Background & Aims

Apelin (Ape-13) is a peptide hormone acting as one of the endogenous ligands of the class A G protein-coupled receptor, APJ (6). Tissue expression of APJ includes many central nervous system (CNS) structures involved in pain transmission and modulation, like dorsal root ganglia, periaqueductal grey and rostroventral medulla. Consequently, intrathecal administration of Ape-13 induces antinociception suggesting that APJ is a potential drug target (3,4,8). However, Ape-13 exhibits poor brain penetration and resistance to proteolytic degradation further limiting its therapeutic use. To increase plasma stability and drug penetration into the CNS following systemic administration, we designed and synthesized a linear analog of Ape-13 (2-Nal) conjugated to Angiopep-2 (An2). Yet, the An2-2-Nal conjugate retains a hypotensive effect that limits its clinical use as an analgesic. We therefore decided to conjugate to An2 a macrocyclic analog of Ape-13, exhibiting reduced hypotensive action.

Methods

We designed and synthesized a linear analog with C-terminal substitution of Phe13 with the unnatural amino acid 2-Nal (2-Nal) (5) and conjugated it with Angiopep-2 (An2), a brain-penetrant peptide that targets LRP1 receptors expressed on vascular endothelial cells to cross the blood-brain barrier (2). We also decided to conjugate KT04-44, a macrocyclic analog of Ape-13, H2N-c[X-R-L-S-X]-K-G-P-(D-2Nal) (7) to An2. We used radioactive ligand binding assays and BRET-based biosensors to determine receptor binding affinity and signaling profiles. Resistance to enzymatic degradation was assessed using a plasma stability assay coupled with UPLC analysis of peptide integrity in multi-time point 96-well plate. Their ability to induce blood pressure lowering and reduce pain behaviors was assessed by monitoring the change in mean arterial pressure and pain behaviors in a tonic pain model (formalin test) respectively, in male Sprague-Dawley rats.

Results

KT04-44 and An2-KT04-44 exhibit excellent affinity for APJ (Ki = 13 nM and 1.4 nM, respectively) compared to Ape13 (Ki = 0.6 nM). The intracellular signaling profiles of KT04-44 and its An2 conjugated version are similar with regard to G?i1 activation (EC50 = 18 and 19 nM and Emax = 102% and 100%, respectively) and ?-arrestin 2 recruitment (EC50 = 378 nM and 364 nM and Emax = 62% and 64%, respectively). These ligands also retain a high plasma half-life of >24h (KT04-44) and >7h (An2-KT04-44). They exhibit a reduced hypotensive action correlated with their negative bias for the ?-arrestin 2 pathway, which has been linked to hypotensive action (1). Furthermore, both compounds administered intrathecally were effective in reversing the nociceptive behaviors observed after intraplantar injection of formalin. As expected, only An2-KT044 retained its analgesic property following systemic administration, demonstrating that conjugation of KT04-44 with An2 increases its therapeutic use.

Conclusions

Development of linear or macrocyclic apelinergic analogs conjugated to An2 represents a promising approach for the effective treatment of pain, given their excellent affinity for their pharmacological target, their biased signaling limiting the occurrence of adverse effects, and their increased plasma stability for prolonged analgesia. Their development offers a promising alternative to controversial opioid analgesics.

References

1.Besserer-Offroy, É. and al. The hypotensive effect of activated apelin receptor is correlated with ?-arrestin recruitment. Pharmacological Research 131, 7–16 (2018).

2.Demeule, M. and al. Conjugation of a Brain-Penetrant Peptide with Neurotensin Provides Antinociceptive Properties. J. Clin. Invest. 124, 1199-1213 (2014).

3.Lv, S.-Y. and al. Supraspinal antinociceptive effect of apelin-13 in a mouse visceral pain model. Peptides 37, 165–170 (2012).

4.Lv, S. and al. Intravenous Administration of Pyroglutamyl Apelin-13 Alleviates Murine Inflammatory Pain via the Kappa Opioid Receptor. Front. Neurosci. 14, 929 (2020).

5.Murza, A. and al. Elucidation of the Structure-Activity Relationships of Apelin: Influence of Unnatural Amino Acids on Binding, Signaling, and Plasma Stability. ChemMedChem 7, 318–325 (2012).

6.O’Carrol, A-M. and al. The Apelin Receptor APJ : Journey from an Orphan to a Multifaceted Regulator of Homeostasis. J. of Endocrinology 219, R13-R35 (2013).

7.Tran, K. and al. Size-Reduced Macrocyclic Analogues of [Pyr1]-apelin-13 Showing Negative G?12 Bias Still Produce Prolonged Cardiac Effects. J. Med. Chem. 65, 531-551 (2022).

8.Xu, N. and al. Supraspinal administration of apelin-13 induces antinociception via the opioid receptor in mice. Peptides 30, 1153–1157 (2009).

Presenting Author

Alexandre Tremblay

Poster Authors

Alexandre Tremblay

BSc

Université de Sherbrooke

Lead Author

Kien Tran

PhD

Lead Author

Magali Chartier

PhD

Lead Author

Léa Théroux BSc

Institut de pharmacologie de Sherbrooke, Université de Sherbrooke

Lead Author

Jérôme Côté

PhD

Lead Author

Marc-André Bonin

PhD

Lead Author

Isabelle Brochu

MSc

Lead Author

Alexandre Murza

PhD

Lead Author

Jean-Michel Longpré PhD

Institut de pharmacologie de Sherbrooke, Université de Sherbrooke

Lead Author

Pierre-Luc Boudreault

PhD

Lead Author

Philippe Sarret PhD

Institut de pharmacologie de Sherbrooke, Université de Sherbrooke

Lead Author

Topics

  • Treatment/Management: Pharmacology: Novel Targets