Open questions: •  Pharmacophore filtering •  Specific “hot spots” interactions

•  Mutant docking score comparison

•  Fragments and scaffolds

•  Novel drug design

Fragment Based Drug Design is being performed to find lead compounds. It is based on identifying small chemical fragments, which bind to the biological target, and then combining them to produce a lead with a higher affinity. As a first approach to FBDD we used FK506 and some of the molecules docked so far. The molecules selected for this strategy were the best in terms of docking score ranking and with specific interactions with the PfFKBD residues “hot spot”. To calculate the relevant fragments from the molecules RECAP - REtrosynthetic Combinatorial Analysis Procedure [5] was used. RECAP is a powerful analysis which allows the fragmentation of the molecule following the synthesis chemistry rules. The fragments identified will be solved In crystals of PfFKBD.

In silico strategies for Plasmodium falciparum FKBP35 (PfFKBP35)

inhibition Alessandra Bianchin1, Angus Bell2, Denis Shields1 and Anthony Chubb1.

1School of Medicine and Medical science, University College Dublin, Belfield, DUBLIN 4, IRELAND. 2School of Genetics and Microbiology, Trinity College Dublin, DUBLIN 2, IRELAND.

DOCKING  PHASE  AND  RESULTS  

STRATEGY  –  VIRTUAL  SCREENING  (VS)  

INTRODUCTION  

HOT  SPOT  RESIDUES  –  MUTANT  ANALYSIS    

ACKNOWLEDGEMENTS      

REFERENCES  

CONCLUSION  

The target of this research project is a Plasmodium falciparum protein PfFKBP35 [1,2]. This protein is the only P.falciparum FKBP type immunophilin, and has been suggested to be a potential drug target. PfFKBP35 presents foldase and chaperone activities and it is composed of three TPR domains, a calmodulin binding domain and a FK506-binding domain (FKBD). FK506 is better known as an immunosuppressant but it also has potent antimalarial activity [1]. Humans present several FKBP isoforms. hFKBP12 is a single domain protein and its inhibition of calcineurin (in complex with FK506) causes immunosuppression. At the moment attention is focused on the FKBD domain to build an inhibitor which does not cause immunosuppression in the human body.

PDB  structures   ACTIVE  SITES  

Differences in the binding domains of human FKBP12 and PfFKBD were exploited and highlighted in virtual screening studies. The structure of FK506 co-crystalized with FKBD allows one to study the active sites and the specific interactions. Virtual screening has been performed through the design of different pharmacophores (Ph4). Ph4s developed are based on FK506 and on the structure of the P. falciparum protein active site.

Original DB Confab DB lig.PH4 RMSDmin. rec.PH4 RMSDmin.

Zinc11_n13 148831 3853483 163787 27920 1212 488FDA_approved 1391 13943 493 111 45 16GSK_P. fal. 13504 773700 2595 393 2461 407GSK_CHEMBLndt 26784 58598 2090 354 99 4Zinc_50% sim. 24668 360208 2557 275 334 107Zinc_70% sim. 15 94 44 4 0 0XXXX_nat_CycloPs 56393 - 12285 - 24 -CXXXXC_nat_CycloPs 425152 - 35103 - 1210 -

11.  Roles of peptidyl-prolyl cis-trans isomerase and calcineurin in the mechanisms of antimalarial action of

cyclosporin A, FK506, and rapamycin. Biochem Pharmacol 1994, 48: 495-503. Bell et al. 2.  A Plasmodium falciparum FK506-binding protein (FKBP) with peptidyl-prolyl cis-trans isomerase and chaperone

activities. Molecular and Biochemical Parasitology, 2005 139: 185- 195. Monaghan et al. 3.  Clustal W and Clustal X version 2.0 . Bioinformatics 2007 23(21): 2947-2948. Larkin et al. 4.  Confab – Systematic generation of diverse low-energy conformers. Journal of Cheminformatics 2011, 3: 8.

O’Boyle et al. 5.  RECAP-Retrosynthetic Combinatorial Analysis Procedure: A Powerful New Technique for Identifying Privileged

Molecular Fragments with Useful Applications in Combinatorial Chemistry. J. Chem. Inf. Comput. Sci. 1998, 38, 511-522. Qing Lewell et al.

PHARMACOPHORES  

Figure 1. ClustalW alignment of hFKBP12 with PfFKBP35 [3]. Identical residues are highlighted with a star.

Figure 2. The first box shows Plasmodium falciparum FKBD35 crystal structures, 2OFN “apo form” in blue and 2VN1 “holo form” in red. An electrostatic map of the binding site and FK506 (fuchsia) are displayed. In the second box is highlighted the active site and the most important non-conserved residues between hFKBP12 (yellow) and PfFKBD (red). The third box shows the interaction of FK506 into the PfFKBD binding site. The last box presents the pharmacophores developed for the screening.

Several compound databases were used for the screening (first column of the table). The molecules were processed using MOE to guarantee their correct structure. Numerous molecule conformations were generated using Confab [4] (not performed for cyclical peptides). Pharmacophore-based virtual screening was applied as a first process to identify the possible molecules and/or the most interacting fragments for this binding site. These molecules were then used for the docking phase.

RECAP  ANALYSIS  

INTERACTIONS  

HOT  SPOT  

STRATEGY  –  FRAGMENT  BASED  DRUG  DESIGN  (FBDD)  

Here reported as an example is the database of FDA approved drug.

Figure 3. Alignment of PfFKBD35 crystal structure 2VN1 (in red) and hFKBP12 crystal structure 1FKJ (in yellow). Presented also is the electrostatic map of the active site and in fuchsia the drug FK506. Highlighted in red and in yellow, respectively, the residues “hot spot” for the specific interactions.

Figure 4. Right: Plots of FDA database docking score results. (A) Plot of MOE docking scores for FDA database filtered with the pharmacophore based on FK506. (B) Plot of MOE docking scores for the same database filtered with the pharmacophore based on PfFKBD. (C) Plot of AutoDock Vina docking scores for the filtered molecules as (B).

(A)

(B) (C)

Figure 3. Representation of ligand volume in the binding site. (A) In fuchsia, FK506’s original position in the 2VN1 crystal structure. (B) Docked pose of FK506 in the wild-type structure 2VN1 (RMSD 0.7281) (C) Modified PfFKBD with a single mutation at A106. (D) Docked pose of FK506 into the mutant A106 active site (RMSD 8.1690).

(A) (C)

(B) (D)

Figure 5. RECAP – Retrosynthetic Combinatorial Analysis Procedure. The molecule presented as example is FK506. On the right some of FK506 fragments are shown.

Several strategies can be used in computational drug discovery. We present here two different main strategies: Pharmacophore based virtual screening and Fragment based drug design. Virtual screening and docking analysis optimization was performed using human FKBP12 as control. The human protein presents a similar structure and the same interactions as the P. falciparum protein. On this basis we were able to determine the specific interactions expected for PfFKBP35. As a rationale for the fragment based drug design strategy docked molecules were fragmented and will be tested. The strategies presented here are the starting point for a efficient and selective in silico drug design.

(A) (B) (C)

Figure 4. Plots of FDA database MOE docking score results on Mutant A106. (A) Docking scores plot of FDA database filtered with the pharmacophore based on FK506. (B) Plot of FDA database filtered with a second Ph4 based on FK506, docking scores. (C) Plot of FDA database filtered with Ph4 based on PfFKBD docking scores.

Every molecule (minimum RMSD conformation) obtained after the screening was used for the docking phase. Docking was performed for PfFKBD and for hFKBP12 as control. The software used for docking are MOE and AutoDock Vina. Each programme has its own unique method. AutoDock Vina uses a genetic algorithm and precompiled energy grid while MOE calculates the binding energy of conformation ensemble in the active site.