Investigating and characterizing the direct binding interactions between chemotherapeutic cisplatin and pattern recognition receptor, Toll-like Receptor 4

• Author / Creator

  Domingo, Ivan K

• Cisplatin is one of the most effective chemotherapeutic agents available. Unfortunately, adverse drug reactions - cisplatin-induced toxicities - often limit the use of cisplatin. These toxicities develop in at least half of those treated, are diverse, and are especially problematic for pediatric patients. Research into the development of these toxicities has repeatedly implicated the innate immune receptor, TLR4 (Toll-Like Receptor 4), in the process. TLR4 usually helps promote immune response to bacterial pathogens, but it is also known to do the same towards metal allergens, like nickel. Because cisplatin-induced toxicities are associated with TLR4-dependent immune responses and cisplatin has a metal (platinum) core, I explored the possibility of direct binding interactions between TLR4 and cisplatin.
  I first probed for direct binding interactions (cisplatin:TLR4 interactions) by pre-treating HEK293-hTLR4 cells with soluble mouse (mTLR4) or human TLR4 (hTLR4). I then examined the effect on TLR4 activation by several agonists. Here, I found that both mTLR4 and hTLR4 can impede endogenous TLR4 activation in response to cisplatin. Through microscale thermophoresis, I acquired evidence for direct binding interactions between hTLR4, nickel, and cisplatin. To my knowledge, this is the first explicitly direct demonstration of TLR4-metal allergen binding. To ascertain whether known metal-binding TLR4 residues, H456 and H458, were responsible for cisplatin binding and pro-inflammatory IL-8 secretion in response to platinum ions, I created and tested the responsiveness of mutant hTLR4 constructs. In doing so, I discovered that these residues only enhance an underlying capacity for TLR4s to bind and respond to cisplatin. Returning to microscale thermophoresis, I determined that mTLR4 could also bind cisplatin directly, despite being incapable of binding nickel.
  
  Ultimately, I not only provide evidence for direct binding events between hTLR4 and cisplatin but also characterize some of the features involved in this process. As a result, I raise the possibility that there are additional, conserved, residues specific and sufficient for binding cisplatin and platinum ions.

• Subjects / Keywords

  • cisplatin
  • TLR4
  • pattern-recognition receptor
  • Toll-Like Receptor 4

• Graduation date

  Fall 2022

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/r3-3vvr-bp55

• License

  This thesis is made available by the University of Alberta Library with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.