An in silico Appraisal to Identify High Affinity Anti-Apoptotic Synthetic Tetrapeptide Inhibitors Targeting the Mammalian Caspase 3 Enzyme

Apoptosis is a general phenomenon of all multicellular organisms and caspases form a group of important proteins central to suicide of cells. Pathologies like cancer, Myocardial infarction, Stroke, Sepsis, Alzheimer’s, Psoriasis, Parkinson and Huntington diseases are often associated with change in caspase 3 mediated apoptosis and therefore, caspases may serve as potential inhibitory targets for drug development. In the present study, two series of synthetic acetylated tetrapeptides containing aldehyde and fluromethyl keto groups respectively at the C terminus were proposed. All these compounds were evaluated for binding affinity against caspase 3 structure. In series 1 compound Ac-DEHD-CHO demonstrated appreciable and high binding affinity (Rerank Score: -138.899) against caspase 3. While in series 2 it was Ac-WEVD-FMK which showed higher binding affinity (Rerank Score: -139.317). Further these two compounds met ADMET properties and demonstrated to be non-toxic.


Introduction
Caspase, initially called interleukin 1β converting enzyme (ICE), was identified not because of its involvement in cell death, but because it was responsible for cleaving (Miao et al., 2011) and thereby activating interleukin 1β, a pro-inflammatory cytokine (Leist et al., 2011), later caspases were accomplished to be the key executioners of the cell death program (Denault et al., 2002;Danial et al., 2004;Gomez et al., 2005;Turk et al., 2007;Noy et al., 2010).The evidence of involvement of caspases in cell death comes from the pioneering study wherein the inhibitors designed against caspases prevented worm cells from killing themselves.The above observation also added to the speculation that because the mechanisms of programmed cell death in the worm were similar to those for apoptosis of mammalian cells (Li et al., 2012;Sankari et al., 2012), caspase inhibitors could presumably prevent the death of mammalian cells as well (Hengartner et al., 2000).
During the last decade, major progress has been made to further understand caspase structure and function, providing a unique basis for drug design (Lavrik et al., 2005;Le et al., 2006;Takai et al., 2012;An et al., 2013, Zou et al., 2013;Liu et al., 2014).Caspases belong to the family of cysteinyl aspartate-specific proteases (Chai
Most of the synthetic peptide caspase inhibitors were developed based on the tetrapeptide caspase recognition motif (Fischer et al., 2005, Kuhn et al., 2011).Therefore, the selectivity of inhibitors matches the caspase substrate specificities, the introduction of an aldehyde group at the C terminus of the tetrapeptide results in the generation of reversible inhibitors (Powers et al., 2002;Grawert et al., 2012), whereas a fluoromethyl ketone (fmk), a chloromethyl ketone (cmk) (Shi et al., 2002;Huang et al., 2001), or a diazomethyl ketone (dmk) (Concha et al., 2002) at this position irreversibly inactivates the enzyme.
In the view of above, we have proposed two series of 25 synthetic acetylated tetrapetides compounds flanked by aldehyde or fluromethyl ketone at C terminus and observed their binding affinity, drug likeness and tested for toxicity, which we anticipate can form potent inhibitor targeting caspase 3.

Selection of tetrapeptide compound
In the study we proposed two series of 25 tetrapeptide compounds anticipated to have appreciable inhibitory potential against caspase 3.In one of the series all the tetra peptides were flanked by acetyl group at N terminus and aldehyde group at C terminus.In the second series the compounds were same as in series one, except the C terminus was flanked by fluromethyl group.The series of tetrapeptide compounds selected in the study and their corresponding sequence is shown in Table 1.

Structure preparation and minimization
The twenty five tetrapetide with their unique sequence was built using Accelyrys Dicovery Studio 3.0 visualization software.Further at the N terminal the acetyl, and at C terminal aldehyde groups (for series 1 compound) and fluromethyl ketone (for series2 compounds) groups were added to the tetrapetide sequence with Marvin Sketch 5.6.0.2.
The three-dimensional structure of caspase-3 [PDB: 1RHK] ( Becker et al., 2004) was retrieved from the Protein Data Bank.Before docking of the compounds, the protein was optimized and prepared by removing all bound crystal water molecules and adding hydrogen bonds.Explicit hydrogen was created and bond orders, hybridizations and charges were assigned wherever missing.The resulting structure was saved in .pdbformat for docking studies.(Nayarisseri et al., 2013).Docking parameters were set to 0.20 Å as grid resolution, maximum iteration of 1500 and maximum population size of 50.Energy minimization and hydrogen bonds were optimized after the docking.Simplex evolution was set at maximum steps of 300 with neighborhood distance factor of 1.After the ligand was docked the total energy was minimized using Nelder Mead Simplex Minimization (using non-grid force field and H bond directionality).

Flexible molecular docking of tetrapeptide compounds
Binding affinity and interactions of the compound with receptor was evaluated on the basis of the internal electrostatic, hydrogen bond interactions and sp2-sp2 torsions.On the basis of rerank score, best compound in each series with optimal binding affinity was selected against caspase.
Lipinski's drug-likeness and toxicity screening Lazar an online server (Hardy et al., 2010) was used to predict toxicity and Lipinski filter were applied to test the drug-likeness of the compounds.

Analysis of ligand binding affinities
Evident from rerank scores (Table 2), both Series 1 compounds (Acetyl Tetrapeptide Aldehyde) and 2 (Acetyl Tetrapeptide Fluro Methyl Ketone) demonstrated appreciable binding affinity against Caspase 3. A closer  perusal into the rerank scores showed that Ac-DEHD-CHO (Figure 1) had high binding affinity (Rerank Score: -138.899) in Series 1 compounds while, in case of series 2, Ac-WEVD-FMK (Figure 2) demonstrated higher binding affinity (Rerank Score: -139.317).Though both Ac-DEHD-CHO and Ac-WEVD-FMK had almost similar binding affinity, the latter though not significant, but had marginally higher binding affinity.In the series 2, Ac-WEVD-FMK demonstrated higher binding affinity and it is also marginally greater than Series1 Ac-DEHD-CHO.Keen investigation on ligand receptor interactions shows that seven hydrogen bonds are formed in the caspase with residues viz, three Hbonds with Arg 341, two H bonds with Arg 179 and one each with Ser 339 and His237.Further the good affinity between receptor ligand is reflected from electrostatic interactions between Ser 343 and 339, Asn342, Arg 179,     8).The electrostatic interactions and solvent accessible surface area of caspase 3 on ligand binding is shown in Figure 9 and 10 respectively.

Pharmacophoric identification of Ac-DEHD-CHO and
Drug likeness and toxicity screening.
Both the compounds Ac-DEHD-CHO and Ac-WEVD-FMK passed through Lipinski and Vebers filters implying the compounds are drugs likely.Further the compounds were screened for toxicity by Lazar Toxicity screening program.Both the compounds proved to be non toxic (Table 3).
Since both the compounds are non-toxic and passed

Discussion
Caspase inhibitors have now surfaced as important targets to prevent the death of mammalian cells and the research is now speeding up to bring down needless apoptotic events that kills the normal cells.The tetrapeptide caspase inhibitors have taken their stand in potentially inhibiting caspases from triggering cell death.Supplementing to the ongoing research, we in possible attempt proposed two series of 25 acetylated tetrapeptide compounds flanked by Aldehyde or Fluromethyl keto groups.Compound Ac-DEHD-CHO and Ac-WEVD-FMK showed remarkable interactions with caspase 3, further which proved to be non toxic.We anticipate that these two compounds can be put to pharmacodynamic and pharmacokinetic studies in way ahead for successful inhibition of pointless apoptotic events.

Figure
Figure 3. Interactions of Ac-DEHD-CHO with Amino Acids of Caspase 3 Structure

Figure 10 .
Figure 10.Electrostatic Interactions of Ac-WEVD-FMK with Caspase 3 structure drug likeness rules and further shows good affinity, the compounds now can be designated as a potent inhibitor nevertheless, need to be tested in vitro for further drug development.