The African black mamba’s bite has been called “the kiss of death”—and for good reason. It has been estimated that just 10 to 15 mg of venom from the world’s deadliest snake, triggering severe pain, vomiting, shock, and limb paralysis, can kill a person within hours.
Yet within the black mamba’s venomous brew of toxins, a French research team has discovered a small fraction of peptides with surprising properties. Called “mambalgins,” they are not toxic, and, at least in mice, they produce a potent analgesia that equals the pain-relieving effect of morphine—but with fewer adverse effects.
Eric Lingueglia and his colleagues at the Institut de Pharmacologie Moléculaire et Cellulaire in Valbonne, France, including Sylvia Diochot—an engineer specializing in animal venoms and toxins—and Anne Baron, reported the findings in the October 3 Nature.
The new study shows the mambalgins inhibit the activity of multiple acid-sensing ion channels (ASICs), which are major players in pain pathways responding to tissue acidification—a common feature of many pain-generating conditions including acute heat pain and inflammation. The peptides suppress ASIC channel activity both in central neurons and in peripheral nociceptors.
In addition to their potential for yielding novel painkillers with superior side effect profiles, the mambalgins are already revealing unappreciated functions of ASIC channels and new therapeutic targets.
ASICs, first cloned in the 1990s, are cationic channels activated by extracellular protons. They are expressed both in the central nervous system and in peripheral nerves. In rodents, six types are known, arising from four genes—ASIC1-4. Four subtypes are splice variants: ASIC1a, 1b, 2a, and 2b. ASICs can be homomeric—containing one channel type—or heteromeric, made up of different combinations of variants. The roles of ASIC types and subtypes in pain sensation are being explored by the Lingueglia group and others, using snake, spider, and other venoms to both activate and inhibit them.
“Mambalgins have the unique property of being potent, rapid, and reversible inhibitors of recombinant homomeric ASIC1a and heteromeric ASIC1a/2a or ASIC1a/2b channels—that is, all the ASIC subtypes expressed in the central nervous system,” the authors note. Mambalgins had no effect on ASIC2a, ASIC3, ASIC1a/3, and ASIC1b/3 channels.
The functional studies were done in mice, but the investigators showed that the peptides inhibited human ASICs in vitro. “We don’t know if they will have the same effects in humans,” says Lingueglia, “but we are confident, because the peptides act on the human channels, and most of the pain pathways we studied are highly conserved between mice and humans.” Lingueglia reported the peptides have been licensed to a pharmaceutical company in Valbonne for development of human applications.
The Lingueglia group previously used two other venom toxins, PcTx1 (from tarantula) and APETx2 (from sea anemone), to functionally define certain ASIC channels. Now, in a screen for additional ASIC-blockers, the team identified black mamba venom as a potent, reversible inhibitor of ASIC1a expressed in Xenopus oocytes. Two active fractions were collected—isopeptides they named mambalgin-1 and mambalgin-2. They belong to the class of snake venoms termed “three-finger “ peptides, reflecting their distinctive structure.
When injected intrathecally, mambalgins produced strong analgesia against heat pain as assessed with the tail-flick and paw-flick tests, but the effect disappeared in ASIC1a knockout mice, demonstrating the essential involvement of ASIC1a-containing channels. The effect was as potent as that of morphine, but was not reduced by naloxone, indicating that the inhibited channels were independent of opioid pathways. Moreover, in repeated administration, morphine was associated with tolerance such that after five days, its analgesic powers had all but vanished, while mambalgin-1 continued to be effective—additional evidence of a non-opioid mechanism of action.
In experiments looking at peripheral action, injections of mambalgin-1 into mouse paws reduced acute pain and reversed or prevented inflammatory hyperalgesia created by intraplantar injection of carrageenan. Curiously, the researchers found, mambalgin-1 continued to produce analgesia in peripheral pain pathways in the ASIC1a knockouts. If ASIC1a was not responsible, the scientists wondered, what was? Peripheral nociceptors express both ASIC1a and ASIC1b, but the latter’s role in pain was not known. Mambalgin-1 blocks both ASIC1a and ASIC1b in dorsal root ganglion neurons, the scientists noted; therefore, analgesia induced by mambalgin-1 in the ASIC1a knockouts suggests a critical role of ASIC1b in pain sensation. To prove that, the researchers knocked down ASIC1b, and showed that loss of the channel mimicked the effect of mambalgin to reduce pain in the mice.
“ASIC1b at the periphery—nociceptors—was never associated with any type of pain before,” notes Lingueglia. Also, he says, “the heteromeric channel made of ASIC1a+ASIC2a subunits in central neurons was also not known to be involved in pain before this work.”
“Our results indicate that mambalgins have analgesic effects by targeting both primary nociceptors and central neurons, but through different ASIC subtypes,” the authors say.
The effects of mambalgins stand in contrast to those of the tarantula PcTx1 toxin, which also induces analgesia via inhibition of central ASIC1a, but in an opioid-dependent manner (Mazzuca et al., 2007). The mambalgin experiments, together with these previous findings, indicate that “different pathways involving different subtypes of ASIC channels can lead to different types of central analgesia (opioid sensitive or insensitive),” the authors note.
Other snake venoms that target ASIC channels activate them to produce intense pain (see PRF related news story). Therefore, it’s surprising that “one of the most venomous snakes on Earth makes a few molecules of analgesics with no toxicity,” says Lingueglia. Some spider venoms contain analgesic fractions apparently to keep the prey as quiet as possible while the toxic enzymes do their work. But given that the mamba’s venom is so potent and rapidly acting, he says, “I’m not sure it needs something to keep its victims still.”
Richard Saltus is a science journalist based in Boston, Massachusetts, US.
Image: Tad Arensmeier, Wikimedia Commons