Structure-based modelling for overcoming multi-drug resistance

  • Alessandro Barbieri

Student thesis: Phd

Abstract

This Ph.D. project provided new insights into ABCB1/P-glycoprotein, a crucial transporter involved in multi-drug cancer resistance, currently one of the major causes of chemotherapy failure for Cancer patients. In order to get more structural information about this transporter, two analyses with single-particle cryo-electron microscopy were carried out. Murine ABCB1 was first trapped in a post-hydrolytic state, revealing novel features (Chapter 2). In our cryo-EM study multiple extra-densities corresponding to nucleotides, portions of the N-terminal region and of the internal linker were observed even at relatively low resolution (7.9A). Secondly, different conformations of ABCB1 in bound- and apo-states were studied simultaneously in the presence of sub-stoichiometric levels of Ivacaftor, a known competitive inhibitor (Chapter 3). This compound is a multi-billion dollar drug employed as a potentiator for treating people affected by cystic fibrosis. High contrast cryo-electron microscopy imaging with a Volta phase plate was employed and the presence of ivacaftor was associated with the appearance of an additional density in one of the conformational states detected. Knowledge acquired from structural data were subsequently applied to several computational studies with Molecular Dynamics and Docking. An improved cryo-EM map of the post-hydrolytic state (7.9A > 5-6A) showing more details of portions of the linker , Single Nucleotide Polymorphisms and microseconds molecular dynamics were combined and employed to study the linker functionality. WT human P-glycoprotein and R669C, a known gain-of-function mutations localized in the linker sequence were reconstituted in a lipid environment through biological assays and reproduced in silico via Homology Modelling and MD simulations. MD simulations of the unstructured reconstituted linker were associated with the formation of molten globules, compatible with experimental evidence. More investigations on the length of the linker were conducted based on known experiments or X-ray crystal structures reported in the literature. Molecular Dynamics was also employed to study the interaction between the PDZ2 domain of Na+/H+ exchange regulatory cofactor (NHERF1) and ABCC7 (CFTR), a protein complex pertinent to the genetic disease cystic fibrosis (Chapter 4). The obtained X-ray crystallographic structure revealed novel patterns of interaction with respect to known studies and was confirmed via site-specific mutagenesis. A complete panel of MD simulations and energy calculations were carried out and it was found that the ionisation state of histidine residues involved in the binding of the CFTR PDZ motif was crucial for interaction. This combination of approaches identified positions in the PDZ motif of CFTR previously thought to be irrelevant.
Date of Award1 Aug 2022
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorRobert Ford (Supervisor) & Alexander Golovanov (Supervisor)

Keywords

  • P-glycoprotein
  • CFTR
  • ABC transporters
  • MD simulations
  • Cryo-EM
  • ABCB1

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