rational design of new COXs binders, might miss crucial bits of information and one cannot compare medicinal chemists have to pay attention to the selectivity proper- the results of modeling with the experiments. In fact, our model ties of the designed drugs, and a fine-tuning of the COX selec- of ligand binding allows one to rationalize many previously tivity profile might be necessary to generate novel effective drugs with reduced side effects. In the rational modulation of the bin- Author contributions: V.L., L.M., F.L.G., A.C., E.N., and M.P. designed research; V.L. ders selectivity precious help comes from the x-ray crystallogra- performed research; M.B. contributed new reagents/analytic tools; V.L., M.B., and M.P. phy. In fact, the crystallographic structures show that selective analyzed data; and V.L. and M.P. wrote the paper. and nonselective inhibitors generally bind in two different pat- The authors declare no conflict of interest. terns. In COX-1, the space of the selectivity pocket is reduced This article is a PNAS Direct Submission. due to the presence of Ile523, while in COX-2 the presence of 1To whom correspondence may be addressed. E-mail: [email protected]. Val523 augments the available space providing a more stable ethz.ch or [email protected]. binding possibility for selective inhibitors (6). The kinetics of This article contains supporting information online at www.pnas.org/cgi/content/full/ selective and nonselective inhibitors are also different in general, 0913377107/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.0913377107 PNAS Early Edition ∣ 1of6 Downloaded by guest on September 27, 2021 unexplained experimental results, for instance, the key role hypothesis that the group of helices A–D forms the door through played by some residues in the binding path of the ligand as well which the arachidonic acid and other ligands pass (14). as the kinetic models for the binding mechanism of closely related This is a slow mode that needs to be sampled in a biased MD by analogues of SC-558 (8, 9, 16). Additionally, our study highlights the introduction of an appropriate CV. In order to address this the importance of the three alpha-helices’ flexibility at the effect we use a path collective variable (21) constructed with entrance of the enzyme, in line with what has been discussed the contact map (22) between the residues that determine the in literature (15). Finally, we contrast the behavior of COX-2 with gate flexibility (see Methods and SI Text). In addition we use a that of COX-1, in which only one binding mode for SC-558 has distance and a dihedral angle CV to identify the position and been found. orientation of the ligand relative to the enzyme (SI Text). This last set of CVs has been already successfully used in several me- Results tadynamics simulations of undocking processes of small ligands Biased MD Simulations. The well-tempered metadynamics is an from their binding sites (23, 24). The final choice of CV is the evolution of standard metadynamics (13), able of enhancing the result of a lengthy investigation reported in SI Text. sampling and reconstructing the free-energy profile of the pro- cess of interest by adding an adaptive bias on a selected number SC-558 Dissociation Process in COX-2. Under the action of metady- of collective variables (CVs) (12). The user defined CVs must be namics, the ligand leaves the starting position, which corresponds able to discriminate the initial and final states of the system and to the x-ray structure of the SC-558/COX-2 complex (PDB ID take into account all the slow modes of the process (17–19). One code 1cx2) (6), and explores the whole binding site and finally of the very first problems encountered in our undocking study, takes its way out from the enzyme through the helices gate. which has been also one of the major topics in COX inhibition, We describe in detail the relevant minima found along the exiting was the identification of the part of the protein that allows COX path in the following paragraphs.