ALE-0540

Nerve growth factor (NGF), a member of the neurotrophin family, acts to influence the survival and differentiation of neurons in both the central and peripheral nervous systems via its binding to the p75(NTR) and TrkA receptors. Its precursor, proNGF, has been shown to be the dominant form of NGF in the central nervous system, suggesting a biological function beyond its role as a precursor. Like NGF, proNGF is known to bind the p75(NTR) receptor. The dysregulation of both NGF and proNGF have been implicated in several pathologies, including neurodegenerative diseases linked to p75(NTR)-mediated apoptotic signaling. Therefore, the identification of small molecule inhibitors capable of inhibiting both NGF and proNGF-p75(NTR) interactions may be of therapeutic interest. In the present study, we examine the inhibitory action of known small molecule-based inhibitors PD90780, ALE-0540, Ro 08-2750, and PQC 083, as well as novel derivatives of these compounds, using surface plasmon resonance (SPR) spectroscopy.

Nerve growth factor (NGF) is known to regulate the development and survival of select populations of neurons via its binding/activation of the TrkA and p75(NTR) receptors. However, in some physiological circumstances NGF dysregulation can result in debilitating pathologies, including diabetic neuropathies, interstitial cystitis and fibromyalgia. Thus, the identification of small molecules which inhibit NGF signalling have significant therapeutic potential. PD 90780, Ro 08-2750, and ALE 0540 are small molecules that have been reported to bind and inhibit NGF activity. Importantly, the docking site of these compounds is hypothesised to occur at the loop I/IV cleft of NGF-a region which is required for efficient and selective binding of this neurotrophin to its receptor(s). Molecular modelling predicts a number of previously reported NGF antagonists (PD 90780, ALE 0540, and Ro 08-2750) share conserved molecular features, and these drug-like small molecules have the ability to bind and modify the molecular topology of NGF. In order to understand the putative mechanism of binding, we synthesised a pyrazoloquinazolinecarboxilate analogue series and tested each compound in an NGF-dependent PC12 cell differentiation assay. In vitro data confirms that the pyrazoloquinazolinecarboxilate analogues functionally inhibit NGF's effects on PC12 cell differentiation. The results of this study provide evidence to refine the docking mode of pyrazoloquinazolinecarboxilate based compounds for the purposes of inhibiting NGF in vitro. In addition, we identified series analogue PQC 083 (IC50=7.0 µM; CI=5.4-10.1 µM) which displays markedly higher potency than previously described NGF antagonists.