A Quantitative Bioassay to Determine the Inhibitory Potency of NGF-TrkA Antagonists

Abstract

In this article, we demonstrate and validate a new bioassay named the NTAB [NGF-TrkA (nerve growth factor-tropomyosin receptor kinase A) antagonist bioassay] for the determination of the inhibitory potency of NGF-TrkA antagonists, based on the inhibition of NGF-dependent proliferation of the human TF1 erythroleukemic cell line.It is well known that NGF holds great therapeutic potential due to its neurotrophic and neuroprotective properties. NGF is also involved in some pathways, however, principally driven by TrkA that, if not correctly regulated, can lead to unwanted pathological outcomes linked to pain, angiogenesis, and cancer.Indeed, there is an increasing interest, from a therapeutic perspective, in designing new effective molecules (antibodies, antibody fragments, or small molecules) able to inhibit the undesired NGF-TrkA pathway. For these reasons, there is an interest to develop functional cell-based assays for determination of the inhibition potency of compounds inhibiting the NGF-TrkA axis. The NTAB presents significant advantages over other published NGF-TrkA functional bioassays, for these reasons: (1) It is quantitative, (2) it measures a pure TrkA response, (3) it is simpler, (4) it is based on a natural biological response, and (5) it is easily scalable from a lab scale to an automated industrial assay.The NTAB assay was validated with a panel of well-characterized NGF-TrkA inhibitors, yielding characteristic dose-response curves, from which the relative strength of the inhibitors was quantitatively determined and used for comparisons. This new bioassay will be very useful to assist in the validation and prioritization of the best inhibitors among a large number of candidates.

Keywords: NGF; TF1 bioassay; TrkA; antagonist; inhibitor.