Downregulation of Fidgetin-Like 2 Increases Microglial Function: The Relationship Between Microtubules, Morphology, and Activity.

The microtubule cytoskeleton regulates microglial morphology, motility, and effector functions. The microtubule-severing enzyme, fidgetin-like 2 (FL2), negatively regulates cell motility and nerve regeneration, making it a promising therapeutic target for central nervous system injury. Microglia perform important functions in response to inflammation and injury, but how FL2 affects microglia is unclear. In this study, we investigated the role of FL2 in microglial morphology and injury responses in vitro. We first determined that the pro-inflammatory stimulus, lipopolysaccharide (LPS), induced a dose- and time-dependent reduction in FL2 expression associated with reduced microglial ramification. We then administered nanoparticle-encapuslated FL2 siRNA to knockdown FL2 and assess microglial functions compared to negative control siRNA and vehicle controls. Time-lapse live-cell microscopy showed that FL2 knockdown increased the velocity of microglial motility. After incubation with fluorescently labeled IgG-opsonized beads, FL2 knockdown increased phagocytosis. Microglia were exposed to low-dose LPS after nanoparticle treatment to model injury-induced cytokine secretion. FL2 knockdown enhanced LPS-induced cytokine secretion of IL-1α, IL-1ß, and TNFα. These results identify FL2 as a regulator of microglial morphology and suggest that FL2 can be targeted to increase or accelerate microglial injury responses.

Novel Benzofuran Derivatives Induce Monoamine Release and Substitute for the Discriminative Stimulus Effects of 3,4-Methylenedioxymethamphetamine.

3,4-Methylenedioxymethamphetamine (MDMA) has shown efficacy as a medication adjunct for treating post-traumatic stress disorder (PTSD). However, MDMA is also used in non-medical contexts that pose risk for cardiovascular and neurological complications. It is well established that MDMA exerts its effects by stimulating transporter-mediated release of the monoamines, 5­hydroxytryptamine (5-HT), norepinephrine, and dopamine. Current research efforts are aimed at developing MDMA-like monoamine releasers with better efficacy and safety profiles. To this end, we investigated neurochemical and behavioral effects of novel analogs of the designer drug, 5-(2-methylaminopropyl)benzofuran (5-MAPB). We used in vitro transporter assays in rat brain synaptosomes to examine transmitter uptake inhibition and releasing properties for enantiomers of 5-(2-methylaminobutyl)benzofuran (5-MABB) and 6-(2-methylaminobutyl)benzofuran (6-MABB) as compared to MDMA. We then tested these same compounds in male Sprague-Dawley rats trained to discriminate MDMA (1.5 mg/kg) from saline. In vitro results revealed that S isomers of 5- and 6-MABB are efficacious releasing agents at transporters for 5-HT (SERT), norepinephrine (NET), and dopamine (DAT). By contrast, R isomers are efficacious releasers at SERT, partial releasers at NET, but lack releasing activity at DAT. In vivo results showed that all compounds produce dose-dependent increases in MDMA-lever responding and full substitution at the highest dose tested. The diminished NET and DAT releasing activities for R isomers of 5- and 6-MABB are associated with reduced potency for inducing behavioral effects. Collectively, these findings indicate that the aminoalkyl benzofuran scaffold may be a viable template for developing compounds with MDMA-like properties. Significance Statement Despite the clinical utility of MDMA, the drug is associated with certain cardiovascular risks and metabolic side effects. Developing a therapeutic alternative with MDMA-like monoamine releasing activity is of interest. Our in vitro and in vivo findings indicate that the aminoalkyl benzofuran scaffold may be useful for developing compounds with MDMA-like properties.

A covalent fragment-based strategy targeting a novel cysteine to inhibit activity of mutant EGFR kinase.

Several generations of ATP-competitive anti-cancer drugs that inhibit the activity of the intracellular kinase domain of the epidermal growth factor receptor (EGFR) have been developed over the past twenty years. The first-generation of drugs such as gefitinib bind reversibly and were followed by a second-generation such as dacomitinib that harbor an acrylamide moiety that forms a covalent bond with C797 in the ATP binding pocket. Resistance emerges through mutation of the T790 gatekeeper residue to methionine, which introduces steric hindrance to drug binding and increases the Km for ATP. A third generation of drugs, such as osimertinib were developed which were effective against T790M EGFR in which an acrylamide moiety forms a covalent bond with C797, although resistance has emerged by mutation to S797. A fragment-based screen to identify new starting points for an EGFR inhibitor serendipitously identified a fragment that reacted with C775, a previously unexploited residue in the ATP binding pocket for a covalent inhibitor to target. A number of acrylamide containing fragments were identified that selectively reacted with C775. One of these acrylamides was optimized to a highly selective inhibitor with sub-1 µM activity, that is active against T790M, C797S mutant EGFR independent of ATP concentration, providing a potential new strategy for pan-EGFR mutant inhibition.