Wed. Dec 1st, 2021

Chronic pain, which may be associated with nerve damage or metastatic cancer, represents an unmet medical need. Researchers at Duke University Medical Center and colleagues at the University of California (UC), Irvine, have now trawled a “junkyard of cancer drugs” to identify a compound, kenpaullone (KP), that can be reused as a powerful painkiller. . Studies have shown that kenpaullone works by increasing the levels of an ion transporter gene involved in maintaining inhibitory GABA-ergot neurotransmission, with tests in rodent models of nerve damage and bone cancer confirming that the drug effectively reduced pathological pain-like behavior.

“New drugs and other treatments for chronic pain need to be safe, ie the fewer side effects, the better,” said research leader Wolfgang Liedtke, PhD, who has been practicing pain medicine for the past 17 years at Duke University Medical Center and previously headed Liedtke-Lab. to illuminate basic pain mechanisms. “It is especially important that they are non-addictive and non-sedative, while being effective against nerve damage pain and cancer pain, preferably with a minimal amount of time for official approval. Because chronic pain, like many chronic diseases, has an important root in the fact that genetic switches are reprogrammed in a bad way, a disease-modifying treatment of chronic pain should reset the genetic switches, not just cover the pain, as with opioid and aspirin / Tylenol -like painkillers. “

Liedtke and colleagues reported on their studies in Nature communication, in a paper entitled, “Repurposing cancer drugs identify kenpaullone, which relieves pathological pain in preclinical models via normalization of inhibitory neurotransmission.”

In the central nervous system (CNS) of the mature vertebrate, γ-aminobutyric acid (GABA) acts primarily as an inhibitory neurotransmitter, and this signaling molecule is essential for normal CNS function, the authors wrote. It also represents a potential starting point for identifying potentially safe new approaches to pain management. “In chronic pain, GABA-ergic transmission is compromised, causing circulatory failure and disrupting inhibitory neural networks,” the team continued. “Therapeutic approaches to restoring physiological GABA-ergic transmission would enable us to meet the unmet medical need for chronic pain with safer and more effective alternatives to opioids.”

Inhibitory GABA-ergic neurotransmission requires low chloride concentration in neurons, and this is maintained by KCC2, a neuroprotective ion transporter that effectively expels chloride from neurons. When inhibitory neurotransmission is robust and strong in pain pathways, pain signals are attenuated. However, in chronic pathological pain, KCC2 expression is weakened in specific neurons, and in virtually all forms of chronic pain studied in experimental animals and also in human spinal cord models, KCC2 disappears from the neurons that constitute the primary pain port in the dorsal spinal cord.

“In chronic pathological pain, KCC2 expression is attenuated in the primary sensory port of spinal cord dorsal horn (SCDH) neurons,” the authors wrote. “This key pathophysiological mechanism contributes to an excitation / inhibition imbalance because it corrupts inhibitory neurotransmission, leading to inhibitory circulatory failure … we reasoned that if we could boost Kcc2 / KCC2 gene expression … we could normalize inhibitory transmission to alleviation. of chronic pain. “

To try to identify potential Kcc2 gene expression enhancing candidates, the team screened 1,057 compounds contained in two National Cancer Institute libraries in cultured primary cortical neurons from mice. The researchers were particularly interested in researching cancer drugs because many of these affect the epigenetic regulation of genes. In addition to stopping rapidly dividing cancer cells from multiplying, such epigenetic effects can reset maladaptive genetic switches in non-dividing nerve cells. “We therefore performed an objective screening of cell growth regulating compounds,” the investigators commented. “We searched among these compounds because we assumed that a significant number of them function by interfering with epigenetic and transcriptional machinery to inhibit cell division. Since mature neurons do not divide, these compounds are attractive candidates for upregulating gene expression of Kcc2 / KCC2 via epigenetic mechanisms, thereby lowering intraneuronal chloride levels and restoring normal GABAergic inhibitory function… ”

To identify potential candidates for pain medication from this starting pool, Liedtke’s team screened the connections in neurons derived from genetically engineered mice. These cells have a knock-in modification that enables them to serve as a convenient reporter gene system. Specifically, compounds that increase expression of the Kcc2 gene trigger these cells to generate a measurable bioluminescent signal.

Their screen featured 137 compounds that enhanced the expression of Kcc2. Iterative genetic testing then pointed to four very promising candidates, and among these, kenpaullone was selected for further study because it has a strong record in protecting neurons in several experimental models.

Further studies in mice showed that kenpaullone worked effectively against pain caused by nerve constriction damage and pain caused by cancer cell inoculation in the femur. The pain relief was deep, prolonged and with a prolonged onset, consistent with the fact that the drug had an effect on gene regulation. “In a nerve injury pain model, KP restored Kcc2 expression and GABA-induced chloride reversal potential in the dorsal horn of the spinal cord,” the authors commented. Liedtke further noted, “At this stage, we knew we had met the basic requirements for our off-the-shelf cancer drug screening, namely identified Kcc2 gene expression enhancers, and demonstrated that they are analgesics in valid preclinical pain models.”

Top right: Screening of compounds in the “junkyard of cancer drugs”, similar to aiming through sand and looking for gold nuggets. Kenpaullone was identified as a promising candidate because of its ability to turn on the Kcc2 gene, which has been predicted to relieve chronic pain. Top left: Unmanageable chronic pain is a serious and urgent unmet medical need. Kenpaullone has been shown to be extremely effective in preclinical models of nerve injury pain and bone cancer pain. Left box (untreated pain): Pain-inducing events such as narrowing of nerve damage or cancerous cell expansion in bone activate GSK3ß, an enzyme that labels proteins with phosphate. In spinal pain neurons, GSK3ß labels δ-catenin (δ-CAT) and directs δ-CAT to the cellular trash can. Without δ-CAT in the nucleus of the cell, Kcc2 remains off, allowing the maintenance of high chloride levels in the cell, making the cell electrically more nervous and thus resulting in refractory chronic pain. Right box (treatment): Kenpaullone treatment inhibits GSK3β phosphate labeling, allowing unlabeled δ-CAT to enter the nucleus of a pain-mediating neuron. It binds the DNA region of the Kcc2 gene that turns it on and off, the promoter. By binding the promoter, δ-CAT changes expression of Kcc2, thereby inducing the production of KCC2 protein. KCC2 pumps chloride ions out of pain-relay neurons, making them less nervous. This stability enables circulatory repair and pain relief, based on resetting genetic contacts. In this way, Kenpaullone, or δ-CAT delivered via gene therapy, can upregulate KCC2 and thereby reduce the pain transmission in the spinal cord. [Wolfgang Liedtke, MD, PhD]

The researchers then assessed whether kenpaullone affects the spinal cord treatment of pain and subsequently whether treatment using the drug could reduce nerve damage-induced increase in chloride levels in pain-relay neurons. Both sets of experiments yielded encouraging positive results, prompting the team to look into how exactly kenpaullone increases Kcc2 gene expression. They discovered the underlying signaling mechanism of which a key element had not been previously described, through which kenpaullone inhibits GSK3-beta, an enzyme that adds phosphate tags to proteins; phosphate labels have a potent function-changing effect. They found that GSK3beta adds phosphate labels to delta-catenin (δ-cat), which, when labeled in this way, are destined for cell destruction. In association with chronic pain, activation of GSK3 beta leads to loss of δ-cat in pain-relay neurons.

Liedtke’s team demonstrated an original function of δ-cat in relation to Kcc2 expression and transmission of pain signals. That is, they showed that non-phosphorylated delta-CAT is transported into the nucleus of the cell, where it binds directly to the Kcc2 gene, in its promoter region, which turns on the expression of an off-target Kcc2 gene.

To investigate the relevance of this pathway to pain, Liedtke and colleagues then devised a gene therapy approach in which they loaded an AAV9 viral vector with phosphorylation-resistant δ-cat. To infect the spinal cord’s dorsal horn neurons with AAV9, which drives phosphorylation-resistant delta-CAT, they injected it into the cerebrospinal fluid of mice. Remarkably, they found that this experimental gene therapy had analgesic effects similar to those of kenpaullone. “… we observed that spinal transgenesis of δ-cat (S276A) was sufficient to reverse neuropathic pain and to repair impaired Kcc2 mRNA expression in SCDH,” they said. “Transient spinal overexpression of delta-catenin mimicked KP analgesia.”

The results indicate that kenpaullone and similar active kinase inhibitors, as well as δ-cat gene therapy, have the potential to become new tools in the toolbox for chronic refractory pain, including nerve injury pain and cancer bone pain, and probably for other forms of chronic pain (trigeminal pain). with low Kcc2 expression. This approach can also be effective against other neurological and psychiatric disorders where this mechanism appears to contribute to the disease.

The report’s lead author Michele Yeo, PhD, worked with Liedtke for more than a decade to shed light on basic regulation of the Kcc2 gene. Co-author Yong Chen, PhD, now has his own research lab at Duke. Co-senior author Ru-Rong Ji, PhD, director of translational pain research at Duke, and his team studied spinal cord pain relay neurons. Collaboration between Liedtke’s laboratory and the laboratory of Jorge Busciglio, PhD, at UC Irvine was the key to validating the human applicability of kenpaullone.

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