The use of surface plasmon resonance spectroscopy in characterizing small molecule-based nerve growth factor inhibitors

Abstract

Neurotrophins are well known for their effects on neuronal survival and growth. Over the past two decades, considerable evidence has accumulated from both human and animal models that one neurotrophin, nerve growth factor (NGF), is a peripheral pain mediator, particularly in inflammatory pain states. NGF is upregulated in a wide variety of inflammatory conditions, and NGF-neutralizing molecules are effective analgesic agents in many models of persistent pain. Such molecules are now being evaluated in clinical trials and although seemingly effective in early clinical studies, many deteriorating side effects are now being brought to light. Thus, a need still remains for novel NGF-quenching therapeutics. In the past two decades, a handful of small molecule NGF-inhibitors have been described, however, their effects are still quite minimal compared to other inhibitory agents. The central aim of this thesis was to use novel screening strategies to identify small molecule NGF-inhibitors capable of modulating NGF signalling with greater efficiency than previously reported compounds for the purpose of therapeutic development. Using surface plasmon resonance (SPR) spectroscopy, established NGF-inhibitors and a series of novel compounds were analyzed for their specificity to NGF and their inhibitory properties of NGF binding to the TrkA receptor. In vitro techniques confirmed the high nanomolar inhibitory properties of a novel compound, BVNP-0197. Molecular modeling techniques also described a putative binding domain for BVNP-0197 to NGF at the loop II/IV cleft, an alternative domain than previously described by small molecule compounds. This binding domain describes an area with higher specificity for TrkA over binding for p75NTR. Serum albumin was also described as having a role in the binding events that occur between small molecule compounds to NGF in vitro. SPR kinetic analysis established that the addition of serum albumin into solution with small molecule compounds increased their specificity for NGF, as well as decreased their potential toxicity. The findings presented in this thesis will contribute to the future development of small molecule NGF-inhibitors.