Inhibition of dual leucine zipper kinase prevents chemotherapy-induced peripheral neuropathy and cognitive impairments.

ABSTRACT: Chemotherapy-induced peripheral neuropathy (CIPN) and chemotherapy-induced cognitive impairments (CICI) are common, often severe neurotoxic side effects of cancer treatment that greatly reduce quality of life of cancer patients and survivors. Currently, there are no Food and Drug Administration-approved agents for the prevention or curative treatment of CIPN or CICI. The dual leucine zipper kinase (DLK) is a key mediator of axonal degeneration that is localized to axons and coordinates the neuronal response to injury. We developed a novel brain-penetrant DLK inhibitor, IACS'8287, which demonstrates potent and highly selective inhibition of DLK in vitro and in vivo. Coadministration of IACS'8287 with the platinum derivative cisplatin prevents mechanical allodynia, loss of intraepidermal nerve fibers in the hind paws, cognitive deficits, and impairments in brain connectivity in mice, all without interfering with the antitumor activity of cisplatin. The protective effects of IACS'8287 are associated with preservation of mitochondrial function in dorsal root ganglion neurons and in brain synaptosomes. In addition, RNA sequencing analysis of dorsal root ganglia reveals modulation of genes involved in neuronal activity and markers for immune cell infiltration by DLK inhibition. These data indicate that CIPN and CICI require DLK signaling in mice, and DLK inhibitors could become an attractive treatment in the clinic when coadministered with cisplatin, and potentially other chemotherapeutic agents, to prevent neurotoxicities as a result of cancer treatment.

Application of High-Throughput Automated Patch-Clamp Electrophysiology to Study Voltage-Gated Ion Channel Function in Primary Cortical Cultures.

Conventionally, manual patch-clamp electrophysiological approaches are the gold standard for studying ion channel function in neurons. However, these approaches are labor-intensive, yielding low-throughput results, and are therefore not amenable for compound profiling efforts during the early stages of drug discovery. The SyncroPatch 384PE has been successfully implemented for pharmacological experiments in heterologous overexpression systems that may not reproduce the function of voltage-gated ion channels in a native, heterogeneous environment. Here, we describe a protocol allowing the characterization of endogenous voltage-gated potassium (Kv) and sodium (Nav) channel function in developing primary rat cortical cultures, allowing investigations at a significantly improved throughput compared with manual approaches. Key neuronal marker expression and microelectrode array recordings of electrophysiological activity over time correlated well with neuronal maturation. Gene expression data revealed high molecular diversity in Kv and Nav subunit composition throughout development. Voltage-clamp experiments elicited three major current components composed of inward and outward conductances. Further pharmacological experiments confirmed the endogenous expression of functional Kv and Nav channels in primary cortical neurons. The major advantages of this approach compared with conventional manual patch-clamp systems include unprecedented improvements in experimental ease and throughput for ion channel research in primary neurons. These efforts demonstrated feasibility for primary neuronal ion channel investigation with the SyncroPatch, providing the foundation for future studies characterizing biophysical changes in endogenous ion channels in primary systems associated with disease or development.

Lead optimization of purine based orally bioavailable Mps1 (TTK) inhibitors.

Efforts to optimize biological activity, novelty, selectivity and oral bioavailability of Mps1 inhibitors, from a purine based lead MPI-0479605, are described in this Letter. Mps1 biochemical activity and cytotoxicity in HCT-116 cell line were improved. On-target activity confirmation via mechanism based G2/M escape assay was demonstrated. Physico-chemical and ADME properties were optimized to improve oral bioavailability in mouse.

Characterization of the cellular and antitumor effects of MPI-0479605, a small-molecule inhibitor of the mitotic kinase Mps1.

Mps1 is a dual specificity protein kinase that is essential for the bipolar attachment of chromosomes to the mitotic spindle and for maintaining the spindle assembly checkpoint until all chromosomes are properly attached. Mps1 is expressed at high levels during mitosis and is abundantly expressed in cancer cells. Disruption of Mps1 function induces aneuploidy and cell death. We report the identification of MPI-0479605, a potent and selective ATP competitive inhibitor of Mps1. Cells treated with MPI-0479605 undergo aberrant mitosis, resulting in aneuploidy and formation of micronuclei. In cells with wild-type p53, this promotes the induction of a postmitotic checkpoint characterized by the ATM- and RAD3-related-dependent activation of the p53-p21 pathway. In both wild-type and p53 mutant cells lines, there is a growth arrest and inhibition of DNA synthesis. Subsequently, cells undergo mitotic catastrophe and/or an apoptotic response. In xenograft models, MPI-0479605 inhibits tumor growth, suggesting that drugs targeting Mps1 may have utility as novel cancer therapeutics.