MT-125 Inhibits Non-Muscle Myosin IIA and IIB, Synergizes with Oncogenic Kinase Inhibitors, and Prolongs Survival in Glioblastoma.

We have identified a NMIIA and IIB-specific small molecule inhibitor, MT-125, and have studied its effects in GBM. MT-125 has high brain penetrance and retention and an excellent safety profile; blocks GBM invasion and cytokinesis, consistent with the known roles of NMII; and prolongs survival as a single agent in murine GBM models. MT-125 increases signaling along both the PDGFR- and MAPK-driven pathways through a mechanism that involves the upregulation of reactive oxygen species, and it synergizes with FDA-approved PDGFR and mTOR inhibitors in vitro . Combining MT-125 with sunitinib, a PDGFR inhibitor, or paxalisib, a combined PI3 Kinase/mTOR inhibitor significantly improves survival in orthotopic GBM models over either drug alone, and in the case of sunitinib, markedly prolongs survival in ∼40% of mice. Our results provide a powerful rationale for developing NMII targeting strategies to treat cancer and demonstrate that MT-125 has strong clinical potential for the treatment of GBM. Highlights: MT-125 is a highly specific small molecule inhibitor of non-muscle myosin IIA and IIB, is well-tolerated, and achieves therapeutic concentrations in the brain with systemic dosing.Treating preclinical models of glioblastoma with MT-125 produces durable improvements in survival.MT-125 stimulates PDGFR- and MAPK-driven signaling in glioblastoma and increases dependency on these pathways.Combining MT-125 with an FDA-approved PDGFR inhibitor in a mouse GBM model synergizes to improve median survival over either drug alone, and produces tumor free, prolonged survival in over 40% of mice.

Discovery of Selective Inhibitors for In Vitro and In Vivo Interrogation of Skeletal Myosin II.

Myosin IIs, actin-based motors that utilize the chemical energy of adenosine 5'-triphosphate (ATP) to generate force, have potential as therapeutic targets. Their heavy chains differentiate the family into muscle (skeletal [SkMII], cardiac, smooth) and nonmuscle myosin IIs. Despite the therapeutic potential for muscle disorders, SkMII-specific inhibitors have not been reported and characterized. Here, we present the discovery, synthesis, and characterization of "skeletostatins," novel derivatives of the pan-myosin II inhibitor blebbistatin, with selectivity 40- to 170-fold for SkMII over all other myosin II family members. In addition, the skeletostatins bear improved potency, solubility, and photostability, without cytotoxicity. Based on its optimal in vitro profile, MT-134's in vivo tolerability, efficacy, and pharmacokinetics were determined. MT-134 was well-tolerated in mice, impaired motor performance, and had excellent exposure in muscles. Skeletostatins are useful probes for basic research and a strong starting point for drug development.

Methamphetamine Learning Induces Persistent and Selective Nonmuscle Myosin II-Dependent Spine Motility in the Basolateral Amygdala.

Nonmuscle myosin II inhibition (NMIIi) in the basolateral amygdala (BLA), but not dorsal hippocampus (CA1), selectively disrupts memories associated with methamphetamine (METH) days after learning, without retrieval. However, the molecular mechanisms underlying this selective vulnerability remain poorly understood. A known function of NMII is to transiently activate synaptic actin dynamics with learning. Therefore, we hypothesized that METH-associated learning perpetuates NMII-driven actin dynamics in synapses, leading to an extended window of vulnerability for memory disruption. We used time-lapse two-photon imaging of dendritic spine motility in acutely prepared brain slices from female and male mice following METH-associated learning as a readout of actin-myosin dynamics. Spine motility was persistently increased in the BLA, but not in CA1. Consistent with the memory disrupting effect of intra-BLA NMII inhibition, METH-induced changes to BLA spine dynamics were reversed by a single systemic injection of an NMII inhibitor. Intra-CA1 NMII inhibition, on the other hand, did not disrupt METH-associated memory. Thus, we report identification of a previously unknown ability for spine actin dynamics to persist days after stimulation and that this is under the control of NMII. Further, these perpetual NMII-driven spine actin dynamics in BLA neurons may contribute to the unique susceptibility of METH-associated memories.SIGNIFICANCE STATEMENT There are no Food and Drug Administration-approved pharmacotherapies to prevent relapse to the use of stimulants, such as methamphetamine (METH). Environmental cues become associated with drug use, such that the memories can elicit strong motivation to seek the drug during abstinence. We previously reported that the storage of METH-associated memories is uniquely vulnerable to immediate, retrieval-independent, and lasting disruption by direct actin depolymerization or by inhibiting the actin driver nonmuscle myosin II (NMII) in the BLA or systemically. Here we report a potential structural mechanism responsible for the unique vulnerability of METH-associated memories and METH-seeking behavior to NMII inhibition within the BLA.

The role of nonmuscle myosin II in polydrug memories and memory reconsolidation.

Using pharmacologic and genetic approaches targeting actin or the actin-driving molecular motor, nonmuscle myosin II (NMII), we previously discovered an immediate, retrieval-independent, and long-lasting disruption of methamphetamine- (METH-) and amphetamine-associated memories. A single intrabasolateral amygdala complex infusion or systemic administration of the NMII inhibitor Blebbistatin (Blebb) is sufficient to produce this disruption, which is selective, having no retrieval-independent effect on memories for fear, food reward, cocaine, or morphine. However, it was unclear if Blebb treatment would disrupt memories of other stimulants and amphetamine class drugs, such as nicotine (NIC) or mephedrone (MEPH; bath salts). Moreover, many individuals abuse multiple drugs, but it was unknown if Blebb could disrupt polydrug memories, or if the inclusion of another substance would render Blebb no longer able to disrupt METH-associated memories. Therefore, the present study had two primary goals: (1) to determine the ability of Blebb to disrupt NIC- or MEPH-associated memories, and (2) to determine the ability of METH to modify other unconditioned stimulus (US) associations' susceptibility to Blebb. To this end, using the conditional place preference model, mice were conditioned to NIC and MEPH alone or METH in combination with NIC, morphine, or foot shock. We report that, unlike METH, there was no retrieval-independent effect of Blebb on NIC- or MEPH-associated memories. However, similar to cocaine, reconsolidation of the memory for both drugs was disrupted. Further, when combined with METH administration, NIC- and morphine-, but not fear-, associated memories were rendered susceptible to disruption by Blebb. Given the high rate of polydrug use and the resurgence of METH use, these results have important implications for the treatment of substance use disorder.

Memory disrupting effects of nonmuscle myosin II inhibition depend on the class of abused drug and brain region.

Depolymerizing actin in the amygdala through nonmuscle myosin II inhibition (NMIIi) produces a selective, lasting, and retrieval-independent disruption of the storage of methamphetamine-associated memories. Here we report a similar disruption of memories associated with amphetamine, but not cocaine or morphine, by NMIIi. Reconsolidation appeared to be disrupted with cocaine. Unlike in the amygdala, methamphetamine-associated memory storage was not disrupted by NMIIi in the hippocampus, nucleus accumbens, or orbitofrontal cortex. NMIIi in the hippocampus did appear to disrupt reconsolidation. Identification of the unique mechanisms responsible for NMII-mediated, amygdala-dependent disruption of memory storage associated with the amphetamine class may enable induction of retrieval-independent vulnerability to other pathological memories.

Nonmuscle myosin II inhibition disrupts methamphetamine-associated memory in females and adolescents.

Memories associated with drug use can trigger strong motivation for the drug, which increases relapse vulnerability in substance use disorder (SUD). Currently there are no treatments for relapse to abuse of psychostimulants, such as methamphetamine (METH). We previously reported that storage of memories associated with METH, but not those for fear or food reward, and the concomitant spine density increase are disrupted in a retrieval-independent manner by depolymerizing actin in the basolateral amygdala complex (BLC) of adult male rats and mice. Similar results are achieved in males through intra-BLC or systemic inhibition of nonmuscle myosin II (NMII), a molecular motor that directly drives actin polymerization. Given the substantial differences in physiology between genders, we sought to determine if this immediate and selective disruption of METH-associated memory extends to adult females. A single intra-BLC infusion of the NMII inhibitor Blebbistatin (Blebb) produced a long-lasting disruption of context-induced drug seeking for at least 30days in female rats that mirrored our prior results in males. Furthermore, a single systemic injection of Blebb prior to testing disrupted METH-associated memory and the concomitant increase in BLC spine density in females. Importantly, as in males, the same manipulation had no effect on an auditory fear memory or associated BLC spine density. In addition, we established that the NMII-based disruption of METH-associated memory extends to both male and female adolescents. These findings provide further support that small molecular inhibitors of NMII have strong therapeutic potential for the prevention of relapse to METH abuse triggered by associative memories.

The Actin Cytoskeleton as a Therapeutic Target for the Prevention of Relapse to Methamphetamine Use.

A high rate of relapse is a defining characteristic of substance use disorder for which few treatments are available. Exposure to environmental cues associated with previous drug use can elicit relapse by causing the involuntary retrieval of deeply engrained associative memories that trigger a strong motivation to seek out drugs. Our lab is focused on identifying and disrupting mechanisms that support these powerful consolidated memories, with the goal of developing therapeutics. A particularly promising mechanism is regulation of synaptic dynamics by actin polymerization within dendritic spines. Emerging evidence indicates that memory is supported by structural and functional plasticity dendritic spines, for which actin polymerization is critical, and that prior drug use increases both spine and actin dynamics. Indeed we have found that inhibiting amygdala (AMY) actin polymerization immediately or twenty-four hours prior to testing disrupted methamphetamine (METH)-associated memories, but not food reward or fear memories. Furthermore, METH training increased AMY spine density which was reversed by actin depolymerization treatment. Actin dynamics were also shifted to a more dynamic state by METH training. While promising, actin polymerization inhibitors are not a viable therapeutic, as a multitude of peripheral process (e.g. cardiac function) rely on dynamic actin. For this reason, we have shifted our focus upstream of actin polymerization to nonmuscle myosin II. We and others have demonstrated that myosin IIb imparts a mechanical force that triggers spine actin polymerization in response to synaptic stimulation. Similar to an actin depolymerizing compound, pre-test inhibition of myosin II ATPase activity in the AMY produced a rapid and lasting disruption of drug-seeking behavior. While many questions remain, these findings indicate that myosin II represents a potential therapeutic avenue to target the actin cytoskeleton and disrupt the powerful, extinction-resistant memories capable of triggering relapse.

Methamphetamine-associated memory is regulated by a writer and an eraser of permissive histone methylation.

BACKGROUND: Memories associated with drugs of abuse, such as methamphetamine (METH), increase relapse vulnerability to substance use disorder by triggering craving. The nucleus accumbens (NAc) is essential to these drug-associated memories, but underlying mechanisms are poorly understood. Posttranslational chromatin modifications, such as histone methylation, modulate gene transcription; thus, we investigated the role of the associated epigenetic modifiers in METH-associated memory. METHODS: Conditioned place preference was used to assess the epigenetic landscape in the NAc supporting METH-associated memory (n = 79). The impact of histone methylation (H3K4me2/3) on the formation and expression of METH-associated memory was determined by focal, intra-NAc knockdown (KD) of a writer, the methyltransferase mixed-lineage leukemia 1 (Mll1) (n = 26), and an eraser, the histone lysine (K)-specific demethylase 5C (Kdm5c) (n = 38), of H3K4me2/3. RESULTS: A survey of chromatin modifications in the NAc of animals forming a METH-associated memory revealed the global induction of several modifications associated with active transcription. This correlated with a pattern of gene activation, as revealed by microarray analysis, including upregulation of oxytocin receptor (Oxtr) and FBJ osteosarcoma oncogene (Fos), the promoters of which also had increased H3K4me3. KD of Mll1 reduced H3K4me3, Fos and Oxtr levels and disrupted METH-associated memory. KD of Kdm5c resulted in hypermethylation of H3K4 and prevented the expression of METH-associated memory. CONCLUSIONS: The development and expression of METH-associated memory are supported by regulation of H3K4me2/3 levels by MLL1 and KDM5C, respectively, in the NAc. These data indicate that permissive histone methylation, and the associated epigenetic writers and erasers, represent potential targets for the treatment of substance abuse relapse, a psychiatric condition perpetuated by unwanted associative memories.

Selective, retrieval-independent disruption of methamphetamine-associated memory by actin depolymerization.

BACKGROUND: Memories associated with drugs of abuse, such as methamphetamine (METH), increase relapse vulnerability to substance use disorder. There is a growing consensus that memory is supported by structural and functional plasticity driven by F-actin polymerization in postsynaptic dendritic spines at excitatory synapses. However, the mechanisms responsible for the long-term maintenance of memories, after consolidation has occurred, are largely unknown. METHODS: Conditioned place preference (n = 112) and context-induced reinstatement of self-administration (n = 19) were used to assess the role of F-actin polymerization and myosin II, a molecular motor that drives memory-promoting dendritic spine actin polymerization, in the maintenance of METH-associated memories and related structural plasticity. RESULTS: Memories formed through association with METH but not associations with foot shock or food reward were disrupted by a highly-specific actin cycling inhibitor when infused into the amygdala during the postconsolidation maintenance phase. This selective effect of depolymerization on METH-associated memory was immediate, persistent, and did not depend upon retrieval or strength of the association. Inhibition of non-muscle myosin II also resulted in a disruption of METH-associated memory. CONCLUSIONS: Thus, drug-associated memories seem to be actively maintained by a unique form of cycling F-actin driven by myosin II. This finding provides a potential therapeutic approach for the selective treatment of unwanted memories associated with psychiatric disorders that is both selective and does not rely on retrieval of the memory. The results further suggest that memory maintenance depends upon the preservation of polymerized actin.

Differential activation of amygdala Arc expression by positive and negatively valenced emotional learning conditions.

Norepinephrine is released in the amygdala following negatively arousing learning conditions. This event initiates a cascade of changes including the transcription of activity-regulated cytoskeleton-associated protein (Arc) expression, an early-immediate gene associated with memory encoding. Recent evidence suggests that the valence of emotionally laden encounters may generate lateralized, as opposed to symmetric release of this transmitter in the right or left amygdala. It is currently not clear if valence-induced patterns of selective norepinephrine output across hemispheres are also reproduced in downstream pathways of cellular signaling necessary for memory formation. This question was addressed by determining if Arc expression is differentially distributed across the right and left amygdala following exposure to positively or negatively valenced learning conditions respectively. Male Sprague Dawley rats were randomly assigned to groups exposed to the Homecage only, five auditory tones only, or five auditory tones paired with footshock (0.35 mA) during Pavlovian fear conditioning. Western blot analysis revealed that Arc expression in the right amygdala was elevated significantly above that observed in the left amygdala 60 and 90 min following fear conditioning. Similarly, subjects exposed to a negatively valenced outcome consisting of an unexpected reduction in food rewards showed a greater level of Arc expression in only the right, but not left basolateral amygdala. Presenting a positively valenced event involving an unexpected increase in food reward magnitude following bar pressing, resulted in significantly greater Arc expression in the left, but not right basolateral amygdala (p < 0.01). These findings indicate that the valence of emotionally arousing learning conditions is reflected at later stages of synaptic plasticity involving the transcription of immediate early genes such as Arc.

MicroRNA-182 regulates amygdala-dependent memory formation.

De novo protein synthesis supports long-lasting functional and structural plasticity and is a molecular requirement for new memory formation. Recent evidence has suggested that microRNAs may be involved in regulating the molecular mechanisms underlying neural plasticity. MicroRNAs are endogenous, noncoding RNAs capable of post-transcriptional repression of their mRNA targets. To explore the potential for microRNA-mediated regulation of amygdala-dependent memory formation, we performed expression profiling of microRNAs in the lateral amygdala of rats 1 h after auditory fear conditioning. Microarray analysis revealed that over half of all known microRNAs are endogenously expressed in the lateral amygdala, with 7 microRNAs upregulated and 32 downregulated by auditory fear training. Bioinformatic analysis identified several of the downregulated microRNAs as potential repressors of actin-regulating proteins known to be involved in plasticity and memory. Downregulation of one of these microRNAs by auditory fear conditioning, miR-182, was confirmed by quantitative real-time PCR. Overexpression of miR-182 within the lateral amygdala resulted in decreased expression of the protein but not mRNA of two synapse-enriched regulators of actin known to modulate structural plasticity, cortactin and Rac1. The overexpression of miR-182 also disrupted long-term but not short-term auditory fear memory. These data indicate that learning-induced suppression of miR-182, a microRNA previously uncharacterized in the brain, supports long-term memory formation in the amygdala and suggests it does so, at least in part, through the derepression of key actin-regulating proteins. These findings further indicate that microRNAs may represent a previously underappreciated mechanism for regulating protein synthesis during memory consolidation.

Valence dependent asymmetric release of norepinephrine in the basolateral amygdala.

Emerging evidence from literature on humans suggests the valence of emotionally laden environmental stimuli may dictate whether amygdala activation is greater in one hemisphere relative to the contralateral side. However, only a paucity of animal studies attempt to unravel the mechanisms underlying the selective, valence-dependent initiation of activity in the amygdala of opposing hemispheres. The present studies assessed whether exposure to positive or negative appetitive conditions in an operant learning task differentially impacts norepinephrine activation of the left versus right amygdala, respectively. Dialysate samples of norepinephrine were collected from the basolateral nucleus of male Sprague-Dawley rats. Fluctuations in norepinephrine activity were sampled during training conditions involving an abrupt shift (i.e., increase or decrease) in the magnitude of food rewards normally provided for bar press responses. In a subsequent study, dialysate samples of norepinephrine were collected before, during, and after presentation of a negatively valenced stimulus involving footshock delivery during Pavlovian fear conditioning. Norepinephrine concentrations in the left but not right basolateral amygdala were elevated in groups presented with a positive experience of an unexpected increase in food reward after bar pressing (p < .01), relative to respective controls. In contrast, exposure to negatively valenced events involving a reduction in expected food rewards after bar pressing or presentation of a footshock during fear conditioning produced significant increases in norepinephrine output sampled from only the right but not left basolateral amygdala. These findings demonstrate that the valence of a learning event is selective in initiating asymmetric activation of the basolateral amygdala.