Hemopressin as a breakthrough for the cannabinoid field

Abstract

Hemopressin (PVNFKFLSH in rats, and PVNFKLLSH in humans and mice), a fragment derived from the α-chain of hemoglobin, was the first peptide described to have type 1 cannabinoid receptor activity. While hemopressin was shown to have inverse agonist/antagonistic activity, extended forms of hemopressin (i.e. RVD-hemopressin, also called pepcan-12) exhibit type 1 and type 2 cannabinoid receptor agonistic/allosteric activity, and recent studies suggest that they can activate intracellular mitochondrial cannabinoid receptors. Therefore, hemopressin and hemopressin-related peptides could have location-specific and biased pharmacological action, which would increase the possibilities for fine-tunning and broadening cannabinoid receptor signal transduction. Consistent with this, hemopressins were shown to play a role in a number of physiological processes including antinociceptive and anti-inflammatory activity, regulation of food intake, learning and memory. The shortest active hemopressin fragment, NFKF, delays the first seizure induced by pilocarpine, and prevents neurodegeneration in an experimental model of autoimmune encephalomyelitis. These functions of hemopressins could be due to engagement of both cannabinoid and non-cannabinoid receptor systems. Self-assembled nanofibrils of hemopressin have pH-sensitive switchable surface-active properties, and show potential as inflammation and cancer targeted drug-delivery systems. Upon disruption of the self-assembled hemopressin nanofibril emulsion, the intrinsic analgesic and anti-inflammatory properties of hemopressin could help bolster the therapeutic effect of anti-inflammatory or anti-cancer formulations. In this article, we briefly review the molecular and behavioral pharmacological properties of hemopressins, and summarize studies on the intricate and unique mode of generation and binding of these peptides to cannabinoid receptors. Thus, the review provides a window into the current status of hemopressins in expanding the repertoire of signaling and activity by the endocannabinoid system, in addition to their new potential for pharmaceutic formulations.

Keywords: Cannabinoid; Cannabinoid receptors; Endocannabinoid; Hemopressin.

Fig. 1.

The conservation map and stability of hemopressin peptides. (A) The conservation map of hemoglobin α-chain from Rattus norvegicus, based on the Protein Data Bank (RCSB PDB) crystal structure 3HF4. This map was generated using the ConSurf server (Ashkenazy et al., 2016) and Chimera script. The amino acids were colored by their conservation grades, using the color-coding bar with turquoise-through-maroon indicating variable-to-conserved residues. The hemopressin peptides show an overall conserved sequence pattern. (B) The stability of the hemopressin peptides from human and Rattus norvegicus was calculated using the PROSS program (Goldenzweig et al., 2018) and their RCSB PDB maps 4N8T and 3DHT. Eight possible designs for the most stable peptides are shown; mutated positions are easily recognized by having more than one letter in the sequence logo and more than one color per column. Overall, hemopressin is a very stable peptide since the serine residue was the only one suggested to be mutated to alanine, and only in the last design.

Fig. 2.

Amino acid sequence of human hemoglobin α-chain (Wilson et al., 1980). In silico prediction of proteasomal hydrolysis sites (Keşmir et al., 2002) are indicated by small arrows on the hemoglobin amino acid sequence. RVD-HP and additional hemopressin-related fragments, possibly generated in vivo by proteasomal cleavage, are highlighted in red. No hemopressins with C-terminal amino acid extensions have been described, while several N-terminal extended hemopressin-containing peptides have been identified (i.e. SALSDLHAHKLRVDPVNFKLLSH, pepcan23) (Bauer et al., 2012; Gomes et al., 2009). To be noted, in silico the cleavage of the His-Cys bond is not predicted to occur by proteasomal degradation. One possibility is that in vivo the proteasome cleaves the His-Cys bond of hemoglobin α-chain. Another possibility is that additional intracellular peptidases cleave the hemopressin C-terminus right after it is released from the proteasome.

Fig. 3.. In silico docking of hemopressin or NFKF at CB₁R.

Docking of hemopressin (A and B) or NFKF (C and D) at CB₁R (PDB 5TGZ) (de Araujo et al., 2019; Hua et al., 2016). B and D, show amino acid in CB₁R possibly interacting with hemopressin or NFKF, respectively. CB₁R transmembrane hydrophobic helix is shown in red, and hydrophilic intra or extracellular regions are shown in white and blue. Hemopressin and NFKF are shown as a multicolored electronic amino acid shadows within the upper region of CB₁R (located close to the E face of the plasma membrane region). Data obtained using the MOE platform (MOE v2006.08 Chemical Computing Group Inc., 2006; Park et al., 2005).

Fig. 4.

Hemopressins are endocannabinoids derived from the hemoglobin α-chain. Hemopressin functions as a CB₁R antagonist in that it blocks synthetic cannabinoid-mediated signaling. But on its own, it is an inverse agonist in that it decreases KCl-stimulated Ca⁺² increase (Toniolo et al., 2014) like the CB₁R antagonist AM251. Hemopressin, behaving as CB₁R inverse agonist, is able to antagonize the high basal inhibitory G-protein activity and high basal ERK phosphorylation (Heimann et al., 2007). Hemopressin acting on CB₁R inhibits Na⁺/K⁺ ATPase activity in renal epithelial cells LLC-PK1 (Sampaio et al., 2015). On its own, RVD-HP and VD-HP after being secreted (Gelman et al., 2013), can behave as agonists by increasing CB₁R-mediated ERK phosphorylation and Ca²⁺ release (Gomes et al., 2009). RVD-HP also functions as a positive allosteric modulator of CB₂R, and in the presence of CP55,940 or 2-AG, it induces a significant potentiation of cAMP inhibition and [³⁵S]GTPγS binding (Hofer et al., 2015; Petrucci et al., 2017).