السيرة الذاتية

POST-GRADUATION PROJECT (Ph.D.):

Reversing Multi-drug Resistance with Efflux Pump Inhibitors

Drug efflux pumps confer multidrug resistance (MDR) to bacteria against a wide range of unrelated, antiseptics and detergents. Among the different types of efflux pumps, the resistance nodulation division (RND) confer MDR to various species of Gram- negative bacteria. The AcrAB-TolC multidrug efflux pump from E. coli is the best-characterized example and an excellent model for understanding MDR because of an abundance of functional and structural data on this complex. Small molecule inhibitors that target AcrAB-TolC represent a new solution to reversing MDR in clinically relevant pathogenic bacteria.

Structures of AcrAB-TolC in complex with substrates and inhibitors using X Ray-crystallography and/or cryo-electron microscopy has significantly contributed in defining the mechanism of substrate binding and understanding the pharmacological inhibition of multidrug efflux. These structural studies have defined an inhibitor-binding pocket in the AcrB subunit, known as the hydrophobic trap. This has a significant result that has contributed to the effective development of small molecule efflux pump inhibitors (EPIs) that target AcrB using structure-guided design.

Our laboratory has previously reported phytochemicals that function as EPIs that were identified through in silico screening and subsequent evaluation of their biological properties. These studies also predicted that the binding mechanism for these EPIs exploited the hydrophobic trap of AcrB. This screening identified new EPIs that are relevant to this thesis (i) the naphthoquinone shikonin from the traditional Chinese herb Lithospermum erythrorhizon, (ii) the antioxidant nordihydroguaretic acid (NDGA) from creosote bush (iii) pyranopyridine, MBX2319 that has reported activity against RND efflux pumps from Enterobacteriaceae. This work has performed in collaboration Professor Shutao Ma, Shandong University, China, Prof Ma and his team of medicinal chemists designed and synthesized 153 putative EPIs for assesse their ability to inhibit the AcrAB-TolC drug efflux pump from Escherichia coli. Specifically, the biological assays investigated the ability of the compounds to synergize with various classes of antimicrobials, to inhibit the AcrB subunit of the drug efflux pump, and to test for off target activities, such as permeabilisation of the bacterial inner- and outer membrane.

In this project, a total of 47 compounds (out of the 153 tested) were identified as promising lead compounds. This is a significant advance towards the pharmacological reversal of MDR in clinically relevant bacterial pathogens.

Breast cancer is considered as the most common cancer amongst women. Though the rates of breast cancer are increasing daily, death rates are declining due to the enhancement of screening and treatments. The main risk factors for this disease are the age, sex and family history. The hereditary factors are reported 5-10% between women for developing breast cancer and this is due to mutations which includes germ line mutations in the developed BC susceptibility gene-1 (BRCA1). Recently, scholars reported 80 % of risk for developing breast cancer because of mutations in the BRCA-1 gene germ line mutations. Additionally, women with mutations in BRCA-1 get an increased risk of developing ovarian cancer.

The BRCA1 gene is known to undergo over 600 mutations, many of which tend to become cancerous. BRCA1 (Breast Cancer Susceptibility Gene 1) has an N-terminal Ring domain and C-terminal BRCT motifs. The RING domain has E3 ubiquitin ligase activity. More identification of new binding partners with the RING domain and protein- protein interactions is very vital for understanding the RING function and regulation. In this Thesis we aimed to find or identify new or unknown binding partners for the BRCA-1 RING domain through cloning the RING into a Halo tag vector in order to create a protein fusion construct. The RING domain cDNA was amplified from a clone and successfully ligated into a pFN22A construct. This construct can now be transfected into mammalian cells and binding partners identified.

Publications;

1- *Jin, C., *Alenazy, R., Wang, Y., Mowla, R., Qin, Y., Tan, J.Q.E., Modi, N.D., Gu, X., Polyak, S.W., Venter, H. and Ma, S., 2019. Design, synthesis and evaluation of a series of 5-methoxy-2, 3-naphthalimide derivatives as AcrB inhibitors for the reversal of bacterial resistance. Bioorganic & medicinal chemistry letters, 29(7), pp.882-889.

= joint first authors

2- *Wang, Y., *Alenzy, R., Song, D., Liu, X., Teng, Y., Mowla, R., Ma, Y., Polyak, S.W., Venter, H. and Ma, S., 2020. Structural optimization of natural product nordihydroguaretic acid to discover novel analogues as AcrB inhibitors. European Journal of Medicinal Chemistry, 186, p.111910.

= joint first authors

3- Wang, Y., Alenazy, R., Gu, X., Polyak, S.W., Zhang, P., Sykes, M.J., Zhang, N., Venter, H. and Ma, S., 2020. Design and structural optimization of novel 2H-benzo [h] chromene derivatives that target AcrB and reverse bacterial multidrug resistance. European Journal of Medicinal Chemistry, p.113049.