G&I Genomics & Informatics eISSN 2234-0742 Genomics Inform 2016;14(2):53-61 http://dx.doi.org/10.5808/GI.2016.14.2.53 ORIGINAL ARTICLE Received March 21, 2016; Revised April 6, 2016; Accepted April 8, 2016 *Corresponding author: Tel: +919840054575, Fax: +91-44-28254180, E-mail: vumashankar@gmail.com † These two authors contributed equally to this work. Copyright © 2016 by the Korea Genome Organization CC It is identical to the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/). Multilevel Precision-Based Rational Design of Chemical Inhibitors Targeting the Hydrophobic Cleft of Toxoplasma gondii Apical Membrane Antigen 1 (AMA1) Umashankar Vetrivel 1 * † , Shalini Muralikumar 1† , Mahalakshmi B 2 , Lily Therese K 2 , Madhavan HN 2 , Mohamed Alameen 1 , Indhuja Thirumudi 1 1 Centre for Bioinformatics, Kamalnayan Bajaj Institute for Research in Vision and Ophthalmology, Vision Research Foundation, Sankara Nethralaya, Chennai 600-006, India, 2 L&T Microbiology Research Centre, Kamalnayan Bajaj Institute for Research in Vision and Ophthalmology, Vision Research Foundation, Sankara Nethralaya, Chennai 600-006, India Toxoplasma gondii is an intracellular Apicomplexan parasite and a causative agent of toxoplasmosis in human. It causes encephalitis, uveitis, chorioretinitis, and congenital infection. T. gondii invades the host cell by forming a moving junction (MJ) complex. This complex formation is initiated by intermolecular interactions between the two secretory parasitic proteins—namely, apical membrane antigen 1 (AMA1) and rhoptry neck protein 2 (RON2) and is critically essential for the host invasion process. By this study, we propose two potential leads, NSC95522 and NSC179676 that can efficiently target the AMA1 hydrophobic cleft, which is a hotspot for targeting MJ complex formation. The proposed leads are the result of an exhaustive conformational search-based virtual screen with multilevel precision scoring of the docking affinities. These two compounds surpassed all the precision levels of docking and also the stringent post docking and cumulative molecular dynamics evaluations. Moreover, the backbone flexibility of hotspot residues in the hydrophobic cleft, which has been previously reported to be essential for accommodative binding of RON2 to AMA1, was also highly perturbed by these compounds. Furthermore, binding free energy calculations of these two compounds also revealed a significant affinity to AMA1. Machine learning approaches also predicted these two compounds to possess more relevant activities. Hence, these two leads, NSC95522 and NSC179676, may prove to be potential inhibitors targeting AMA1-RON2 complex formation towards combating toxoplasmosis. Keywords: apical membrane antigen 1, drug design, hydrophobic interaction, molecular docking analyses, rhoptry neck protein 2, toxoplasmosis Introduction Toxoplasma gondii is an Apicomplexan intracellular parasite and is a causative agent of toxoplasmosis in human. It also plays a key role over a broad spectrum of clinical syndromes, like encephalitis, uveitis, chorioretinitis, and congenital infections [1]. It infects the host by various modes, such as intake of undercooked meat containing T. gondii cysts, con- sumption of contaminated water, and food defilement with feces from infected cats, and may also spread through blood transfusion [2, 3]. The sexual reproduction of T. gondii occurs in domestic cats, which are considered a definite host [4]. The asexual reproduction of this parasite occurs in inter- mediate hosts—namely, humans, cattle, and birds [5]. The establishment of host-parasite interactions is crucial for parasite survival, as it depends on host nutritive resources. Apicomplexan parasites implement a unique host cell