Examination of 2’4-Dimethoxy-4’alyloxycalcon (CMAM) using Various Organic Solvents as a Candidate of Antitubercular Drug targetting the PhoR Cytoplasmic Domain of Mycobacterium tuberculosis using Dimer-Based Screening System

• Main Author: Winona Gracia - NIM : 10414033 , Rafaella
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/30109
• Institution: Institut Teknologi Bandung

<p align="justify">The constant expansion of antibiotic resistance on Mycobacterium tuberculosis requires the development of antitubercular drug with new mode of action. One of which is targetting the PhoP-PhoR two-component system which regulates essential genes for the survival of M.tuberculosis in host cells thus the disruption of the system can cause in attenuation of M.tuberculosis. In this TCS, PhoR is used as the target for treatment, for example using substances that can inhibit its homodimerization. Screening of such substances can be done using dimer-based screening system that involves the cytoplasmic domain of PhoR fused with repressor protein AraC (AraC-PhoRMtb). Substance that inhibit the homodimerization of PhoR causes AraC repressor unable to bind araC promoter located upstream of the reporter gene EmGFP, thus it can fluoresce. One of the substances that can inhibit the homodimerization is 2’,4-dimethoy-4’-alyloxycalcon (CMAM), but problems regarding the solubility has been reported. In this experiment, three differnet organic solvents are used (DMSO, ethanol, lecithin), and the purpose is to determine the best solvent in dissolving CMAM by quantifying the increment of EmGFP fluorescence in Escherichia coli BL21(DE3) pAraC-PhoRMtb. pAraC-PhoRMtb transformant which has AraC-PhoRMtb fusion was confirmed with PCR, sequencing, and SDS-PAGE. PCR and sequencing results show that the fused protein is identical to known sequence, and SDS-PAGE result shows a band that indicates the fused protein was expressed. Next, from testing and adjusting the system it was observed that the fluorescence of pAraC-PhoRMtb is less than pRSET-AraCDBD (control plasmid without PhoR), which means the PhoR homodimerization system to repress EmGFP in pAraCPhoRMtb works well. Afterwards, CMAM dissolved in DMSO, ethanol, and incorporated into liposome using lecithin are being tested towards pAraC-PhoRMtb. Treatment using CMAM dissolved in DMSO shows increment in the fluorescence quantification as CMAM concentration increases. Treatment using CMAM dissolved in ethanol shows anomaly in the fluorescence quantification as a result of biological effects of ethanol towards the cell. Meanwhile CMAM in a form of liposome using lecithin unable to enter the cell because no optimation of liposome size nor lecithin concentration has been done in this experiment. From these results it can be concluded that the best solvent to dissolve CMAM in this system is DMSO.<p align="justify">