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Spontaneous activity in dorsal root ganglion (DRG) neurons is a key driver of neuropathic pain in preclinical models and in patients suffering from this largely untreated disease. While many intracellular signaling mechanisms have been examined in preclinical models that drive this spontaneous activity (SA), none of these have been tested directly on spontaneously active human nociceptors. Using cultured DRG neurons recovered during thoracic vertebrectomy surgeries, we show that inhibition of mitogen activated protein kinase interacting kinase (MNK) with eFT508 (25 nM) reverses SA in human sensory neurons associated with painful dermatomes. MNK inhibition in spontaneously active nociceptors decreased action potential amplitude and produced alterations in the magnitude of afterhyperpolarizing currents suggesting modification of Na+ and K+ channel activity downstream of MNK inhibition. The effects of MNK inhibition on SA took minutes to emerge and were reversible over time with eFT508 washout. MNK inhibition with eFT508 led to a profound loss of eIF4E Serine 209 phosphorylation, a specific target of the kinase, within 2 min of drug treatment, consistent with the rapid action of the drug on SA in electrophysiology experiments. Our results create a compelling case for the future testing of MNK inhibitors in clinical trials for neuropathic pain.
Antisense oligonucleotides (ASOs) are an emerging class of drugs that target RNAs. Current ASO designs strictly follow the rule of Watson-Crick base pairing along target sequences. However, RNAs often fold into structures that interfere with ASO hybridization. Here we developed a structure-based ASO design method and applied it to target severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our method makes sure that ASO binding is compatible with target structures in three-dimensional (3D) space by employing structural design templates. These 3D-ASOs recognize the shapes and hydrogen bonding patterns of targets via tertiary interactions, achieving enhanced affinity and specificity. We designed 3D-ASOs that bind to the frameshift stimulation element and transcription regulatory sequence of SARS-CoV-2 and identified lead ASOs that strongly inhibit viral replication in human cells. We further optimized the lead sequences and characterized structure-activity relationship. The 3D-ASO technology helps fight coronavirus disease-2019 and is broadly applicable to ASO drug development.
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