Chronic pain that might be associated with nerve injury, or with 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 through a “junkyard of cancer drugs” to identify a compound, kenpaullone (KP), that might be repurposed as a powerful pain medication. Studies indicated that kenpaullone acts by boosting levels of an ion transporter gene that is involved in maintaining inhibitory GABA-ergic neurotransmission, with tests in rodent models of nerve injury and bone cancer confirming that the drug effectively reduced pathologic pain-like behavior.
“New drugs and other therapies against chronic pain need to be safe, i.e., the fewer side effects the better,” said research lead Wolfgang Liedtke, PhD, who has practiced pain medicine for the last 17 years at Duke University Medical Center and directed the former Liedtke-Lab to elucidate basic pain mechanisms. “It is especially important that they be non-addictive and non-sedative, while being effective against nerve injury pain and cancer pain, preferably with a minimal time to official approval. Because chronic pain, like many chronic diseases, has an important root in genetic switches being reprogrammed in a bad way, a disease-modifying treatment for chronic pain should reset the genetic switches, not just cover up the pain, as with opioid and aspirin/Tylenol-like painkillers.”
Liedtke and colleagues reported on their studies in Nature Communications, in a paper titled, “Repurposing cancer drugs identifies kenpaullone which ameliorates pathologic pain in preclinical models via normalization of inhibitory neurotransmission.”
In the mature vertebrate central nervous system (CNS), γ-aminobutyric acid (GABA) acts primarily as an inhibitory neurotransmitter, and this signaling molecule is critical for normal CNS functioning, the authors wrote. It also represents a potential starting point for identifying potentially safe new approaches to treating pain. “In chronic pain, GABA-ergic transmission is compromised, causing circuit malfunction and disrupting inhibitory neural networks,” the team continued. “Therapeutic approaches for restoring physiologic GABA-ergic transmission would enable us to address the unmet medical need of 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 silenced. But in chronic pathologic pain, KCC2 expression is attenuated in specific neurons, and in essentially all forms of chronic pain studied in experimental animals and also in human spinal cord models, KCC2 disappears from the neurons that make up the primary pain gate in the dorsal spinal cord.
“In chronic pathologic pain, KCC2 expression is attenuated in the primary sensory gate in spinal cord dorsal horn (SCDH) neurons,” the authors wrote. “This key pathophysiological mechanism contributes to an imbalance of excitation/inhibition because it corrupts inhibitory neurotransmission, leading to inhibitory circuit malfunction … we reasoned that if we could boost Kcc2/KCC2 gene expression … we could re-normalize inhibitory transmission for relief of chronic pain.”
To try and identify potential Kcc2 gene expression-enhancing candidates, the team screened 1,057 compounds contained in two National Cancer Institute libraries, in cultured mouse primary cortical neurons. The researchers were particularly interested in examining cancer drugs because many of these influence 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, conducted an unbiased screen of cell growth-regulating compounds,” the investigators commented. “We searched among these compounds because we assumed that a sizable 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 to upregulate gene expression of Kcc2/KCC2 via epigenetic mechanisms, thus lowering intraneuronal chloride levels and re-establishing normal GABAergic inhibitory functioning …”
To identify potential candidate anti-pain drugs from this starting pool, Liedtke’s team screened the compounds 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 enhance expression of the Kcc2 gene trigger these cells to generate a measurable bioluminescent signal.
Their screen highlighted 137 compounds that enhanced the expression of Kcc2. Iterative retesting then pointed to four highly promising candidates, and among these, kenpaullone was selected for further study because it has a strong record of protecting neurons in multiple experimental models.
Further studies in mice showed that kenpaullone functioned effectively against pain caused by nerve constriction injury and pain caused by cancer cell seeding in the femur. The pain relief was profound, long lasting, and with a protracted onset, consistent with the drug having an impact on gene regulation. “In a nerve-injury pain model, KP restored Kcc2 expression and GABA-evoked chloride reversal potential in the spinal cord dorsal horn,” the authors commented. Liedtke further remarked, “At this stage, we knew we had met the basic requirement of our screen of shelved cancer drugs, namely identified Kcc2 gene expression-enhancers, and demonstrated that they are analgesics in valid preclinical pain models.”
The researchers next assessed whether kenpaullone affects spinal cord processing of pain and, subsequently, whether treatment using the drug could reduce nerve injury-induced elevation of chloride levels in pain-relaying neurons. Both sets of experiments yielded encouragingly positive results, leading the team to look at clarifying how exactly kenpaullone augments Kcc2 gene expression. They discovered the underlying signaling mechanism, a key element of which had not been described previously, through which kenpaullone inhibits GSK3-beta, an enzyme that adds phosphate tags to proteins; phosphate tags have a potent function-switching effect. They found that GSK3beta adds phosphate tags to delta-catenin (δ-cat), which, when tagged in this way, is fated for destruction by the cell. In the context of chronic pain, activation of GSK3-beta leads to loss of δ-cat in pain-relaying neurons.
Liedtke’s team demonstrated an original function of δ-cat in relation to Kcc2 expression and the relaying of pain signals. That is, they showed that non-phosphorylated delta-CAT is transported into the cell’s nucleus where it binds directly to the Kcc2 gene, in its promoter region, which switches on the expression of a switched-off Kcc2 gene.
To probe the relevance of this pathway for pain, Liedtke and colleagues then devised a gene-therapeutic approach wherein they loaded an AAV9 viral vector, with phosphorylation-resistant δ-cat. To infect spinal cord dorsal horn neurons with AAV9 driving 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 attenuated Kcc2 mRNA expression in the SCDH,” they stated. “Transient spinal over-expression of delta-catenin mimicked KP analgesia.”
The findings indicate that kenpaullone and similarly-acting kinase-inhibitory compounds, as well as δ-cat gene therapy, have the potential to become new tools in the toolbox against chronic refractory pain, including nerve injury pain and cancer bone pain, and likely against other forms of chronic pain (trigeminal pain) associated with low Kcc2 expression. This approach may also be effective against other neurologic and psychiatric disorders in which this mechanism appears to contribute to the disease.
The report’s co-first author Michele Yeo, PhD, worked with Liedtke for more than a decade to elucidate basic regulation of the Kcc2 gene. Co-first author Yong Chen, PhD, now has his own research laboratory at Duke. The co-senior author Ru-Rong Ji, PhD, director of translational pain research at Duke, and his team investigated spinal cord pain relay neurons. Collaboration between Liedtke’s laboratory with the lab of Jorge Busciglio, PhD, at UC Irvine was key to validating the human applicability of kenpaullone.