The N-type voltage-gated calcium channel (CaV2.2) plays crucial roles in pain transmission, making it a tantalizing therapeutic target. Blocking the channel directly, however, produces profound side effects. Last week, researchers reported a subtler strategy: They inhibited channel activity by thwarting the interaction of CaV2.2 with collapsin response mediator protein 2 (CRMP-2), a protein that facilitates proper trafficking of the channel to the cell surface in neurons. The investigators, led by Rajesh Khanna, Indiana University, Indianapolis, showed that a cell-permeable peptide derived from CRMP-2 disrupted channel activity in cells, and alleviated pain in an assortment of neuropathic and inflammatory pain models in rats. The results were published online in Nature Medicine on June 5.
Based on rodent neurobehavioral tests thus far, the peptide has a squeaky clean side effect profile when administered at analgesic doses. As a result, inhibiting the CaV2.2–CRMP-2 interaction—either with the peptide or with some other agent—may offer a promising new route to pain therapy.
CaV2.2 is involved in neurotransmitter release from presynaptic terminals at multiple points along pain pathways. One CaV2.2 blocker, ziconotide (a synthetic version of ω-conotoxin), is used to treat pain, but that drug causes severe side effects, including hypotension, memory loss, and ataxia. Nonetheless, Khanna was convinced that CaV2.2 could be a good target for pain treatment. Even a little bit of inhibition ought to go a long way, he said, because previous results have shown that “miniscule changes in calcium can lead to tremendous changes in transmitter release—which means the physiological function of the cell, and the body, is going to be dramatically affected.” So rather than obstructing the channel entirely, Khanna hoped to find a more nuanced approach to modulating its function.
CRMP-2 seemed to be just what he was looking for. “It was in the right place, at the right time, in the right neurons,” Khanna said. “Most importantly, it had an effect on the activity of the channel.” In previous work, Khanna and others had identified CRMP-2 in a screen for proteins that interacted with CaV2.2, and they discovered that CRMP-2 regulated CaV2.2 activity. Overexpression of CRMP-2 increased expression of CaV2.2 at the cell surface of neurons, enhanced calcium currents, and augmented the stimulus-dependent release of the neuropeptide calcitonin gene-related peptide (CGRP) from dorsal root ganglion (DRG) sensory neurons (Brittain and et al., 2009; Chi et al., 2009).
In the current study, first author Joel Brittain and colleagues set out to obstruct the CaV2.2–CRMP-2 interaction using 15-amino acid bits of CRMP-2. One peptide, CBD3 (from CaV-binding domain 3), bound CaV2.2 in vitro and prevented it from interacting with CRMP-2. Expressing the peptide with CaV2.2 in neuronal cells prevented the channel from trafficking to the plasma membrane and led to a decrease in Ca2+ currents. A modified peptide, in which CBD3 was fused to the TAT domain of HIV-1 (TAT-CBD3), was cell permeant and reduced Ca2+ influx in cultured DRG neurons. In spinal cord slices, TAT-CBD3 reduced capsaicin-evoked release of CGRP from sensory neurons and inhibited synaptic transmission in dorsal horn neurons. In rats, the peptide blocked capsaicin-induced, CGRP-dependent changes in meningeal blood flow (a model of migraine mechanisms), indicating that it was capable of inhibiting sensory neuron responses in vivo.
The researchers then went on to see what the TAT-CBD3 peptide could do for pain. They found that the peptide reduced pain-related behaviors in rats after formalin injection to the hind paw (a model of inflammatory pain) or capsaicin applied to the eye (a model of acute pain). The peptide also showed efficacy against a model of long-lasting, painful neuropathy, caused by the AIDS drug 2’,3’ dideoxycytidine (ddC): Intraperitoneal injection of the peptide seven days after an injection of ddC reversed tactile hypersensitivity.
Pharmacokinetic experiments revealed that the injected peptide made it into the DRG, spinal cord, and even the brain. Nonetheless, at doses that reduced pain, TAT-CBD3 produced no ill effects in tests of motor function, spatial memory, and anxiety-associated behaviors.
“This is now at a level where we’ve done some serious preclinical work to show that [TAT-CBD3] is effective in a variety of pain models... It’s pretty exciting,” Khanna told PRF. He and his collaborators are continuing to test the TAT-CBD3 peptide in additional pain models. They have recently seen evidence that it can protect neurons from the effects of stroke and traumatic brain injury, and Khanna says he is also interested in testing its effects on chemically induced itch.
The researchers are also turning to the tactical question of what compound would be best suited to disrupting the CaV2.2–CRMP-2 interaction in clinical practice. Khanna notes that peptide therapeutics are expensive to synthesize and tend to get degraded in vivo. To get around that problem, he is collaborating with co-author Samy Meroueh, a chemist and computational biologist who is also at Indiana University, to design small-molecule peptidomimetics that could reproduce the activity of the TAT-CBD3 peptide.