Epigenetic priming of memory updating during reconsolidation to attenuate remote fear memories.

Traumatic events generate some of the most enduring forms of memories. Despite the elevated lifetime prevalence of anxiety disorders, effective strategies to attenuate long-term traumatic memories are scarce. The most efficacious treatments to diminish recent (i.e., day-old) traumata capitalize on memory updating mechanisms during reconsolidation that are initiated upon memory recall. Here, we show that, in mice, successful reconsolidation-updating paradigms for recent memories fail to attenuate remote (i.e., month-old) ones. We find that, whereas recent memory recall induces a limited period of hippocampal neuroplasticity mediated, in part, by S-nitrosylation of HDAC2 and histone acetylation, such plasticity is absent for remote memories. However, by using an HDAC2-targeting inhibitor (HDACi) during reconsolidation, even remote memories can be persistently attenuated. This intervention epigenetically primes the expression of neuroplasticity-related genes, which is accompanied by higher metabolic, synaptic, and structural plasticity. Thus, applying HDACis during memory reconsolidation might constitute a treatment option for remote traumata.

Histone deacetylases 1 and 2 inhibition suppresses cytokine production and osteoclast bone resorption in vitro.

The regulation of epigenetic factors is an emerging therapeutic target of immune function in a variety of osteolytic pathologies. Histone deacetylases (HDAC) modify core histone proteins and transcriptional processes, in addition to nonhistone protein activity. The activated immune response in rheumatoid arthritis, periodontitis, and prosthetic implant particle release stimulates the catabolic activity of osteoclasts. In this study, we investigated the effects of novel therapeutic agents targeting HDAC isozymes (HDAC 1, 2, and 5), previously shown to be upregulated in inflammatory bone disorders, in cytokine-stimulated human monocytes and osteoclasts in vitro. Inhibiting HDAC 1 and 2 significantly reduced gene expression of IL-1ß, TNF, MCP-1, and MIP-1α in TNF-stimulated monocytes, while suppressing secretions of IL-1ß, IL-10, INF-γ, and MCP-1 (P < .05). Osteoclast formation and bone resorption were also significantly diminished with HDAC 1 and 2 inhibition, through reduced NFATc1 expression and osteoclast specific target genes, TRAF6, CTR, TRAP, and Cathepsin K (P < .05). Similar trends were observed when inhibiting HDAC 1 and to a lesser extent, HDAC 2, in isolation. However, their combined inhibition had the greatest anti-inflammatory and antiosteoclastic effects. Targeting HDAC 5 had minimal effects on these processes investigated in this study, whereas a broad acting HDACi, 1179.4b, had widespread suppressive outcomes. This study demonstrates that targeting HDACs is a potent and effective way of regulating the inflammatory and catabolic processes in human monocytes and osteoclasts. It also demonstrates the importance of targeting individual HDACs with an overall aim to improve efficiency and reduce any potential off target effects.

Enhancer of Zeste Homolog 2 (EZH2) Mediates Glucolipotoxicity-Induced Apoptosis in ß-Cells.

Selective inhibition of histone deacetylase 3 (HDAC3) prevents glucolipotoxicity-induced ß-cell dysfunction and apoptosis by alleviation of proapoptotic endoplasmic reticulum (ER) stress-signaling, but the precise molecular mechanisms of alleviation are unexplored. By unbiased microarray analysis of the ß-cell gene expression profile of insulin-producing cells exposed to glucolipotoxicity in the presence or absence of a selective HDAC3 inhibitor, we identified Enhancer of zeste homolog 2 (EZH2) as the sole target candidate. ß-Cells were protected against glucolipotoxicity-induced ER stress and apoptosis by EZH2 attenuation. Small molecule inhibitors of EZH2 histone methyltransferase activity rescued human islets from glucolipotoxicity-induced apoptosis. Moreover, EZH2 knockdown cells were protected against glucolipotoxicity-induced downregulation of the protective non-canonical Nuclear factor of kappa light polypeptide gene enhancer in B-cells (NFκB) pathway. We conclude that EZH2 deficiency protects from glucolipotoxicity-induced ER stress, apoptosis and downregulation of the non-canonical NFκB pathway, but not from insulin secretory dysfunction. The mechanism likely involves transcriptional regulation via EZH2 functioning as a methyltransferase and/or as a methylation-dependent transcription factor.

Isoform-selective inhibitor of histone deacetylase 3 (HDAC3) limits pancreatic islet infiltration and protects female nonobese diabetic mice from diabetes.

Preservation of insulin-secreting ß-cells is an important goal for therapies aimed at restoring normoglycemia in patients with diabetes. One approach, the inhibition of histone deacetylases (HDACs), has been reported to suppress pancreatic islet inflammation and ß-cell apoptosis in vitro In this report, we demonstrate the efficacy of HDAC inhibitors (HDACi) in vivo We show that daily administration of BRD3308, an isoform-selective HDAC3 inhibitor, for 2 weeks to female nonobese diabetic (NOD) mice, beginning at 3 weeks of age, followed by twice-weekly injections until age 25 weeks, protects the animals from diabetes. The preservation of ß-cells was because of a significant decrease in islet infiltration of mononuclear cells. Moreover, the BRD3308 treatment increased basal insulin secretion from islets cultured in vitro All metabolic tissues tested in vehicle- or BRD3308-treated groups showed virtually no sign of immune cell infiltration, except minimal infiltration in white adipose tissue in animals treated with the highest BRD3308 dose (10 mg/kg), providing additional evidence of protection from immune attack in the treated groups. Furthermore, pancreata from animals treated with 10 mg/kg BRD3308 exhibited significantly decreased numbers of apoptotic ß-cells compared with those treated with vehicle or low-dose BRD3308. Finally, animals treated with 1 or 10 mg/kg BRD3308 had enhanced ß-cell proliferation. These in vivo results point to the potential use of selective HDAC3 inhibitors as a therapeutic approach to suppress pancreatic islet infiltration and prevent ß-cell death with the long-term goal of limiting the progression of type 1 diabetes.

Overlapping and Divergent Actions of Structurally Distinct Histone Deacetylase Inhibitors in Cardiac Fibroblasts.

Inhibitors of zinc-dependent histone deacetylases (HDACs) profoundly affect cellular function by altering gene expression via changes in nucleosomal histone tail acetylation. Historically, investigators have employed pan-HDAC inhibitors, such as the hydroxamate trichostatin A (TSA), which simultaneously targets members of each of the three zinc-dependent HDAC classes (classes I, II, and IV). More recently, class- and isoform-selective HDAC inhibitors have been developed, providing invaluable chemical biology probes for dissecting the roles of distinct HDACs in the control of various physiologic and pathophysiological processes. For example, the benzamide class I HDAC-selective inhibitor, MGCD0103 [N-(2-aminophenyl)-4-[[(4-pyridin-3-ylpyrimidin-2-yl)amino]methyl] benzamide], was shown to block cardiac fibrosis, a process involving excess extracellular matrix deposition, which often results in heart dysfunction. Here, we compare the mechanisms of action of structurally distinct HDAC inhibitors in isolated primary cardiac fibroblasts, which are the major extracellular matrix-producing cells of the heart. TSA, MGCD0103, and the cyclic peptide class I HDAC inhibitor, apicidin, exhibited a common ability to enhance histone acetylation, and all potently blocked cardiac fibroblast cell cycle progression. In contrast, MGCD0103, but not TSA or apicidin, paradoxically increased expression of a subset of fibrosis-associated genes. Using the cellular thermal shift assay, we provide evidence that the divergent effects of HDAC inhibitors on cardiac fibroblast gene expression relate to differential engagement of HDAC1- and HDAC2-containing complexes. These findings illustrate the importance of employing multiple compounds when pharmacologically assessing HDAC function in a cellular context and during HDAC inhibitor drug development.

EHMT1 and EHMT2 inhibition induces fetal hemoglobin expression.

Fetal hemoglobin (HbF, α2γ2) induction is a well-validated strategy for sickle cell disease (SCD) treatment. Using a small-molecule screen, we found that UNC0638, a selective inhibitor of EHMT1 and EHMT2 histone methyltransferases, induces γ-globin expression. EHMT1/2 catalyze mono- and dimethylation of lysine 9 on histone 3 (H3K9), raising the possibility that H3K9Me2, a repressive chromatin mark, plays a role in silencing γ-globin expression. In primary human adult erythroid cells, UNC0638 and EHMT1 or EHMT2 short hairpin RNA-mediated knockdown significantly increased γ-globin expression, HbF synthesis, and the percentage of cells expressing HbF. At effective concentrations, UNC0638 did not alter cell morphology, proliferation, or erythroid differentiation of primary human CD34(+) hematopoietic stem and progenitor cells in culture ex vivo. In murine erythroleukemia cells, UNC0638 and Ehmt2 CRISPR/Cas9-mediated knockout both led to a marked increase in expression of embryonic ß-globin genes Hbb-εy and Hbb-ßh1. In primary human adult erythroblasts, chromatin immunoprecipitation followed by sequencing analysis revealed that UNC0638 treatment leads to genome-wide depletion in H3K9Me2 and a concomitant increase in the activating mark H3K9Ac, which was especially pronounced at the γ-globin gene region. In RNA-sequencing analysis of erythroblasts, γ-globin genes were among the most significantly upregulated genes by UNC0638. Further increase in γ-globin expression in primary human adult erythroid cells was achieved by combining EHMT1/2 inhibition with the histone deacetylase inhibitor entinostat or hypomethylating agent decitabine. Our data provide genetic and pharmacologic evidence that EHMT1 and EHMT2 are epigenetic regulators involved in γ-globin repression and represent a novel therapeutic target for SCD.

Kinetic and structural insights into the binding of histone deacetylase 1 and 2 (HDAC1, 2) inhibitors.

The structure-activity and structure-kinetic relationships of a series of novel and selective ortho-aminoanilide inhibitors of histone deacetylases (HDACs) 1 and 2 are described. Different kinetic and thermodynamic selectivity profiles were obtained by varying the moiety occupying an 11Å channel leading to the Zn(2+) catalytic pocket of HDACs 1 and 2, two paralogs with a high degree of structural similarity. The design of these novel inhibitors was informed by two ligand-bound crystal structures of truncated hHDAC2. BRD4884 and BRD7232 possess kinetic selectivity for HDAC1 versus HDAC2. We demonstrate that the binding kinetics of HDAC inhibitors can be tuned for individual isoforms in order to modulate target residence time while retaining functional activity and increased histone H4K12 and H3K9 acetylation in primary mouse neuronal cell culture assays. These chromatin modifiers, with tuned binding kinetic profiles, can be used to define the relation between target engagement requirements and the pharmacodynamic response of HDACs in different disease applications.

Signal-dependent repression of DUSP5 by class I HDACs controls nuclear ERK activity and cardiomyocyte hypertrophy.

Cardiac hypertrophy is a strong predictor of morbidity and mortality in patients with heart failure. Small molecule histone deacetylase (HDAC) inhibitors have been shown to suppress cardiac hypertrophy through mechanisms that remain poorly understood. We report that class I HDACs function as signal-dependent repressors of cardiac hypertrophy via inhibition of the gene encoding dual-specificity phosphatase 5 (DUSP5) DUSP5, a nuclear phosphatase that negatively regulates prohypertrophic signaling by ERK1/2. Inhibition of DUSP5 by class I HDACs requires activity of the ERK kinase, mitogen-activated protein kinase kinase (MEK), revealing a self-reinforcing mechanism for promotion of cardiac ERK signaling. In cardiac myocytes treated with highly selective class I HDAC inhibitors, nuclear ERK1/2 signaling is suppressed in a manner that is absolutely dependent on DUSP5. In contrast, cytosolic ERK1/2 activation is maintained under these same conditions. Ectopic expression of DUSP5 in cardiomyocytes results in potent inhibition of agonist-dependent hypertrophy through a mechanism involving suppression of the gene program for hypertrophic growth. These findings define unique roles for class I HDACs and DUSP5 as integral components of a regulatory signaling circuit that controls cardiac hypertrophy.

Discovery of a Highly Selective Glycogen Synthase Kinase-3 Inhibitor (PF-04802367) That Modulates Tau Phosphorylation in the Brain: Translation for PET Neuroimaging.

Glycogen synthase kinase-3 (GSK-3) regulates multiple cellular processes in diabetes, oncology, and neurology. N-(3-(1H-1,2,4-triazol-1-yl)propyl)-5-(3-chloro-4-methoxyphenyl)oxazole-4-carboxamide (PF-04802367 or PF-367) has been identified as a highly potent inhibitor, which is among the most selective antagonists of GSK-3 to date. Its efficacy was demonstrated in modulation of tau phosphorylation in vitro and in vivo. Whereas the kinetics of PF-367 binding in brain tissues are too fast for an effective therapeutic agent, the pharmacokinetic profile of PF-367 is ideal for discovery of radiopharmaceuticals for GSK-3 in the central nervous system. A (11) C-isotopologue of PF-367 was synthesized and preliminary PET imaging studies in non-human primates confirmed that we have overcome the two major obstacles for imaging GSK-3, namely, reasonable brain permeability and displaceable binding.

Hydroxamic acid-based histone deacetylase (HDAC) inhibitors can mediate neuroprotection independent of HDAC inhibition.

Histone deacetylase (HDAC) inhibition improves function and extends survival in rodent models of a host of neurological conditions, including stroke, and neurodegenerative diseases. Our understanding, however, of the contribution of individual HDAC isoforms to neuronal death is limited. In this study, we used selective chemical probes to assess the individual roles of the Class I HDAC isoforms in protecting Mus musculus primary cortical neurons from oxidative death. We demonstrated that the selective HDAC8 inhibitor PCI-34051 is a potent neuroprotective agent; and by taking advantage of both pharmacological and genetic tools, we established that HDAC8 is not critically involved in PCI-34051's mechanism of action. We used BRD3811, an inactive ortholog of PCI-34051, and showed that, despite its inability to inhibit HDAC8, it exhibits robust neuroprotective properties. Furthermore, molecular deletion of HDAC8 proved insufficient to protect neurons from oxidative death, whereas both PCI-34051 and BRD3811 were able to protect neurons derived from HDAC8 knock-out mice. Finally, we designed and synthesized two new, orthogonal negative control compounds, BRD9715 and BRD8461, which lack the hydroxamic acid motif and showed that they stably penetrate cell membranes but are not neuroprotective. These results indicate that the protective effects of these hydroxamic acid-containing small molecules are likely unrelated to direct epigenetic regulation via HDAC inhibition, but rather due to their ability to bind metals. Our results suggest that hydroxamic acid-based HDAC inhibitors may mediate neuroprotection via HDAC-independent mechanisms and affirm the need for careful structure-activity relationship studies when using pharmacological approaches.

Chromatin-targeting small molecules cause class-specific transcriptional changes in pancreatic endocrine cells.

Under the instruction of cell-fate-determining, DNA-binding transcription factors, chromatin-modifying enzymes mediate and maintain cell states throughout development in multicellular organisms. Currently, small molecules modulating the activity of several classes of chromatin-modifying enzymes are available, including clinically approved histone deacetylase (HDAC) and DNA methyltransferase (DNMT) inhibitors. We describe the genome-wide expression changes induced by 29 compounds targeting HDACs, DNMTs, histone lysine methyltransferases (HKMTs), and protein arginine methyltransferases (PRMTs) in pancreatic α- and ß-cell lines. HDAC inhibitors regulate several hundred transcripts irrespective of the cell type, with distinct clusters of dissimilar activity for hydroxamic acids and orthoamino anilides. In contrast, compounds targeting histone methyltransferases modulate the expression of restricted gene sets in distinct cell types. For example, we find that G9a/GLP methyltransferase inhibitors selectively up-regulate the cholesterol biosynthetic pathway in pancreatic but not liver cells. These data suggest that, despite their conservation across the entire genome and in different cell types, chromatin pathways can be targeted to modulate the expression of selected transcripts.

Synthesis and profiling of a diverse collection of azetidine-based scaffolds for the development of CNS-focused lead-like libraries.

The synthesis and diversification of a densely functionalized azetidine ring system to gain access to a wide variety of fused, bridged, and spirocyclic ring systems is described. The in vitro physicochemical and pharmacokinetic properties of representative library members are measured in order to evaluate the use of these scaffolds for the generation of lead-like molecules to be used in targeting the central nervous system. The solid-phase synthesis of a 1976-membered library of spirocyclic azetidines is also described.

Inhibition of Histone Deacetylases 1, 2, and 3 Enhances Clearance of Cholesterol Accumulation in Niemann-Pick C1 Fibroblasts.

Niemann-Pick disease type C1 (NPC1) is a rare genetic cholesterol storage disorder caused by mutations in the NPC1 gene. Mutations in this transmembrane late endosome protein lead to loss of normal cholesterol efflux from late endosomes and lysosomes. It has been shown that broad spectrum histone deacetylase inhibitors (HDACi's) such as Vorinostat correct the cholesterol accumulation phenotype in the majority of NPC1 mutants tested in cultured cells. In order to determine the optimal specificity for HDACi correction of the mutant NPC1s, we screened 76 HDACi's of varying specificity. We tested the ability of these HDACi's to correct the excess accumulation of cholesterol in patient fibroblast cells that homozygously express NPC1 I1061T , the most common mutation. We determined that inhibition of HDACs 1, 2, and 3 is important for correcting the defect, and combined inhibition of all three is needed to achieve the greatest effect, suggesting a need for multiple effects of the HDACi treatments. Identifying the specific HDACs involved in the process of regulating cholesterol trafficking in NPC1 will help to focus the search for more specific druggable targets.

HDAC2 targeting stabilizes the CoREST complex in renal tubular cells and protects against renal ischemia/reperfusion injury.

Histone/protein deacetylases (HDAC) 1 and 2 are typically viewed as structurally and functionally similar enzymes present within various co-regulatory complexes. We tested differential effects of these isoforms in renal ischemia reperfusion injury (IRI) using inducible knockout mice and found no significant change in ischemic tolerance with HDAC1 deletion, but mitigation of ischemic injury with HDAC2 deletion. Restriction of HDAC2 deletion to the kidney via transplantation or PAX8-controlled proximal renal tubule-specific Cre resulted in renal IRI protection. Pharmacologic inhibition of HDAC2 increased histone acetylation in the kidney but did not extend renal protection. Protein analysis demonstrated increased HDAC1-associated CoREST protein in HDAC2-/- versus WT cells, suggesting that in the absence of HDAC2, increased CoREST complex occupancy of HDAC1 can stabilize this complex. In vivo administration of a CoREST inhibitor exacerbated renal injury in WT mice and eliminated the benefit of HDAC2 deletion. Gene expression analysis of endothelin showed decreased endothelin levels in HDAC2 deletion. These data demonstrate that contrasting effects of HDAC1 and 2 on CoREST complex stability within renal tubules can affect outcomes of renal IRI and implicate endothelin as a potential downstream mediator.

Selective inhibition of glycogen synthase kinase 3α corrects pathophysiology in a mouse model of fragile X syndrome.

Fragile X syndrome is caused by FMR1 gene silencing and loss of the encoded fragile X mental retardation protein (FMRP), which binds to mRNA and regulates translation. Studies in the Fmr1-/y mouse model of fragile X syndrome indicate that aberrant cerebral protein synthesis downstream of metabotropic glutamate receptor 5 (mGluR5) signaling contributes to disease pathogenesis, but clinical trials using mGluR5 inhibitors were not successful. Animal studies suggested that treatment with lithium might be an alternative approach. Targets of lithium include paralogs of glycogen synthase kinase 3 (GSK3), and nonselective small-molecule inhibitors of these enzymes improved disease phenotypes in a fragile X syndrome mouse model. However, the potential therapeutic use of GSK3 inhibitors has been hampered by toxicity arising from inhibition of both α and ß paralogs. Recently, we developed GSK3 inhibitors with sufficient paralog selectivity to avoid a known toxic consequence of dual inhibition, that is, increased ß-catenin stabilization. We show here that inhibition of GSK3α, but not GSK3ß, corrected aberrant protein synthesis, audiogenic seizures, and sensory cortex hyperexcitability in Fmr1-/y mice. Although inhibiting either paralog prevented induction of NMDA receptor-dependent long-term depression (LTD) in the hippocampus, only inhibition of GSK3α impaired mGluR5-dependent and protein synthesis-dependent LTD. Inhibition of GSK3α additionally corrected deficits in learning and memory in Fmr1-/y mice; unlike mGluR5 inhibitors, there was no evidence of tachyphylaxis or enhanced psychotomimetic-induced hyperlocomotion. GSK3α selective inhibitors may have potential as a therapeutic approach for treating fragile X syndrome.

Lactate is an antidepressant that mediates resilience to stress by modulating the hippocampal levels and activity of histone deacetylases.

Chronic stress promotes depression in some individuals, but has no effect in others. Susceptible individuals exhibit social avoidance and anxious behavior and ultimately develop depression, whereas resilient individuals live normally. Exercise counteracts the effects of stress. Our objective was to examine whether lactate, a metabolite produced during exercise and known to reproduce specific brain exercise-related changes, promotes resilience to stress and acts as an antidepressant. To determine whether lactate promotes resilience to stress, male C57BL/6 mice experienced daily defeat by a CD-1 aggressor, for 10 days. On the 11th day, mice were subjected to behavioral tests. Mice received lactate before each defeat session. When compared with control mice, mice exposed to stress displayed increased susceptibility, social avoidance and anxiety. Lactate promoted resilience to stress and rescued social avoidance and anxiety by restoring hippocampal class I histone deacetylase (HDAC) levels and activity, specifically HDAC2/3. To determine whether lactate is an antidepressant, mice only received lactate from days 12-25 and a second set of behavioral tests was conducted on day 26. In this paradigm, we examined whether lactate functions by regulating HDACs using co-treatment with CI-994, a brain-permeable class I HDAC inhibitor. When administered after the establishment of depression, lactate behaved as antidepressant. In this paradigm, lactate regulated HDAC5 and not HDAC2/3 levels. On the contrary, HDAC2/3 inhibition was antidepressant-like. This indicates that lactate mimics exercise's effects and rescues susceptibility to stress by modulating HDAC2/3 activity and suggests that HDAC2/3 play opposite roles before and after establishment of susceptibility to stress.

2-Hydroxypropyl-ß-cyclodextrin is the active component in a triple combination formulation for treatment of Niemann-Pick C1 disease.

Niemann-Pick type C1 (NPC1) disease is a fatal neurovisceral disease for which there are no FDA approved treatments, though cyclodextrin (HPßCD) slows disease progression in preclinical models and in an early phase clinical trial. Our goal was to evaluate the mechanism of action of a previously described combination-therapy, Triple Combination Formulation (TCF) - comprised of the histone deacetylase inhibitor (HDACi) vorinostat/HPßCD/PEG - shown to prolong survival in Npc1 mice. In these studies, TCF's benefit was attributed to enhanced vorinostat pharmacokinetics (PK). Here, we show that TCF reduced lipid storage, extended lifespan, and preserved neurological function in Npc1 mice. Unexpectedly, substitution of an inactive analog for vorinostat in TCF revealed similar efficacy. We demonstrate that the efficacy of TCF was attributable to enhanced HPßCD PK and independent of NPC1 protein expression. We conclude that although HDACi effectively reduce cholesterol storage in NPC1-deficient cells, HDACi are ineffective in vivo in Npc1 mice.

Exploiting an Asp-Glu "switch" in glycogen synthase kinase 3 to design paralog-selective inhibitors for use in acute myeloid leukemia.

Glycogen synthase kinase 3 (GSK3), a key regulatory kinase in the wingless-type MMTV integration site family (WNT) pathway, is a therapeutic target of interest in many diseases. Although dual GSK3α/ß inhibitors have entered clinical trials, none has successfully translated to clinical application. Mechanism-based toxicities, driven in part by the inhibition of both GSK3 paralogs and subsequent ß-catenin stabilization, are a concern in the translation of this target class because mutations and overexpression of ß-catenin are associated with many cancers. Knockdown of GSK3α or GSK3ß individually does not increase ß-catenin and offers a conceptual resolution to targeting GSK3: paralog-selective inhibition. However, inadequate chemical tools exist. The design of selective adenosine triphosphate (ATP)-competitive inhibitors poses a drug discovery challenge due to the high homology (95% identity and 100% similarity) in this binding domain. Taking advantage of an Asp133→Glu196 "switch" in their kinase hinge, we present a rational design strategy toward the discovery of paralog-selective GSK3 inhibitors. These GSK3α- and GSK3ß-selective inhibitors provide insights into GSK3 targeting in acute myeloid leukemia (AML), where GSK3α was identified as a therapeutic target using genetic approaches. The GSK3α-selective compound BRD0705 inhibits kinase function and does not stabilize ß-catenin, mitigating potential neoplastic concerns. BRD0705 induces myeloid differentiation and impairs colony formation in AML cells, with no apparent effect on normal hematopoietic cells. Moreover, BRD0705 impairs leukemia initiation and prolongs survival in AML mouse models. These studies demonstrate feasibility of paralog-selective GSK3α inhibition, offering a promising therapeutic approach in AML.

Functionally Biased D2R Antagonists: Targeting the ß-Arrestin Pathway to Improve Antipsychotic Treatment.

Schizophrenia is a severe neuropsychiatric disease that lacks completely effective and safe therapies. As a polygenic disorder, genetic studies have only started to shed light on its complex etiology. To date, the positive symptoms of schizophrenia are well-managed by antipsychotic drugs, which primarily target the dopamine D2 receptor (D2R). However, these antipsychotics are often accompanied by severe side effects, including motoric symptoms. At D2R, antipsychotic drugs antagonize both G-protein dependent (Gαi/o) signaling and G-protein independent (ß-arrestin) signaling. However, the relevant contributions of the distinct D2R signaling pathways to antipsychotic efficacy and on-target side effects (motoric) are still incompletely understood. Recent evidence from mouse genetic and pharmacological studies point to ß-arrestin signaling as the major driver of antipsychotic efficacy and suggest that a ß-arrestin biased D2R antagonist could achieve an additional level of selectivity at D2R, increasing the therapeutic index of next generation antipsychotics. Here, we characterize BRD5814, a highly brain penetrant ß-arrestin biased D2R antagonist. BRD5814 demonstrated good target engagement via PET imaging, achieving efficacy in an amphetamine-induced hyperlocomotion mouse model with strongly reduced motoric side effects in a rotarod performance test. This proof of concept study opens the possibility for the development of a new generation of pathway selective antipsychotics at D2R with reduced side effect profiles for the treatment of schizophrenia.

Multiplex quantitative assays indicate a need for reevaluating reported small-molecule TrkB agonists.

Brain-derived neurotrophic factor (BDNF) and its receptor, tropomyosin-related kinase B (TrkB), have emerged as key regulators of brain plasticity and represent disease-modifying targets for several brain disorders, including Alzheimer's disease and major depressive disorder. Because of poor pharmacokinetic properties of BDNF, the interest in small-molecule TrkB agonists and modulators is high. Several compounds have been reported to act as TrkB agonists, and their increasing use in various nervous system disorder models creates the perception that these are reliable probes. To examine key pharmacological parameters of these compounds in detail, we have developed and optimized a series of complementary quantitative assays that measure TrkB receptor activation, TrkB-dependent downstream signaling, and gene expression in different cellular contexts. Although BDNF and other neurotrophic factors elicited robust and dose-dependent receptor activation and downstream signaling, we were unable to reproduce these activities using the reported small-molecule TrkB agonists. Our findings indicate that experimental results obtained with these compounds must be carefully interpreted and highlight the challenge of developing reliable pharmacological activators of this key molecular target.