Binding affinity of asiatic acid derivatives design against Inducible Nitric Oxide Synthase and ADMET Prediction

Article history: Received on: 12/12/2013 Revised on: 08/01/2014 Accepted on: 20/02/2014 Available online: 27/02/2014 Asiatic acid (AA) is a pentacyclic triterpenoid compound isolated from pegagan (Centella asiatica) and is reported to show anti-inflammatory activities by inhibiting inducible nitric oxide synthase (iNOS), an isoenzyme responsible for the catalysis of nitric oxide formation. The aim of this study was to obtain information regarding binding affinity of some potential asiatic acid derivatives to iNOS as well as pharmacokinetic properties including oral absorption, distribution, metabolism, and toxicity (ADME/T) using in silico methods. Twelve AA derivatives that were produced by modeling of AA on Aor C-ring or its carboxylic acid group, were included in this study. The affinities of these compounds were studied using molecular docking methods, while pharmacokinetic properties were studied using the PreADMET online program. The results showed that eight AA derivative designs have lower free energy binding (FEB) in comparison to AA (–9.79 kcal/mol), while four of the compound designs showed higher FEB than AA. 2,3-dioxo-11,13 diene-23-carboxy asiatic acid (7) showed the lowest FEB of -11.33 kcal/mol. This compound has the human intestinal absorption (HIA), Caco-2 cell permeability, and plasma protein binding values of 96.62%, 20.90 (nm/Sec.), and 98.46%, respectively, which are comparable to those of AA and other AA derivatives. It is concluded that 2,3-dioxo-11,13 diene-23-carboxy asiatic acid (7) is an AA derivative with potential to be developed as a potential iNOS inhibitor.


INTRODUCTION
Nitric oxide synthases (NOS) is an enzyme that catalyzes the formation of nitric oxide (NO) from L-arginine.iNOS (inducible nitric oxide synthase), one of three isozymes of NOS, is induced by microbial products, such as lipopolysaccharide (LPS) and inflammatory cytokines such as interleukin-1 (IL-1), tumor necrosis factor-α (TNF-α), and interferon-γ (INF-γ) in macrophages and some other cells (Calixto et al., 2003).NO is a free radical with an unpaired electron and is an important cellular signaling molecule that has a role in septic shock and autoimmune diseases.The high levels of NO formation by iNOS have an important role in the inflammatory response, and it can destroy functional normal tissues during acute and chronic inflammation.There have been several research efforts to identify a selective iNOS inhibitor.Compounds that inhibit expression or activity of iNOS are proposed to be potential anti-inflammatory agents.Antiinflammatory activities of asiatic acid (AA), including acting as an iNOS inhibitor, have been reported using in vitro and in vivo .
. methods, and it is more active against iNOS than COX-2 (Huang et al., 2001;Yun et al., 2008).AA is a pentacyclic triterpenoid compound isolated from Centella asiatica, and its structure is derived from an ursane skeleton that has three hydroxyl at C(2), C(3), and C(23); it also has an olefin at C(12), and one carboxylic acid group function at C(28).The structure-activity relationship of ursolic and oleanolic acid derivatives that have an ursane structure show a modification of the A ring, C ring, and carboxyl group of the structure, which is important for their significant activity as inhibitors of nitric oxide production in mouse macophages (Honda et al., 1997, Honda et al., 2000).Previously, we reported that the affinity of asiatic acid to iNOS is higher than the COX-2 receptor, and the important pharmacophore features are the hydroxyl (ring A) and carboxylic group acting as hydrogen bond acceptor (HBA), and also the olefin group at C(12) as a hydrophobic function (Musfiroh et al., 2013).In the field of molecular modelling, molecular docking is method to explore the interaction between the ligand and receptor, and it can be used to predict the binding affinity.The principle of docking involves docking the ligand into the binding site of receptors based on its form-similarity and characteristics .
including its electrostatic nature (Goodsell et al., 1990).In the drug discovery and development process, the discovery of drugs not only has good activity and bind selectively to target but also have the appropriate physico-chemical properties such as absorption and distribution properties (Zhao et al., 2001), as well as the toxicity to reach the target site when delivered orally.
Computational methods in the early discovery were conducted to reduce the probability of failure at the development stage of drug candidates.As mentioned above, the structure modification of asiatic acid functional groups might provide important information of the structure-activity relationships for the development of novel anti-inflammatory agents.Asiatic acid is a triterpenoid suitable to be selected as the lead compound for development of an improved anti-inflammatory agent, because in addition to its anti-inflammatory effect, it also shows other activities such as being hepatoprotective (Zhao et al., 2007), a wound-healing agent (Jeong et al., 2006), and it suppresses tumor promotion.
Here, we studied the binding affinity and inhibitory parameters of some asiatic acid derivatives against the iNOS enzyme that were better than the lead (AA) by the molecular docking method (structure based) using autodock v.3.05 software (Goodsell et al., 1990), and to predict their ADMET properties using Pre-ADMET software (Lee et al., 2003).The toxicity prediction was generated using Toxtree software.

MATERIAL AND METHODS
The protein crystal structure of iNOS complexed with Arginine (Arg) was downloaded from the RSCB protein Data Bank (PDB ID: 1NSI) (Li et al., 1999).The 3D structure of iNOS was reported by Li et al. (1999) using X-ray diffraction technique with a resolution of 2.55 Å. Twelve structures of asiatic acid derivatives were constructed using Chemdraw, then were optimized using Moleculer Mechanic (MM+), 3000 maximum cycles, followed by conjugate gradient minimization to a Root Mean Square (RMS) energy gradient of 0.01 kcal/(mol Å).

Molecular Docking Simulation Parameters
Molecular docking simulations were performed using the AutoDock 3.0.5.All water molecules and hetero-atoms attached to the proteins were removed.Hydrogen atoms were removed from the protein structure but later added only for polar hydrogen atoms using the programed protonation and charges assigned using the kollua.amberoption of AutoDock 3.0.5.The grid box of 40×40×40 points with a spacing of 0.375 Å were set for AutoGrid computation with grid centered at (x) 9.740; (y) 64.640; (z) 15.986 to cover important residues in the binding site (Musfiroh et al., 2013).Molecular docking was performed employing the Lamarckian genetic algorithm (LGA) with pseudo-Solis and Wets local search and with the following standard parameters: population size of 150 and a total of 50 docking runs .for each ligand.The other parameter was default.The docking results from each of the calculations were clustered based on RMSD and on FEB (Morris, et al., 1998).

Predicting the absorption, distribution and toxicity properties
The PreADMET program was accessed at http://preadmet.bmdrc.org/.Asiatic acid, twelve structure modifications, and its glycoside were used in this study.The structure of all compounds were converted into molfile (*.mol).The program automatically calculated the predictive absorption for Caco-2 cell, HIA (human intestinal absorption), and plasma protein binding (Lee et al., 2003).Predicting the toxicity properties was done using Toxtree free software and using Benigni/Bossa rule-base methods (for mutagenicity and carcinogenicity) (Begini et al.,2008).
The results revealed some interesting structure/activity relationships: the acetylation in the A ring modification (C2, C3, C23) resulted in much higher affinity than asiatic acid.In the Cring modification, a substitution and rearrangement of a double bond at the position of C11 and C13 was important to enhance the affinity into the binding site of iNOS, and the carboxyl group at C28 without a methyl group (e.g., 7) gave much higher affinity than the methylesther group (e.g., 8).Hydrophilic groups appeared to have higher affinity against the iNOS binding site than hydrophobic groups of asiatic acid derivatives.
Previously, the study of interactions against iNOS showed that asiatic acid formed hydrogen bonds from Gln 263 and Trp 372 of iNOS to the carboxyl group of C28 and the hydroxyl of the A Ring (Musfiroh et al., 2013).In this study, the results showed that there are different amino acid residues of iNOS taking part in the interaction between asiatic acid and its derivatives; however they have more ability to compete against iNOS to replace the role of L-Arg than AA (Table 1).These derivative compounds form hydrogen bonds with various amino acid residues in the iNOS binding site (Table 2).

Prediction of absorption, distribution, and toxicity properties
The pharmacokinetic parameters, absorption and distribution, were considered for selection of compounds as drug candidates.In this study, the PreADMET program was used to predict ADME of asiatic acid and its derivatives.The aspect prediction of absorption properties included percentage human intestinal absorption (% HIA) and Caco-2 cell permeability.
Caco-2 cells are derived from colon adenocarcinoma and possess multiple drug transport cycles through the intestinal epithelium.The Caco-2 cell model is reliable in vitro model for prediction of oral drug absorption, while HIA is the sum of bioavailability and absorption evaluated from the ratio of excretion or cumulative excretion in urine, bile, and feces.The distribution properties were calculated using predictive plasma protein binding, which is available in PreADMET software also.It was used because the degree of plasma protein binding of a drug has an important role on its disposition and the drug`s efficacy (Lee et al., 2003).The study of the pharmacokinetic profile of asiatic acid and asiaticoside showed that asiatic acid is well absorbed in human volunteers after oral administration (Grimaldi et al., 1990), while the bioavailability of asiaticoside is very low for intragastric administration (Liu et al., 2010).Our prediction value of HIA showed similar results with that study: AA and asiaticoside have HIA values of 91.23% (well-absorbed categories) and 1.81% (low absorbed), respectively.There is different lipophilicity between asiatic acid (Log P: 5.32) and its glycoside (log P: 1.73).
Prediction HIA value of AA derivatives are comparable to those of AA, and those compounds have a similar level of lipophilicity with AA.However, the plasma protein binding of asiatic acid and its derivatives were very strongly bound.The toxicity risk parameters such as mutagenicity and carcinogenicity of the AA and its derivatives have no risk probability (Table 3).The results showed that compounds (3), ( 5), ( 6), ( 7), ( 8), ( 9), ( 11), ( 12), (13) had a higher affinity than the asiatic acid, as well as good affinity absorption properties.Based on the lipophilicity properties (Log P < 5), compounds 7 and 13 are more likely to be developed further as iNOS inhibitors for oral administration (Lipinski, 2001).

CONCLUSION
The affinity prediction of asiatic acid structure modification indicated that the structures (3), ( 6), ( 7), ( 8), ( 9), ( 11), (12), and (13) (Fig. 1) have lower FEB values than that of AA; however, the structure of (7) shows the lowest FEB.The HIA, Caco-2 cell permeability, and plasma protein binding values are comparable to those of AA and other AA derivatives.Based on the overall results, it is concluded that the structure of 7 (2,3-dioxo-11,13 diene-23carboxy asiatic acid) was an AA derivative structure with the highest possibility of being developed as a potential iNOS inhibitor.

ACKNOWLEDGMENT
This research was supported by Riset KK ITB 2013.
The structure of asiatic acid and its modifications.

Fig. 2 :
Fig. 2:The interaction of asiatic acid derivatives (7) in the iNOS binding site (red line is hydrogen bond).

Table . 1
: The substituent of asiatic acid derivatives, their binding energies, and predicted inhibitory activities to iNOS.Table.2:Theamino acid residues form hydrogen bonds between AA derivatives in the binding site of iNOS.

Table 3 .
Predictive absorption, distribution, toxicity, and some physicochemical properties of asiatic acid derivatives.No.