Quantifying Intrinsic Specificity: A Potential Complement to Affinity in Drug Screening Jin Wang, 1,2, * Xiliang Zheng, 1 Yongliang Yang, 2 Dale Drueckhammer, 2 Wei Yang, 3 Gennardy Verkhivker, 4 and Erkang Wang 1 1 State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences Changchun, Jilin 130022 People’s Republic of China 2 Department of Chemistry and Department of Physics, State University of New York at Stony Brook, Stony Brook, New York 11794-3400, USA 3 Department of Chemistry, Florida State University, Tallahassee, Florida 32306-4390, USA 4 Pfizer Global Research and Development, La Jolla Laboratories, 10777, Science Center Drive, San Diego, California 92121, USA (Received 23 October 2006; published 6 November 2007) We report here the investigation of a novel description of specificity in protein-ligand binding based on energy landscape theory. We define a new term, intrinsic specificity ratio (ISR), which describes the level of discrimination in binding free energies of the native basin for a protein-ligand complex from the weaker binding states of the same ligand. We discuss the relationship between the intrinsic specificity we defined here and the conventional definition of specificity. In a docking study of molecules with the enzyme COX- 2, we demonstrate a statistical correspondence between ISR value and geometrical shapes of the small molecules binding to COX-2. We further observe that the known selective (nonselective) inhibitors of COX-2 have higher (lower) ISR values. We suggest that intrinsic specificity ratio may be a useful new cri- terion and a complement to affinity in drug screening and in searching for potential drug lead compounds. DOI: 10.1103/PhysRevLett.99.198101 PACS numbers: 87.15.v Studying biomolecular recognition is critical in under- standing the fundamental biological metabolism and sig- naling events and is also at the core of drug design [1,2]. The two crucial issues related to the binding process are the affinity of the two molecules for each other and the spe- cificity or tendency of a molecule to bind to its desired target instead of other biomolecules. High affinity is often used as the initial criterion in the screening of drug targets in the pharmaceutical industry. However, high affinity does not guarantee the high specificity which is critical for drug target discrimination. An important lesson comes from inhibitors of the highly homologous cyclooxygenase (pros- taglandin synthase) enzymes COX-1 and COX-2. Inhibition of COX-2 can reduce inflammation and pain (typical COX-2 inhibitors include aspirin and advil) [3 – 5]. However, nonspecific binding to COX can cause serious side effects, with over 16 500 deaths and 103 000 hospital- izations per year in the U.S. [6]. While affinity is readily defined as the free energy difference between associated and dissociated states, the definition of specificity is less clear. We have investigated a new approach to specificity based on energy landscape theory. The conventional definition of specificity is the ability of a specific ligand (by ligand here we mean small molecule) to discriminate against different macromolecular receptors [Fig. 1(a)]. To determine the specificity of a specific ligand for a specific receptor, one has to search all the related receptors and find the set with lowest binding free energies sufficiently separated from the rest (to realize the discrimi- nation in population which is exponentially related to the free energy by Boltzmann law), which is often impractical. An alternate view of specificity is the capability of a particular macromolecular receptor to discriminate be- tween different ligand molecules [Fig. 1(c)]. A new view of specificity addressed in this Letter is the preference for a particular binding state (or mode) or a particular set of binding modes of a ligand to its receptor [Fig. 1(b)] to be much lower in energy than the other weakly binding states. During the process of a ligand binding to its receptor, different intermediate binding states emerge which have FIG. 1 (color online). Illustration of the concept of specificity in ligand binding and relationship between intrinsic specificity and the conventional specificity as well as the corresponding energy spectrum: (a) Different receptors binding to the same ligand; (b) Different binding states (modes) of a particular ligand to its receptor; (c) Different ligands binding to the same receptor. PRL 99, 198101 (2007) PHYSICAL REVIEW LETTERS week ending 9 NOVEMBER 2007 0031-9007= 07=99(19)=198101(4) 198101-1 © 2007 The American Physical Society