The proteolytic and non-proteolytic activation of the catalytic subunit of coagulation Factor XIII (FXIII) i.e. FXIII-A subunit involves major conformational change in the FXIII-A subunit and its domains. The functional regions and residues involved in the conformational change occuring during FXIII-A activation can serve as regions of interest for developing inhibitors against FXIII-A. Inhibition of FXIII is currently seen as a futuristic therapeutic option against thrombosis and prothrombotic conditions. Therefore, in the interest of developing highly specific therapeutic inhibitors against the activation path of FXIII protein, the structures of the zymogenic and activated FXIII-A (PDB ID: 1F13 & 4KTY respectively) along with two previously reported modelled FXIII-A activation path transition state intermediates (4th and 8th) were processed for virtual screening on the InfiniSee and SeeSAR platforms. The binding sites on all these structures were defined automatically on SeeSAR as well as manually (on modelled structures). A non-random library of 18 small molecules (appearing associated with FXIII as search term) was selected from three major open source major medicinal chemistry databases (Chembl, BindingDb and Pubchem) and docked onto these binding sites on all the structures. Binding affinity estimation followed by correction of docking poses, energy minimization, modification and re-docking was performed for all molecules in SeeSAR. The edited molecules were used as a reference molecule to define the chemical space using InfiniSee. The virtual screening pipeline yielded a total of 53 small molecules were discovered against the defined activation path strucures of FXIII-A. This scientific challenge was successfully conducted using BiosolveIT software tools SeeSAR, InfiniSee, and command-line tools of FlexX and HyDe. The present lot of 53 small molecules are currently being investigated from a biochemical, synthetic perspective following which they will be synthesized/purchased and tested on the bench.
After 1 year, Arijit has achieved the following goals:
- Library preparation of small molecules against FXIII-A: A library of small molecule ligands specific to transglutaminases was created by searching the major open access medicinal chemistry dataases of ChEMBL, PubChem, and BindingDB with search terms like FXIII and coagulation Factor XIII. Selections were random and no structural/functional restraints were imposed. Binding efficiency charts, Ki, Kd, RO5 and partition coefficient values integrated within the small molecule details in these databases were used as filters to narrow down only pharmacologically suitable molecules. In total, a non-redundant library of 18 small molecules were selected and imported to SeeSAR platform for further processing. Structures of the zymogenic and activated FXIII-A (PDB ID: 1F13 & 4KTY respectively) along with two previously reported modelled FXIII-A activation path transition state intermediates (4th and 8th) were uploaded tot he SeeSAR platofrm for docking/binding site detection.
- Modification and docking of the ligands to the activation path FXIII-A structures: A total 18, 7 and 12 binding pockets were identified for the zymogenic, activated and 2 transition state FXIII-A structures using Binding Site mode of SeeSAR. The screened library of 18 small molecules were docked onto the binding pockets using the Docking mode of SeeSAR. Each small molecule generated on an average fifty poses corresponding to individual pockets. Molecules exhibiting multiple torsion hindrance, inter and intra clashes were further energy minimized by importing ligand-protein complex poses to YASARA and subsequently their binding affinity was re-evaluated with Hyde scores. Parallely, to improve the affinity of these small molecules, they were subjected to information guided manipulation and modification using Molecular Editor and Inspiratior mode in SeesSAR. The modified ligands were further re-docked onto the FXIII-A structures and screened for high affinity (~ nM) based on Hyde scores.
- Defining chemical space using InfiniSee platform and docking of final set of small molecules against the activation path FXIII-A structures: Along with the library of 18 small molecules, nine structurally modified ligands were used as a reference to find structurally similar molecules in InfiniSee. This resulted in 180 similar small molecules that were exported to SeeSAR for the docking against FXIII forms. It resulted in 41 molecules showing affinity towards zymogenic form of FXIII-A, 27 molecules showing affinity towards activated form of FXIII-A, 29 molecules showing affinity towards activation transition states (2 chosen) of FXIII-A. Therefore, we were able to identify 53 molecules showing high affinity to the activation path structures of FXIII-A with several of them showing affinity towards multiple structures. Additionally, command line tools of FlexX and HyDe were used to develop automated docking, scoring and ranking of the ligands onto forms of FXIII.