Design, docking, synthesis and anti E. coli screening of novel thiadiazolo thiourea derivatives as possible inhibitors of Enoyl ACP reductase (FabI) enzyme

A series of 1-phenyl-[5-substitutedphenyl)-1,3,4-thiadiazol-2-yl]-3-thiourea (3a-h) were synthesized and screened for their anti Escherichia coli potential. The characterization of the newly synthesized compounds was based upon their spectral data. The design of the title compounds was done utilizing the in silico methodology. Virtual screening technique was utilized for the identification of the lead, thiadiazole. The pharmacokinetic behavior was predicted by lead optimization and docking studies helped to analyze the binding interactions. Antibacterial activity of the title compounds were predicted by the PASS prediction software. The anti E. coli screening results showed that the derivatives, 3b and 3h possessed significant activity. DOI: http://dx.doi.org/10.3329/bjp.v9i1.16992 Bangladesh J Pharmacol 2014; 9: 49-53

Selection of the target and lead: The target enzyme selected was enoyl-acyl protein reductase (FabI) and the hits were obtained from the zinc database. The selection of lead from the hits was based on the virtual screening technique using iGEMDOCK v.2. A small molecular library of 10,000 hits was constructed utilizing ZINC database and was docked into the enzyme and the potential leads were discovered based on the binding affinity. Thiadiazole was selected as the potential lead. The binding alignment of the selected moiety is depicted in Figure 1.
Lead optimization: The selected lead was substituted with various substituent and the in silico lead optimization was done to evaluate its pharmacokinetic profile ( Table I) Docking simulations: The energy minimized ligands were subjected to docking studies in order to predict its interaction with the key binding sites on the enzyme. Initially Grid map was set with 60 points and a spacing of 0.375 A° was set. Lamarckian genetic algorithm was used, with a maximum number of 25,000,000 energy evaluations and a maximum number of 5,000 generations, for each run and 150 docking runs were performed. The binding energies obtained through in silico studies are depicted in Table I. The snap shots of the docked structures are shown in Figure 2.
PASS prediction: PASS prediction is based on SAR analysis of the training set containing more than 35,000 compounds which have more than 500 kinds of biological activity. This in silico method is used to predict the activity of the chemical entity before its synthesis. The compounds subjected to docking studies in the present study were subjected to PASS prediction  to confirm its potential as anti E. coli agents. If Pa>0.7 the chance to find the activity in experiment is high, but in many cases the compound may occur to be the close analogue of known pharmaceutical agents. If 0.5 < Pa < 0.7 the chance to find the activity in experiment is less, but the compound is not so similar to known pharmaceutical agents. If Pa<0.5 the chance to find the activity is even more less, but it can be also a new chemical entity (Poroikov et al., 2002). The PASS prediction data is given in Table I.
were synthesized based on the reported procedure (Jatav et al., 2008).
3 mL) was refluxed for 2 hours and the reaction mixture was poured into ice-cold water. The solid separated was filtered and recrystallised from ethanol. The other thiadiazolyl thioureas were prepared in the same way. The completion of the reaction was monitored using solvent system (acetone/benzene: 4:6).  ( (  Antibacterial activity: The antibacterial activity of the newly synthesized compounds, (3a-h) were assessed against E. coli by agar well diffusion method (Cappuccino et al., 1992). Mueller Hinton agar plates were prepared aseptically to get a thickness of 5-6 mm. The plates were allowed to solidify and inverted to prevent condensate falling on the agar surface. The plates were dried at 37°C before inoculation. The sterile swab was dipped in the previously standardized inoculums and excess of inoculums was removed by pressing and rotating the swab firmly against the sides of the culture tube above the level of the liquid. The swab was then streaked all over the surface of the medium three times, rotating the plates through an angle of 60 o after each application. Finally, the swab was pressed round the edge of the agar surface. The inoculated medium was allowed to dry at room temperature, with the lid closed. Cork borer was sterilized by using flame and well was made by using cork borer. By using micropipette, the test sample and standard were added into the well and were refrigerated for one hour to facilitate uniform diffusion of the drug. This was then incubated for 18-24 hours at 37°C. The diameter of the zones of inhibition around the drugs were measured and compared with that of the standard. The antibacterial activity data is shown in Table II. All the synthesized compounds were tested for antibacterial activity against E. coli bacteria.

Results and Discussion
The virtual screening technique predicted that thiadiazole scaffold can possess excellent interaction profile with the target enzyme enoyl ACP reductase of E. coli. For improving the pharmacokinetic profile thiourea side chain was attached to the parent thiadiazole scaffold. The lead optimization technique showed that the thiourea substituted derivatives possessed excellent human intestinal absorption, plasma protein binding, log P value with no signs of hepatotoxicity. Thus ADMEtox data evaluated through Accelerys Accord for Excel predicted that the designed leads are having good pharmacokinetic profile. The selected optimized leads on docking study revealed that there existed excellent interactions of the ligands with the enzyme. The ligands were interacting with the key amino acids Gly 93 and Tyr 158 through NAD (Figure 2 The prediction showed that compound 3h has the highest probability and 3f has the least probability of antibacterial activity. Most of the derivatives possessed a correlation with the PASS prediction.
The derivatives obtained through computational tools  13 CNMR also revealed the successful formation of the title compounds. The molecular ion peak at 312 was corresponding with the molecular formula, C15H12N4S2 of 3a.
The antibacterial results revealed that the derivatives, 3b and 3h showed a zone of inhibition of 28 mm when compared to the standard, ciprofloxacin (30 mm) at 250 µg/mL. Other derivatives also showed significant activity as predicted by the in silico studies. It can be assumed that the significant antibacterial potential exihibited by the above derivatives are due to the presence of electron with drawing groups like chloro and nitro groups.
To conclude with, the present research focussed on the design of enoyl ACP reductase inhibitors of E. coli has resulted in the development of novel thiourea linked 1,3,4-thiadiazole derivatives. The activity profile of the designed compounds indicated that there existed a significant correlation with the computational data. Thus the new 1,3,4-thiadiazolyl thiourea derivatives are excellent candidates in antibacterial drug discovery.