A structural, biophysical and cellular study on CLIC1 as a drug target for Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is the most aggressive and prevalent form of primary brain cancer and its poor prognosis makes GBM a public health concern. Increasingly, evidence points towards Chloride Intracellular Channel 1 (CLIC1) promoting oncogenic development with its high level of activity and expression during tumorigenesis. Strikingly, CLIC1 possesses the ability to transition between cytosolic and membrane-bound forms. CLIC1's unique 'moonlighting' abilities means that it may serve separate functions at both the cytoplasm and membrane. Intriguingly, the metamorphic nature of CLIC1 serves as a biological switch for malignant transformation in which only the membrane-bound form is oncogenic. This distinct feature could pave way for a new selective, conformation-specific cancer therapy which would potentially spare normal cells making CLIC1 a highly promising pharmacological target.

Our research aims are two-fold, firstly to understand the membrane-bound structure of CLIC1 as this is currently unknown. We used the SMALP system to encapsulate CLIC1 in a membrane environment and bypass the need for detergents. In doing so, we successfully produced nanodiscs after optimising reconstitution parameters and found that using the c-terminal GFP-tagged CLIC1 construct resulted in a higher yield of nanodiscs as compared to untagged CLIC1. To visualise the structure of the membrane-bound form, we have used TEM and CryoEM using single particle analysis. Secondly, we aimed to develop selective CLIC1 inhibitors with antiproliferative activity for the treatment of glioblastoma multiforme. Here, we successfully co-crystallised CLIC1 with FDA-approved compounds, and this study revealed for the first time their drug binding sites via X-ray crystallography. 2D 1H-15N NMR TROSY HSQC experiments revealed the chemical shift changes across different FDA-approved drugs corroborated with the drug binding sites found using crystallography. We also performed viability assays in human glioblastoma cells in the presence of these drugs to assess their antiproliferative nature and highlighted some of the challenges of this drug target. This integrative approach using a plethora of biophysical and cellular techniques sheds some light on CLIC1 interaction with drugs for the treatment of glioblastoma multiforme.