Australian scientists have discovered promising new candidate analgesic molecules derived from a Penicillium fungus, which represents a promising resource for the development of safer new analgesics, they reported in the Oct. 14, 2019, edition of Proceedings of the National Academy of Sciences (PNAS).

The researchers discovered three new tetrapeptides, called the bilaids, with a unique stereochemical arrangement of hydrophobic amino acids, which target the mu-opioid receptor (MOPr) and were shown to be potential candidate molecules from which to develop new analgesics.

"Ours is the first study to identify the bilaids as being potential candidates for the development of new analgesics," said study co-leader Robert Capon, a professor at the Institute for Molecular Bioscience at The University of Queensland (UQ) in Brisbane.

Developing drugs targeting G protein-coupled receptors (GPCRs), which account for around 35% of all currently approved drugs, has attracted extensive interest, with such ligands being expected to provide the basis for development of agonists with superior pharmaceutical properties.

Agonists that signal by differentially recruiting G proteins over the signaling protein, beta-arrestin, to the MOPr, could deliver improved analgesics, since down-regulating beta-arrestin recruitment decreases adverse effects.

That is important, since although MOPr agonists are currently the gold standard for analgesia, their safety and therapeutic use is limited due to side effects, including respiratory depression, constipation, tolerance and dependence.

Consequently, "there is a pressing need for new analgesics that are effective, but safer than morphine and its derivatives," study co-leader Paul Alewood, a professor in the Institute for Molecular Bioscience at UQ, told BioWorld.

For example, the G protein-biased MOPr small nonpeptide agonist, oliceridine (Olinvo/TRV-130, Trevena Inc.), is a potent analgesic in rodents and has been shown to have a lower incidence of adverse events than morphine in human testing.

Currently in phase III trials for the treatment of postoperative pain, oliceridine was granted fast track designation in the U.S. for the treatment of moderate to severe acute pain in 2015. In 2016, it achieved FDA breakthrough therapy designation for managing moderate to severe acute pain.

In the new PNAS study, Capon and Alewood, in collaboration with MacDonald Christie, professor of pharmacology at the University of Sydney, studied bilaids derived from the Australian estuarine Penicillium sp isolate MST-MF667.

Discovery of the bilaids was serendipitous, said Capon. "We set out to study unusual natural products in microbes from unusual sources. It was only after isolating and identifying the bilaids that we considered the possibility they may be suitable candidates for opioid receptor analgesics."

The bilaids were found to resemble known short peptide opioid agonists and were weak mu-opioid agonists, prompting the researchers to design the potent and selective MOPr agonist, bilorphin.

In contrast to natural product opioid peptides that recruit beta-arrestin very effectively, bilorphin was shown to be G protein-biased, weakly phosphorylating the MOPr and only marginally recruiting beta-arrestin, with no receptor internalization.

Importantly, bilorphin had a similar G protein bias to oliceridine, while molecular dynamics simulations of bilorphin and the strongly arrestin-biased endomorphin-2 with the MOPr indicate distinct receptor interactions and receptor conformations, which could underlie their large differences in bias.

Those are significant findings, "as they show structurally that bilorphin binds differently to the MOPr than to the endomorphin-2 receptor, which is biased towards beta-arrestin," said Alewood. "This gives us hope that bilorphin will signal differently to all other MOPr agonists and therefore be associated with fewer side effects."

Regarding efficacy, although subcutaneous or intravenous bilorphin failed to inhibit nociception in the hotplate test in mice, intrathecally injected bilorphin was shown to be antinociceptive, suggesting that the lack of systemic activity was due to poor penetration of the blood–brain barrier (BBB).

The researchers therefore developed several bilorphin analogues with substitutions to enhance BBB permeability, among which a diglycosylated analogue, bilactorphin, was shown to be orally active and to have similar in vivo potency to morphine in mice.

Collectively, those findings indicate that bilorphin is both a unique molecular tool to improve our understanding of MOPr-biased signaling and a promising lead candidate for the development of next-generation analgesics.

However, Capon said that before bilorphin or its derivatives can be tested in human trials, "this will necessitate extensive in vivo efficacy studies, and assessment of the levels of residual adverse properties, including addiction, tolerance, respiratory suppression and constipation."

Said Christie, "Those initial trials are now underway and, if the outcomes are positive, bilactorphin can be further developed. If positive, this will further strengthen the evidence that increased G protein bias can produce safer opioids."

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