Background & Aims

Negative allosteric modulators (NAMs) targeting Tropomyosin receptor kinase A (TrkA), a pivotal receptor in neurotrophin signaling, represent a promising avenue in therapeutic intervention. By binding to a site distinct from the active site on TrkA [1-3], NAMs offer a unique approach to attenuate TrkA signaling without directly interfering with the levels of NGF. This modulation holds potential for treating conditions associated with aberrant NGF or TrkA signaling, such as chronic pain conditions. We have during a lead optimization program previously described analgesic and anti-inflammatory effects of TrkA-NAMs, shedding light on the mechanism of action, therapeutic implications, and the prospects this class of molecules hold for addressing unmet medical needs. The aim of the present study was to evaluate the clinical candidate ACD137, a novel TrkA-NAM molecule, for its pharmacological effects in various in vitro and in vivo models.

Methods

IC50 determinations were routinely performed using a cell-based assay. Kinetic profiling was performed at Enzymlogic SL, Spain, using the KINETICfinder® assay, which is based on displacement of an active site directed fluorescent probe. Crystallization was performed at SARomics Biostructures AB using partial TrkA (aa 474-796).
The in vivo models, including chemotherapy-induced peripheral neuropathy (CIPN) and the Brennan model (incisional pain), were performed at Aragen Life Sciences, India, using male Sprague Dawley rats. CIPN was induced by paclitaxel administration (i.p.) on day 0, 2, 4, and 6. Incisional pain was studied by performing a 1 cm incision, involving a longitudinal cut of the plantaris muscle, of the right hind paw. Rats were treated with ACD137 (3, 10, and 30 mg/kg on day 15 for the CIPN model or on day 1 for the Brennan model) via oral route and mechanical allodynia was measured using von Frey filaments at 1h, 3h, and 6h after administration of test compound.

Results

A thorough lead optimization work conducted in our laboratory has led to the discovery of ACD137, a potent, selective, and orally bioavailable TrkA-NAM with an IC50 value of 0.8 nM on TrkA and >31,000-fold selectivity over TrkB. Interestingly, binding of ACD137 reduced the affinity of an active site directed fluorescent probe in a competition assay and yielded a Kd of 0.011 nM for ACD137.
The structure of TrkA in complex with a TrkA-NAM compound was determined to 2.4 Å resolution. The compound was bound in a single conformation at the previously described allosteric site with close to 100% occupancy.
Analgesic efficacy was demonstrated in two models including paclitaxel induced CIPN and incisional pain (Brennan model). Oral administration of ACD137 resulted in a significant analgesic effect corresponding to approximately 70% pain reduction, which was similar to 100 mg/kg of Gabapentin or to 30 mg/kg of Tramadol at 3h and 6h in CIPN or Brennan model, respectively.

Conclusions

TrkA-NAM’s have shown promise in both neuropathic and nociceptive models. Enhanced pain relief is attributed to the inhibition of NGF/TrkA signaling, attenuating peripheral sensitization and inflammation. Identification of selective and potent TrkA-NAM’s could potentially avoid some of the side effects observed for anti-NGF antibodies. ACD137 has previously demonstrated analgesic and anti-inflammatory effects using Hargreave’s test [4]. The crystal structure determination and the competition with the active-site directed probe, suggests that binding of TrkA-NAM’s to the allosteric site induces a conformational change resulting in reduced affinity of TrkA towards ATP. The present study clarifies the mechanism of action and supports the analgesic effect of ACD137 in relevant preclinical models of pain. Overall, TrkA-NAM’s demonstrate potential as a novel therapeutic approach for alleviating different types of pain, highlighting their role in advancing pain management strategies.

References

1.Su, H.-P., et al., Structural characterization of nonactive site, TrkA-selective kinase inhibitors. Proceedings of the National Academy of Sciences, 2017. 114(3).
2.Furuya, N., et al., The juxtamembrane region of TrkA kinase is critical for inhibitor selectivity. Bioorg Med Chem Lett, 2017. 27(5): p. 1233-1236.
3.Bagal, S.K., et al., Discovery of Allosteric, Potent, Subtype Selective, and Peripherally Restricted TrkA Kinase Inhibitors. J Med Chem, 2019. 62(1): p. 247-265.
4.Lidell, V., et al., Analgesic and Anti-inflammatory Effects of Small Molecule Negative Allosteric Modulators of TrkA Poster, 2022. IASP 2022.

Presenting Author

Märta Segerdahl

Poster Authors

Pontus Forsell

Alzecure Pharma AB

Lead Author

Gunnar Nordvall

PhD

Alzecure Pharma AB

Lead Author

Maria Backlund

PhD

Alzecure Pharma AB

Lead Author

Veronica Lidell

MSc

Alzecure Pharma AB

Lead Author

Cristina Parrado-Fernandez

PhD

Alzecure Pharma AB

Lead Author

Johan Sandin

PhD

Alzecure Pharma AB

Lead Author

Märts Segerdahl

MD, PhD

Alzecure Pharma AB

Lead Author