Pain sensitivity after an injury, such as damage to a nerve or an inflammatory insult, involves the synthesis of new proteins in pain neurons. This process is thought to play a role in the transition from acute to chronic pain, suggesting that blocking it may prevent chronic pain from developing in the first place. Now, a new paper reports a novel strategy to inhibit protein synthesis and stop pain in mice.
In a collaborative effort between two labs at the University of Texas at Dallas, US, researchers have developed a new RNA-mimicking molecule, called Poly(A) SPOT-ON, which acts as a competitive inhibitor by binding to and sequestering Poly(A)-binding protein (PABP), a protein required for messenger RNA (mRNA) stabilization. This results in destabilization of mRNA transcripts, preventing the synthesis of new proteins in nociceptive neurons. Further, treating mice with Poly(A) SPOT-ON reduced acute pain and hyperalgesic priming induced by hindpaw incision or the injection of nerve growth factor (NGF), interleukin 6 (IL-6), or capsaicin. Together, this RNA-based “decoy” molecule represents a new class of drugs—RNA-binding protein inhibitors— that may prove useful for the treatment of pain.
“The idea of inhibiting translation by targeting an mRNA-protein interaction with this RNA decoy is extremely interesting and novel. It may even have broad implications in disorders other than pain,” says Cheryl Stucky, Medical College of Wisconsin, Milwaukee, US, who was not involved in the study.
The project was led by Zachary Campbell, a molecular biologist with expertise in RNA-protein interactions who created Poly(A) SPOT-ON, and Theodore Price, a pain researcher who studies the role of protein translation mechanisms in chronic pain. The findings appeared online January 2 in Nature Communications.
The “Achilles’ heel” of protein translation
According to the central dogma of molecular biology, which describes the two-step process by which genes encode proteins, genes are first transcribed into an intermediary, mRNA, which is then translated into protein by ribosomes. Newly translated proteins initiate and sustain sensitization of pain neurons (Khoutorsky and Price, 2018).
mRNA translation, however, first requires two post-transcriptional modifications. The first is placement of a specialized cap on the 5’ end of the mRNA, and the second is a so-called poly(A) tail, consisting of a series of adenosine molecules, added to the 3’ end.
Price’s lab previously demonstrated that inflammation regulates translation via proteins that bind the 5’ cap and that these proteins could be targeted to decrease translation and chronic pain in mice (Moy et al., 2017). In contrast, researchers knew much less about the 3’ tail. What they did know, however, was that the RNA-binding PABPs coat the tail as it grows, which increases the stability of mRNA transcripts and helps guide them to ribosomes for translation into protein.
PABPs and other RNA-binding proteins are essential in regulating what is translated, where in the cell translation occurs, and how much protein is produced. Thus, PABPs appeared as a prime target for blocking the entire process (de la Peña and Campbell, 2018). “We saw this as a potential Achilles’ heel of protein translation, but didn’t have a way to target it,” explained Price.
Unbeknownst to Price at the time, Campbell’s lab, just two floors away, was studying how proteins interact with RNA, from a basic science perspective.
“We were both at a grants club,” said Price. “I had come late and there was one open chair right next to Zak. We didn’t know each other, but quickly realized we were both interested in translation regulation, but for very different reasons.” Over a cup of coffee that lasted hours, they began to put together the basic concept of their study.
“This is really where Ted’s insights were enormous, developing a robust biological context in which to apply my work,” explained Campbell.
They reasoned that if they deciphered the exact sequence that PABP recognizes within mRNA, they could create an artificial RNA molecule with an even higher binding affinity for PABP. Acting as a decoy, this new RNA molecule would sequester PABP away from the mRNA, destabilize it, and prevent the protein translation that sets chronic pain into motion.
Creating Poly(A) SPOT-ON
The first step was to find the mRNA sequence that PABP preferentially binds. “During my postdoc years, I developed a system that allowed us to determine, in an unbiased way, the nucleotide sequence an RNA-binding protein preferentially recognizes,” explained Campbell.
Using the system, called sequence specificity landscapes (SEQRS), Campbell found that, as expected, PABP preferred binding to a homopolymer of adenosines, exactly what is present in a Poly(A) tail. The investigators then made their own RNA molecule that consisted of 10 adenosines in a row, but needed a way to stabilize it.
“RNA is a very ephemeral molecule, so creating drugs that are made of RNA doesn’t work very well, since they would degrade quickly,” said Campbell. “So we borrowed a page from the antisense oligonucleotide community.”
Using some genetic tricks, they made chemical modifications to ribose, the sugar in the RNA backbone, and added one extra adenosine to each end of the RNA with a specialized linker to prevent degradation and cleavage of the molecule. With a new sequence of A*AAAAAAAAAA*A, the asterisks denoting the specialized linker, they named this 12-base RNA Poly(A) SPOT-ON, which mimics the make-up of the Poly(A) tail.
In a series of in vitro experiments, they found that PABP bound more strongly to Poly(A)SPOT-ON than it did to normal, unmodified RNA and that Poly(A) SPOT-ON was much more stable. Indeed, while the unmodified RNA had a half-life of 18 hours, Poly(A) SPOT-ON exhibited a half-life of 10 days.
Moving to cultured dorsal root ganglion (DRG) sensory neurons, the investigators found that Poly(A) SPOT-ON was readily taken up by and distributed throughout cells containing peripherin or TRPV1, proteins that mark pain neurons. Confirming that their proposed strategy did actually interfere with translation, they found that treatment of the cells with Poly(A) SPOT-ON effectively inhibited protein synthesis.
Can Poly(A) SPOT-ON alleviate acute pain?
To test if their newly designed Poly(A) SPOT-ON could relieve acute pain, the group, including first author Paulino Barragán-Iglesias from Price’s lab, injected the proinflammatory cytokines NGF or IL-6 into the hindpaw of mice to cause mechanical hypersensitivity, as measured with von Frey hairs. As hoped, co-injection of Poly(A) SPOT-ON at the same time and location of NGF or IL-6 injection inhibited mechanical hypersensitivity.
They saw similar results with a hindpaw incision model of pain where Poly(A) SPOT-ON was injected at the same time and location of the incision. This not only decreased mechanical hypersensitivity, but also facial grimace and paw guarding, two measures of spontaneous pain.
Although NGF, IL-6, and an incision each result in pain, they affect not only nociceptors but also other cell types. To provide evidence that Poly(A) SPOT-ON relieved pain by specifically targeting nociceptors, the group injected the hindpaw with capsaicin, a chemical that binds and activates TRPV1 channels on pain neurons. This caused robust mechanical and thermal hypersensitivity. Co-injection with Poly(A) SPOT-ON, however, prevented this capsaicin-induced hypersensitivity.
What about chronic pain?
To explore the effects of Poly(A) SPOT-ON on chronic pain, the investigators looked to hyperalgesic priming. Following an injury, pain will in most cases eventually go away. But, still, nociceptors can remain in a “primed” state, such that subsequent exposure to a minimally painful stimulus can cause prolonged hyperalgesia. For instance, naïve mice show only short-lasting pain in response to the proinflammatory chemical prostaglandin E2 (PGE2). But mice that have been primed by receiving NGF or IL-6, but have since recovered, show robust hyperalgesia in response to PGE2, and this lasts for days. “These animals remain susceptible for a very long time,” said Price.
So the researchers looked back to the mice they had injected with NGF, IL-6, or capsaicin, and to those that had received hindpaw incision, nine to 15 days after injection, a time at which their pain had resolved. In these mice, injection with PGE2 caused robust mechanical hypersensitivity, an indication of the presence of hyperalgesic priming. Animals injected with Poly(A) SPOT-ON, however, had only a minimal response to PGE2, showing that Poly(A) SPOT-ON could not only reduce acute pain, but also prevent hyperalgesic priming and the transition to chronic pain.
The undruggable becomes druggable
From a basic science perspective, Poly(A) SPOT-ON represents a first-in-class drug that acts as an RNA mimic to competitively inhibit the interaction between RNA and an RNA-binding protein.
“There are upwards of a couple of thousand RNA-binding proteins,” said Campbell. “But we never had a good pharmacological handle on them. They were always dismissed as undruggable, until now.”
In future studies, the two teams plan to address the mechanism of Poly(A) SPOT-ON pain relief and the question of cell type specificity. Critically, PABP is present in many different cell populations, not only pain neurons. “This approach is going to target lots of cell types,” Stucky said. “Is it affecting pain neurons, keratinocytes, fibroblasts, or maybe even Schwann cells?”
Further, identifying the clinical area where an RNA decoy strategy would be most effective will be an important step.
“Can you use this approach after the injury has already occurred and reverse chronic pain?” asked Stucky. “Because that’s really what you want to do with patients. You can’t inhibit priming if they already have an injury.”
In this regard, Price sees postsurgical pain as a possibility.
“It would be exciting to use this for postsurgical pain where you would give the decoy with a nerve block to relieve pain acutely, but also inhibit protein translation to stop the development of persistent pain,” he explained.
In the meantime, Price and Campbell view their study as a great example of how collaborative research can come about and succeed. “It’s really a fortuitous and accidental meeting that led to this great collaboration,” said Price.
Nathan Fried is a postdoctoral fellow at the University of Pennsylvania, Philadelphia, US.
Image credit: mikrostoker/123RF Stock Photo.
