Then Autodock was employed for site directed docking

Then, Autodock4.2 was employed for site directed docking on AD with the ligands of the cluster(s) having high binding affinities in VS II experiment (Morris et al., 2009). Docking protocols were followed as per our earlier report with 200 GA (genetic algorithm) run (Kalani et al., 2015). Likewise, ligand molecules for docking studies were prepared using ChemBioOffice suite (www.cambridgesoft.com). Best docking position of ligands was selected from the largest cluster of 200 docking poses. Their coordinates were taken out to form protein-ligand complexes. For simulation of complexes, the topology of ligands was separately prepared. Amber topology of ligands were built in a sequential manner using AmberTols and acpype script (Case et al., 2012; Sousa da Silva and Vranken, 2012): required hydrogen atoms were added; partial atomic charges assigned (AM1-BCC) (Jakalian et al., 2002); general amber force field (GAFF) employed for van der Waals and bonded parameters (Wang et al., 2004); and, parmchk and tleap programs used to build final Amber topology followed by its conversion into GROMACS topology. Thereafter, topology and coordinates of protein and various ligands were merged to generate complex structures that in turn subjected for 20 ns long simulations. Then, last 2500 frames were taken out from each simulated complex ensemble structure for binding free phosphodiesterase inhibitor (BFE) calculation of respective ligand with AD by g_mmpbsa approach (Kumari et al., 2014). Prior to MD simulations, protonation state of charged residues in AD as well as in its complexes (with various ligands) was checked by PROPKA script, but no significant changes observed (Suppl. Table S1) (Olsson et al., 2011). Besides, Python (https://www.python.org/), Grace (http://plasma-gate.weizmann.ac.il/Grace/) and Chimera were used for graphical representations (Pettersen et al., 2004).
Results
Discussion Hunt for the new drugs begin with the identification of the target and its modulator, usually, a small molecule called inhibitor, of desired therapeutic effect. To identify these compounds, HTS was introduced in the 1980s. Though it was a very popular approach at that time, immensely deprecated in later years for its high price and complexity (Jorgensen, 2004; Lounnas et al., 2013). And so, the SBVS was introduced in the late 1990s, and since its inception, it has produced many success stories, became one of the integrated tools in preclinical drug discovery programs (Lavecchia and Di Giovanni, 2013). However, most of the studies were focused on the target for which crystal structures were available, or at least have a homologous structure that can serve as a template to delineate its 3D model. In addition, screened compounds were from ZINC or other databases which were synthesized and tested after the hit declaration in VS studies. Here, we have screened PubChem compounds database with reported antifungal activities, and successfully predicted potential inhibitors of Dam1 complex subunit Ask1, a potential antifungal drug target of C. albicans. A fungus specific outer kinetochore Dam1 complex is essential in C. albicans, S. cerevisiae, S. pombe, but non-essential in fission yeast, and absent from metazoans. The reason for this reductive evolution of Dam1 complex is unknown yet. Both C. albicans and S. pombe showed regional centromeres as opposed to short-point centromeres of S. cerevisiae. In both S. cerevisiae and C. albicans, the interaction of one microtubule per kinetochore is established, early during the cell cycle, which contrasts with multiple microtubules per kinetochore in S. pombe, only during mitosis. Prior reports also suggest that Dam1 complex is associated with kinetochore throughout the cell cycle in S. cerevisiae and C. albicans, but only during mitosis in S. pombe. Studies also suggest that Dam1 complex is essential for viability and indispensable for proper mitotic chromosome segregation in C. albicans. Kinetochore localization of the Dam1 complex is independent of kinetochore-microtubule interaction, but the function of this complex is monitored by a spindle assembly checkpoint. Thus, the Dam1 complex is required to prevent precocious spindle elongation in pre-mitotic phases. Prior studies correlated the essentiality of Dam1 complex based on constitutive kinetochore localization associated with the one-microtubule-one-kinetochore type of interaction, but not the length of a centromere (Thakur and Sanyal, 2011). The schema of the strategy is given in Fig. 1.