However, crystal structures of C1and C2 are not available. at the phenyl ring of most active inhibitor produced a great deal of variation in PSI-6206 13CD3 its orientation causing the phosphinate group to interact strongly with residue K40. Dynamics simulations revealed the conformational variation in region of V36-F41upon substrate and inhibitor binding induced a shift in the location of K40 thus changing its interactions with them. Chymase complexes with the most activecompound and substrate were used for development of a hybrid pharmacophore model which was applied in databases screening. Finally, hits which bound well at the active site, exhibited key interactions and favorable electronic properties were CPP32 identified as possible inhibitors for chymase. This study not only elucidates inhibitory mechanism of chymase inhibitors but also provides key structural insights which will aid in the rational design of novel potent inhibitors of the enzyme. In general, the strategy applied in the current study could be a promising computational approach and may be generally applicable to drug design for other enzymes. Introduction Chymase (EC 3.4.21.39) is an enzyme of the hydrolase class that catalyzes the hydrolysis of peptide bonds and it is abundant in secretory granules of PSI-6206 13CD3 mast cells. Chymase is the major extravascular source of vasoactive angiotensin II(Ang II), which is generated very efficiently by human chymase via hydrolysis of the Phe-8CHis-9 bond of angiotensin PSI-6206 13CD3 I(Ang I) [1]. Chymase is stored in mast cells in an inactive form and is released as an active enzyme when mast cells are stimulated by injury or inflammation. Chymase shows enzymatic activity immediately after its release into the interstitial tissues at pH 7.4 following various stimuli in tissues. As chymase has no enzymatic activity in normal tissues, chymase inhibitors have the potential to be safe/non-toxic because specific chymase inhibitors may not have effects on any other targets in normal tissues [2]. Cardiovascular diseases are the leading cause of death in the developed world and are now on course to emerge as the major cause of death in the developing world [3]. One particular manifestation of cardiovascular diseases, heart failure (HF), is dramatically increasing in frequency. A link between heart failure and chymase has been ascribed, and there is an interest to develop a specific chymase inhibitor as a new therapeutic treatment for the disease [4]. The density of cardiac mast cells is remarkably increased in patients with heart failure, and cardiac chymase may play an important role in the development of several cardiovascular diseases [5]. Recently, it was observed that chymase activation was increased in ischemic myocardium following acute myocardial ischemia/reperfusion (AMI-R) compared to non-ischemic and sham myocardial tissue [6]. Chymase is also known to activate matrix metalloproteinase (MMP)-9 by cleaving a specific site of the catalytic domain of MMP-9. MMP-9, known as 92 kDa gelatinase, is correlated with an increase in infarct sizeand left ventricle PSI-6206 13CD3 (LV) fibrosis following experimental AMI [7]. Chymase also converts the precursor of transforming growth factor- (TGF-)to its active form thus contributing to vascular response to injury (Figure 1). Both TGF- and MMP-9 are involved in tissue inflammation and fibrosis, resulting in organ damage [8]. Previous studies have shown the involvement of chymase in the escalation of dermatitis and chronic inflammation following cardiac and pulmonary fibrosis [9]. Therefore, inhibition of chymase is likely to reveal therapeutic methods for the treatment of cardiovascular diseases, allergic inflammation, and fibrotic disorders. Chymase inhibition may also be useful for preventing the progression of type 2 diabetes, along with the prevention of diabetic retinopathy [10]. Moreover, the role of chymase in inflammation has demonstrated its PSI-6206 13CD3 restorative value in diseases such as chronic obstructive pulmonary disease (COPD) and asthma [11]. Open in a separate window Figure 1 Chymase-dependent conversion of angiotensin I to angiotensin II and precursors of TGF- and MMP-9 to their active forms. Over the past 15C20 years, several peptide and non-peptide inhibitors of chymase have been synthesized [12], [13], [14], [15]. In general, chymase inhibitors readily decompose in plasma, thus the stability.