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Identification and transcriptional characterisation of novel inhibitors of NAV1.7 AND TRKB

Student thesis: Doctoral ThesisDoctor of Philosophy

The focus of this thesis is on the discovery of novel neurotherapeutics, more specifically, the identification and characterisation of biologics and small molecules that bind to and selectively inhibit function of two transmembrane proteins that have been validated as disease-modifying targets, i.e., Nav1.7 and TrkB. Importantly, transcriptional profiling has been used in an innovative manner in order to characterise genes that are regulated downstream of these receptors and to establish a bespoke transcriptional signature that may potentially be used to evaluate the efficacy and specificity of putative antagonists.
Nav1.7 is an isoform of voltage-gated sodium channels that is implicated in a spectrum of pain disorders and hypothesised to play a role in cancer metastasis via cell fate determination. We synthesised Nav1.7 peptidomimetics as “bait” and identified two hits capable of binding to the peptides from phage-display libraries of single-domain antibodies (vNARs). However, following extensive characterisation we concluded that they did not display significant binding to the native Nav1.7 protein and were not pursued further. We subsequently generated a transcriptional profile of a Nav1.7-selective inhibitor, ProTx-II, and found that Nav1.7 inhibition elicited moderate transcriptional responses in the H460 cells with enrichment in pathways crucial for cell growth and motility, providing a bespoke transcriptional signature that might be used as a screening tool for future drug discovery programs.
TrkB is one of the three Trk isoforms that exhibits high affinity for BDNF. Dysregulation of TrkB/BDNF has been identified in various CNS pathologies and also leads to cell cycle disruption found in malignant cancers. ANA-12 has been reported as a TrkB-selective small-molecule drug that was discovered via in silico screening targeting the “specificity” patch on the TrkB receptor. A similar strategy has been employed by our lab to identify A3 as a potential TrkB-selective antagonist. To compare the transcriptional effects of ANA-12 and A3, we determined the TrkB transcriptional signature and developed a bespoke multiplex assay which allows for rapid quantification of multiple transcripts. Using this assay, it was found that ANA-12 and A3 induced similar transcriptional responses consistent with a shared mechanism of action. However, the results also showed that at best these compounds were modest antagonists as they were unable to fully block the transcriptional responses elicited by the activated TrkB receptor.
To summarise, we have verified the utility of transcriptional profiling for characterising the effects of selective inhibitors. It is likely that Nav1.7 inhibition affects motility-related transcripts which would explain its integral role in cancer metastases. Furthermore, distinctive transcriptional patterns between the TrkB agonist and antagonists suggest that transcriptional characterisation is useful for assessing compound activity and distinguishing one compound class from another.
Key words: Drug discovery, transcriptional characterisation, selective antagonist, pain, phage display, vNARs, Nav1.7, A3, ANA-12, TrkB, neurotrophin.
Original languageEnglish
Awarding Institution
Award date2018


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