Compound for fatty acid synthase inhibition
10851043 ยท 2020-12-01
Assignee
Inventors
Cpc classification
A61K35/748
HUMAN NECESSITIES
International classification
Abstract
Fatty acids play very important and diverse roles in living organisms, from signal transduction, energy management, maintaining of structural integrity to the normal metabolic processes. Due to its diverse presence and roles, Fatty Acid Synthase (FAS) is an attractive drug target under different pathologic contexts such as cancer, TB, obesity, microbial infection, etc. There are a number of molecules targeting Fatty Acid Synthase (FAS) in the prior art like Cerulenin, Orlistat, Pyrimidine, Triclosan, C75 etc. The present invention relates to a compound of formula I having fatty acid synthase inhibitory properties which was confirmed by an FAS inhibition assay. The present invention also relates to a process for collection, extraction, isolation, purification and characterization of the claimed compound from marine cyanobacterium, Phormidium ambiguum. ##STR00001##
Claims
1. A compound of (3S,5Z,7S,8Z)-7-amino-3,5-dihydroxy-2-methyl-13-[(1s,4s)-4-hydroxycyclohexa-2,5-dien-1-yl]trideca-5,8-dien-4-one represented by Formula II: ##STR00008##
2. The compound as claimed in claim 1, wherein the compound has a .sup.1H and .sup.13C NMR spectra with signals at H 7.26 correlated with C 67.62; H at 2.46 to 2.62 and C at 29.64; H at 1.25 to 1.67; and 5.23 to 5.27.
3. A process for preparing the compound of the formula II as claimed in claim 1, comprising the steps of: a. collecting cyanobacterial biomass by scrapping off the cyanobacteria from semi submerged rock pits and washing with water; b. squeezing and grinding the biomass with ethanol to obtain a fine paste of the biomass; c. drying and solidifying the paste obtained in step (b) by spreading a thin layer of the paste; d. grinding the solidified paste obtained in step (c) into a powder; e. defatting the biomass powder obtained in step (d) using hexane; f. extracting the defatted powder obtained in step (e) using soxhlet at 60 C. to 70 C. for 6 to 8 hours using chloroform to obtain a pale white precipitate; g. concentrating the extract obtained in step (f) to .sup.th of its original volume using a rotary vacuum evaporator operated at 145 rpm-155 rpm/and at 35 C.-45 C.; h. precipitating the concentrated extract obtained in step (g) using methanol and re-dissolving in chloroform; i. repeating step (h) 3 to 4 times or until precipitate of pale white color are obtained; j. purifying the precipitate obtained in step (i) using silica gel column chromatography carried out using ethyl acetate, dichloromethane and methanol as solvents for gradient elution and wherein elution is initiated with 100% ethyl acetate followed by attaining 100% dichloromethane in the middle and finally using 100% methanol in the last stage; k. purifying the compound obtained in step (j) using a vacuum liquid chromatography using 100% dichloromethane to 100% methanol for elution; and l. purifying the compound obtained in step (k) using a reverse phase High Pressure Liquid Chromatographic (R-HPLC) with a gradient elution using acetonitrile to water at a constant flow rate of 0.2 mL/min with a constant column temperature of 30 C.
4. The process as claimed in claim 3, wherein the collected cyanobacterial biomass is processed within 1-2 hours.
5. The process as claimed in claim 3, wherein the extraction of defatted powder in step (f) is carried out at 65 C.
6. The process as claimed in claim 3, wherein the concentration of extract in step (g) is carried out using a rotary vacuum evaporator operated at 150 rpm at 40 C.
7. A pharmaceutical composition produced by the process as claimed in claim 3, the pharmaceutical composition comprising said compound.
8. The pharmaceutical composition as claimed in claim 7, further comprising pharmaceutically acceptable excipients, diluents, or additives.
9. A pharmaceutical composition produced by the process as claimed in claim 5, the pharmaceutical composition comprising said compound.
10. A pharmaceutical composition produced by the process as claimed in claim 6, the pharmaceutical composition comprising said compound.
Description
BRIEF DESCRIPTION OF DRAWINGS
(1)
(2)
(3)
DETAILED DESCRIPTION OF THE INVENTION
(4) While the invention is susceptible to various modifications and alternative forms, specific embodiments thereof is described in detail in examples section below. It should be understood, however that it is not intended to limit the invention to the particular forms disclosed, but on the contrary, the invention is to cover all modifications, equivalents, and alternative falling within the scope of the invention as defined by the appended claims.
(5) The present invention relates to a general compound of Formula I.
(6) ##STR00006##
(7) wherein R1, R2 and R3 are independently selected from the group consisting of alkyl halides; halide esters; allylation reaction products of primary, secondary, tertiary and higher alcohols including benzylic, propargylic, allylic, and other aliphatic alcohols; nucleophilic substitution products of the hydroxyl group in stereogenic alcohols; stereospecific-nucleophilic-substitution-of-hydroxyl groups; electrophilic aromatic substitution products of oxygen of hydroxyl groups formed by aromatic nitration, aromatic halogenation, aromatic sulfonation, and acylation and alkylating reaction to yield products such as alkyl/aryl halides; products formed by ester formation with electrophilic derivatives of carboxylic and sulfonic acids; amides and R4 is NH.sub.2.
(8) The present invention relates to a specific compound, (3S,5Z,7S,8Z)-7-amino-3,5-dihydroxy-2-methyl-13-[1s,4s)-4-hydroxycyclohexa-2,5-dien-1-yl]trideca-5,8-dien-4-one, represented by the Formula II
(9) ##STR00007##
(10) Collection of Marine Cyanobacterium for Preparation of Extract
(11) Cyanobacteria samples were collected during the period of January to May. Cyanobacterial cell biomass was found at semi submerged rock pits. During the high tide these pits remain submerged around a depth of about 0.5 to 1 meter but at low tide time seawater moves back and the pits become exposed. Although exposed these pits are always filled with sea water. Cyanobacterial biomass was collected from the rocks and moved to a plastic container. Collected cell biomass was worked on within 1 to 2 hours of collection to avoid microbial degradation which would affect the intended compound yield. The collected sample was then washed under tap water to remove off any sand, mud and other small sea creatures associated with the cell biomass. Then, the cleaned biomass was squeezed well to get rid of excess water and ground with a small volume of ethanol in a laboratory blender to form a fine paste. This paste was then spread as thin layer on the surface of a large glass plate and refrigerated till it dried and solidified.
(12) Preparation of Compound
(13) Dried cell biomass obtained was reduced to fine powder using a laboratory blender. Approximately 400-600 g of the powdered cyanobacterial cell mass was defatted with hexane. The defatted powder was then dried and subjected to Soxhlet extraction at 60 C. to 70 C. with chloroform for about 6 to 8 hrs. The chloroform extract was concentrated to .sup.th the volume using a rotary vacuum evaporator at 145 rpm-155 rpm and 35 C. to 45 C. The concentrate was then added to excess of methanol in a separating funnel, stirred and kept overnight till a greenish precipitate was formed. The greenish precipitate was removed and re-dissolved in minimal amount of chloroform. This step was repeated 3 to 4 times until the precipitate turned to pale white in color.
(14) Purification
(15) The Pale white waxy compound obtained above was further purified using silica gel column chromatography. The column was subjected to gradient elution using ethyl acetate to methanol, and finally dichloromethane. The column was subjected to gradient elution using 100% ethyl acetate to 100% methanol with attainment of 100% dichloromethane in the middle of the chromatography. Elution was started with ethyl acetate alone and subsequently dichloromethane was added in an increments of 5 to 20% (volume/volume) thus making a total of 100% of eluent. Similarly, in the middle of the chromatographic protocol, the attained 100% dichloromethane was diluted to zero with the third solvent methanol which was added in increments of 10% in volume/volume yielding 100% methanol at the end. Elution and fraction collected was not based on time interval but on volume basis. The compound was eluted at dichloromethane:methanol 1:1 and was precipitated out as clear white waxy form when excess methanol was added to the eluent. A second purification step was carried out using vacuum liquid chromatography with TLC grade silica. Elution was carried out with dichloromethane to methanol gradient and the compound was eluted at a ratio of dichloromethane:methanol 1:1. Final purification was carried out using reverse phase High Pressure Liquid Chromatographic (R-HPLC). Gradient elution was performed with acetonitrile to water at a constant flow rate of 0.2 mL/min with a constant column temperature of 30 C. The compound finally eluted exhibits a retention time (RT) of 2.9 in preparative HPLC under the said conditions (
EXAMPLES
(16) The following examples are set forth below to illustrate the methods and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods, compositions, and results. These examples are not intended to exclude equivalents and variations of the present invention, which are apparent to one skilled in the art.
Example 1
(17) Preparation of Compound of Formula II
(18) Cyanobacteria samples were collected during the period of January to May from Ezhara beach (GPS coordinates 11 4909.9N 75 2502.9E), Kannur district, Kerala, India. Cyanobacterial biomass was collected from submerged rock pits by scrapping off the cyanobacteria using a metal chisel with wide end to detach the cyanobacterial cells from the rocks. The floating cell biomass from the pits was collected using a small net and moved to a plastic container. Collected cell biomass was worked on within 1 to 2 hours of collection to avoid microbial degradation which would affect the intended compound yield. If the collected biomass is used immediately for extraction, a 40% increase in yield was observed compared to delayed extraction. The collected sample was washed and then squeezed well to get rid of excess water and ground with a small volume of ethanol in a laboratory blender to form a fine paste. This paste was spread as thin layer on the surface of a large glass plate and refrigerated till it dried and solidified. Dried cell biomass obtained was reduced to a fine powder using a laboratory blender. Approximately 500 g of the powdered cyanobacterial cell mass was defatted with hexane. The defatted powder was then dried and subjected to Soxhlet extraction at 65 C. with chloroform for about 6 to 8 hrs. The chloroform extract was concentrated to th the volume using a rotary vacuum evaporator at 150 rpm/40 C. The concentrate was then added to excess of methanol in a separating funnel, stirred and kept overnight till a greenish precipitate was formed. The greenish precipitate was removed and re-dissolved in minimal amount of chloroform. This step was repeated 3 to 4 times until the precipitate turned to pale white in color. The Pale white waxy compound was further purified using silica gel column chromatography (530 mm column) using 240 mesh size silica. The column was subjected to gradient elution using 100% ethyl acetate to 100% methanol with attainment of 100% dichloromethane in the middle of the chromatography. Elution was started with ethyl acetate alone and subsequently dichloromethane was added in 10% increments (volume/volume) as shown in the table, thus making a total of 100 mL of eluent. Similarly, in the middle of the chromatographic protocol, the attained 100% dichloromethane was diluted to zero with the third solvent methanol which was added in increments of 10% in volume/volume yielding 100% methanol at the end (Elution and fraction collected was not based on time interval but on volume basis). The compound was eluted at dichloromethane:methanol 1:1 and was precipitated out as clear white waxy form when excess methanol was added to the eluent.
(19) TABLE-US-00001 TABLE 1 Solvent gradient system for elution in silica gel chromatography Solvent A Solvent B Column loading and 100% Ethyl 0% Dichloro- starting of elution acetate methane 90% 10% 80% 20% 70% 30% 60% 40% 50% 50% 40% 60% 30% 70% 20% 80% 10% 90% 0% 100% Dichloromethane Methanol 100% 0% 90% 10% 80% 20% 70% 30% 60% 40% The compound 50% 50% was eluted at dichloromethane: methanol 1:1. 40% 60% 30% 70% 20% 80% 10% 90% 0% 100%
(20) A second purification step was carried out using vacuum liquid chromatography with TLC grade silica 60H (1.515 mm column). Elution was carried out with dichloromethane to methanol gradient and the compound was eluted at a ratio of dichloromethane:methanol 1:1.
(21) TABLE-US-00002 TABLE 2 Solvent gradient system for elution in vacuum liquid chromatography Column loading and 100% 0% starting of elution Dichloromethane Methanol 90% 10% 80% 20% 70% 30% 60% 40% Compound eluted 50% 50% out of the column 40% 60% 30% 70% 20% 80% 10% 90% Final elution 0% 100% of the column
(22) Final purification is carried out using reverse phase High Pressure Liquid Chromatographic, R-HPLC system equipped with ExtendC18 column of 1.8 m, 2.150 mm, Diode Array Detector in combination with Chem32, Chemstation software. Gradient elution was performed with acetonitrile to water at a constant flow rate of 0.2 mL/min with a constant column temperature of 30 C. The compound finally eluted exhibits a retention time (RT) of 2.9 in preparative HPLC under the said conditions (
Example 2
(23) Characterization of the Eluted Compound
(24) Structure Elucidation
(25) 1. Fourier-Transform Infrared Spectroscopy
(26) Infrared (IR) spectra was taken on a FTIR spectrophotometer as neat. The Fourier Transform Infrared Spectroscopy (FTIR) spectrum exhibited the presence of alkenes, alkanes, hydroxy and aldehyde groups is shown in
(27) 2. Mass Spectrometry
(28) Molecular weight of the compound was determined by LCMS/MS equipped with Aquity BEH c 18 1.7 M column having 2.150 mm dimensions. QTOF MS/MS data revealed m/z value of 349.093 from which its molecular Formula was determined to be C.sub.20H.sub.31NO.sub.4. This was consistent with the molecular composition calculated by CHN analysis which gave a value of C: 0.687, H: 0.089, N: 0.040, O: 0.183. The molecular weight of the compound was later calculated to be 349.475 g/mol (
(29) 3. Nuclear Magnetic Resonance Spectroscopy
(30) .sup.1H NMR spectra were recorded and the chemical shifts were expressed in (ppm) with trimethylsilane as an internal reference. .sup.1H NMR and .sup.13C NMR spectra were recorded operating at 500 MHz for .sup.1H NMR and 500 MHz for .sup.13C spectra. The chemical shifts were given in ppm () and were referenced relative to CDCl3 (7.26 and 77.24 ppm for .sup.1H and .sup.13C NMR respectively). From the .sup.1H NMR and .sup.13C NMR spectra, the isolated compound was confirmed as (3S,5Z,7S,8Z)-7-amino-3,5-dihydroxy-2-methyl-13-[(1s,4s)-4-hydroxycyclohexa-2,5-dien-1-yl] trideca-5,8-dien-4-one as observed from
Example 3
(31) Anti-Tumor Assay
(32) 1. Determination of In-Vitro Cytotoxic Effect on Cultured L929 Cells
(33) Cultures of L929 (Fibroblast cells) were maintained in Dulbecco's modified eagles media supplemented with 10% fetal bovine serum (FBS) and grown to confluence at 37 C. and 5% CO.sub.2 in a humidified atmosphere using a CO.sub.2 incubator. The cells were trypsinised (500 L of 0.025% Trypsin in PBS/0.5 mM Ethylene diamine tetraacetic acid (EDTA) solution for 2 minutes and passed to T flasks under complete aseptic conditions. Test compounds were added to grown cells at a final concentration of 6.25, 12.5, 25, 50 and 100 g/mL from a stock of 1 mg/mL and incubated for 24 hours. The percentage difference in viability was determined by standard MTT assay after 24 hours of incubation. The MTT assay is a colorimetric assay for assessing cell metabolic activity. NAD (P) H-dependent cellular oxidoreductase enzymes under defined conditions, reflect the number of viable cells present.
(34) Cytotoxicity test revealed that the compound of Formula II had an LD.sub.50 of about 89.65 (g/mL) against L929 cell lines. Viability percentage was calculated using the Formula,
% viability=(OD of Test/OD of control)100, where control OD was 0.993.
(35) TABLE-US-00003 TABLE 3 Result of cytotoxic assay against L929 cells using compound of Formula II Sample Average Percentage Concentration (g/mL) Absorbance @ 540 nm Viability Control 0.993 99.9 6.25 0.901 90.821 12.5 0.852 85.793 25 0.741 74.680 50 0.638 64.262 100 0.554 55.768
(36) 2. In-Vitro Anti-Proliferative Property on Cultured A549 Cells
(37) A549 lung carcinoma cells were maintained in Dulbecco's modified eagles media (Himedia, India) supplemented with 10% FBS (Invitrogen, India) and grown to confluence at 37 C. and 5% CO.sub.2 in a humidified atmosphere using a CO.sub.2 incubator (NBS, Eppendorf, Germany). The cells were trypsinized (500 L of 0.025% trypsin in PBS/0.5 mM EDTA solution (Himedia, India) for 2 minutes and transferred to T flasks under aseptic condition. Test compounds were added to grown cells with a final concentration of 6.25, 12.5, 25, 50, 100 g/mL from a stock of 1 mg/mL and incubated for 24 hours. The % difference in viability was determined by standard MTT assay (Arung et al. 2000) after 24 hours of incubation. Viability percentage was calculated using the Formula,
% viability=(OD of Test/OD of Control)100
(38) Compared to the LD50 of about 89.65 (g/mL) against L929 cell lines, A549 cells showed higher susceptibility to the compound of Formula II. It means that the compound of Formula II is required in lesser amount to obtain equivalent level of cytotoxicity. In other words, A549 cells need less amount of the compound of Formula II compared to L929 cell lines to obtain complete destruction of cancer cells of type A549. This may be assumed to be still less in certain other cancer cells. In addition, it is observed that more amounts of Cerulenin and C75 are required as compared to the compound of Formula II to achieve the same level of cancer cell destruction.
(39) TABLE-US-00004 TABLE 4 In-vitro anti-proliferative effect of compound of Formula II, C75 and Cerulenin on cultured A549 cells Sample Percentage Viability Percentage Percentage Concentration Compound of Viability Viability (g/mL) Formula II Cerulenin C75 6.25 92.302 100.0 100.0 12.5 89.669 96.869 100.0 25 67.385 72.007 80.110 50 55.654 62.247 66.483 100 42.615 48.066 55.985 IC.sub.50 g/mL 76.034 86.419 99.034
Example 4
(40) FAS Assay
(41) Preparation of Bacterial FAS II Enzyme for FAS Assay
(42) Crude FAS containing protein fraction was isolated according to the modified protocol of Ahmad et al. (1981). Stock culture of Bacillus subtilis was inoculated in 100 mL LB broth pH 7.2 and cultured for 24 hours at room temperature. It was used for further inoculation of four 1000 mL flasks with same media and culture conditions. The flasks were placed in a rotator shaker to ensure adequate suspension of cells. Bacteria were harvested during the late-exponential phase of growth by centrifugation at 10000g for 10 minutes and washed twice with 50 mM sodium phosphate buffer (pH 7.0) to provide cell pastes which were stored at 20 C. until use. Frozen cell pastes (25-35 g) were thawed and suspended in 50 mM sodium phosphate buffer (pH 7.0). The cell suspension was disrupted by sonication (12 W, 615 pulses with 15 second intervals) in 2 mL Eppendorf tubes containing 1.5 mL of cell suspension, supplemented with lysozyme (250 g/mL) in a sonicator, operating at maximum power output while maintaining the temperature below 4 C. Unbroken cells and cell debris were removed by centrifugation at 30000g for 30 minutes at 4 C. The pellets were re-suspended in 50 mM sodium phosphate buffer (pH 7.0) and made up to appropriate volume and subjected to ammonium sulphate precipitation. Solid ammonium sulphate was added and the protein precipitate at 40-60% saturation was collected by centrifugation, dialyzed and used for further assay.
(43) Extraction of FAS I Enzyme from Bovine Liver
(44) Crude preparation of FAS I enzyme was made according to modified protocol of Dulter et al. (1971). Liver from freshly slaughtered Indian water buffalo was collected and transported in ice bucket and kept at 4 C. till processing. The liver was processed at the earliest by removing connective tissues and sliced into small pieces. Using a blender, about 750 g of liver was minced to form a suspension with 0.05 M potassium phosphate buffer pH 7.4 containing 1 mM EDTA to make up to a volume of 2.5 liters. The thick solution was centrifuged at 7500g for 20 minutes to remove meat debris and the supernatant obtained was further centrifuged at 30000g for 30 minutes to get a translucent solution. The supernatant was further diluted to specific volume using 0.05 M Potassium phosphate buffer (pH 7.4) with 1 mM EDTA and brought initially to 10% and then to 40% saturation with solid ammonium sulfate at 4 C. The 40% ammonium sulfate fraction was centrifuged and protein precipitate was re-dissolved in 0.05 mM Tris HCl buffer, pH 7.4, dialyzed against same buffer. The FAS I enzyme was further purified by ion exchange chromatography with Diethylaminoethyl (DEAE) cellulose column as described above (Dulter et al. 1971).
(45) FAS Assay
(46) FAS assay for FAS I enzyme was performed according to a modified process of Li et al. 2002. Crude protein was made up to a concentration of 0.5 mg/mL of protein and used for the assay. Assay was carried out in 96 well plates with the final working volume of 100 L. Compounds to be tested were incubated with previously made crude protein extract for 30 minutes at 25 C. 50 L of this mixture was added to 50 of reaction mixture containing 1 mM each of malonyl CoA and Nicotinamide adenine dinucleotide phosphate (NADPH), 40 M acetyl CoA, and 2 mM Dithiothreitol (DTT) in phosphate buffer (250 mM).
(47) The microplate was read immediately for 10 minutes at 340 nm. FAS activity was calculated by subtracting the optical density (OD) value obtained after 1 minute from the OD value obtained after 10 minutes. FAS activity was calculated and compared with that of control and test compounds. Dimethyl sulfoxide (DMSO) was taken as control as it was used to dilute the compounds. DMSO (1%) was also tested and did not interfere with FAS activity. The negative control consisted of protein plus reaction buffer. Another control consisted of buffer reaction without protein. The IC.sub.50 was determined by linear regression. FAS activity was determined by the rate of oxidation of NADPH to NADP which was monitored at 340 nm. The change in concentration of NADPH during oxidation was calculated using the following equation:
C=A/E
(48) Where, C was change in the concentration of NADPH, A was change in absorbance, and E was extinction coefficient of NADPH (E340 nm=6.22 mM.sup.1.Math.cm.sup.1). FAS activity was expressed as n mol NADPH oxidized/min/mg protein.
(49) Assay using partially purified enzyme from Bacillus subtilis was also performed to verify the FAS II inhibitory activity of compound of Formula II. At lower concentrations, compound of Formula II showed results comparable with Cerulenin and C75 and higher inhibition at higher concentration of compound of Formula II. The graph was plotted with change in concentration of NADP to NADPH against time. Each point in graph was the concentration of NADPH measured on a spectrophotometer at 340 nm (
(50) TABLE-US-00005 TABLE 5 FAS inhibition activity of compound of Formula II vis-a-vis Cerulenin and C75 taken as standard inhibitors FAS I assay compared with known inhibitors Compound of Control Cerulenin Formula II C75 Average C 0.051383 0.032476 0.030064 0.036125 Inhibition compared to control 36.49% 41.49% 29.60% FAS II assay compared with known inhibitors Compound of Control Cerulenin Formula II C75 Average C 0.05037 0.03955 0.034984 0.042476 Inhibition compared to control 21.48% 30.54% 15.67%
Example 5: Evaluation of ADME and Docking Studies
(51) ADME Prediction
(52) In a drug design point of view the ADME (absorption, distribution, metabolism, and excretion) properties of the compound of Formula II compared to the standard, Cerulenin and C75 was predicted using the Schrdinger suite's Qikprop module (QikProp, version 3.5, Schrdinger, LLC, New York,). Also, Qikprop can predict any possible drug leads by comparing the compound scaffolds with known database and analyzing similarity within a class of compounds (Schrdinger Release 2015: QikProp, Schrdinger, LLC, New York, N.Y., 2016.). Table 6 demonstrates that the Compound of Formula II exhibits values which fall well within the acceptable ranges for the ADME parameters as tested.
(53) TABLE-US-00006 TABLE 6 ADME predictions of compound of Formula II vis-a-vis Cerulenin and C75 predicted using QikProp module of Schrodinger suit. Compound SASA.sup.c Donor Accept QPlogPo/ PSA.sup.h ID #stars.sup.a #rotor.sup.b () HB.sup.d HB.sup.e w.sup.f QPlogKp.sup.g () Compound of 0 10 726.16 5 7 1.882 5.27 106.23 Formula II Cerulenin 0 7 223.27 1 5 0.697 3.59 90.39 C75 0 9 254.32 1 5 2.616 3.22 87.84 Recommended ranges: .sup.aNumber of descriptor values fall outside values of 95% known drugs: 0 to 5 .sup.bNumber of rotatable bonds: 0-15 .sup.cTotal solvent accessible surface area (SASA) in square angstroms: 300 to 1000 .sup.dNumber of hydrogen-bond donors: 0 to 6 .sup.eNumber of hydrogen-bond acceptors: 2 to 20 .sup.fPredicted octanol/water partition coefficient: 2 to 6.5 .sup.gPredicted skin permeability: 8.0 to 1.0 .sup.hvan der Waals surface area of polar nitrogen and oxygen atoms and carbonyl carbon atoms: 7-200
(54) Molecular Docking Studies
(55) Molecular docking studies were carried out to explain the possible binding mode of the purified compound to FAS. Since the structure of the purified compound of Formula II was similar to Cerulenin, crystal structure with protein data bank (PDB) structure no. 4LS7 was selected as receptor which is of Bacillus subtilis FAS II beta-ketoacyl-ACP synthase II (FabF) domain with non-covalently bonded Cerulenin. Similarly, for studying the interactions of the ligand with mammalian FAS-I, PDB structure no. 3HHD (Pappenberger et al, 2010) consisting of human FAS-I's KS-MAT domain was taken as a framework. Prior to the docking studies, the receptor structure was prepared by deleting the crystallographic water molecules and adding hydrogen to polar atoms. Later, the disulphide bond lengths were corrected and a minimization was performed by applying a RMSD cut off 0.30 using a force field OPLS 2001. In PDB structure no. 4LS7 (Trajtenberg et al, 2014), Cerulenin at the active site was taken as reference and grid was generated with 151515 volume surrounding the ligand. In case of PDB structure no. 3HHD, according to Wettstein-Knowles et al. (2006), active site residues CYS161, HIS331 and HIS293 play crucial roles in FAS I's activity, hence these residues were taken as the center and a grid was generated with a box dimensions of 151515 . Docking simulation was performed using extra precision (XP) docking method implemented in Schrodinger version 9.4.0. (Friesner et al, 2006).
(56) Calculation of Binding Free Energies
(57) The docked poses with highest glide score were selected and binding free energies for the protein-ligand complex was calculated using Prime MM-GBSA. Prime MM-GBSA generates energy properties for the ligand, the receptor, and complex structure along with energy differences relating to strain and binding. Prime calculates the binding free energy using the equation:
G (bind)=E_complex (minimized)(E_ligand (minimized)+E_receptor (minimized))
(58) Inference
(59) Binding result was interpreted based on the score obtained in docking studies as well as the theoretical binding energies calculated from the docked poses. Possible amino acid residues involved in binding and the type of interactions made by compound of Formula II with active site residues are shown in
(60) TABLE-US-00007 TABLE 7 Comparison of docking of Compound of Formula II, Cerulenin and C75 against Bacillus FAS II XP Glide Score MMGBSA dG Bind Compound (kcal/mol) (kcal/mol) Compound of Formula II 8.612 54.679 Cemlenin 7.589 48.109 C75 5.040 30.549
(61) TABLE-US-00008 TABLE 8 Number of interactions at 5 distance from active site with docked ligands in Bacillus beta-ketoacyl-ACP synthase II domain Number of Number of Number of Hydrogen Hydrophobic residues van der Waals Compound bonds at 5 distance interactions Compound of 3 18 465 Formula II Cemlenin 2 17 347 C75 3 13 341
(62) TABLE-US-00009 TABLE 9 Comparison of docking of Compound of Formula II, Cerulenin and C75 against human FAS I XP Glide Score MMGBSA dG Bind Compound (kcal/mol) (kcal/mol) Compound of Formula II 5.093 43.936 Cemlenin 4.850 39.420 C75 3.900 26.489
(63) TABLE-US-00010 TABLE 10 Number of interactions at 5 distance from active site with docked ligands in human FAS IKS-MAT di-domain Number of Number of Number of Hydrogen Hydrophobic residues van der Waals Compound bonds at 5 distance interactions Compound of 4 9 632 Formula II Cerulenin 3 8 363 C75 1 8 454
(64) The above analyses convincingly demonstrate that compound of Formula II is a better inhibitor of human and bacterial or eukaryotic and prokaryotic FAS enzymes in comparison to the inhibitors used currently as drugs.