Chemically targeting the redox switch in AP1 transcription factor ΔFOSB.

The AP1 transcription factor ΔFOSB, a splice variant of FOSB, accumulates in the brain in response to chronic insults such as exposure to drugs of abuse, depression, Alzheimer's disease and tardive dyskinesias, and mediates subsequent long-term neuroadaptations. ΔFOSB forms heterodimers with other AP1 transcription factors, e.g. JUND, that bind DNA under control of a putative cysteine-based redox switch. Here, we reveal the structural basis of the redox switch by determining a key missing crystal structure in a trio, the ΔFOSB/JUND bZIP domains in the reduced, DNA-free form. Screening a cysteine-focused library containing 3200 thiol-reactive compounds, we identify specific compounds that target the redox switch, validate their activity biochemically and in cell-based assays, and show that they are well tolerated in different cell lines despite their general potential to bind to cysteines covalently. A crystal structure of the ΔFOSB/JUND bZIP domains in complex with a redox-switch-targeting compound reveals a deep compound-binding pocket near the DNA-binding site. We demonstrate that ΔFOSB, and potentially other, related AP1 transcription factors, can be targeted specifically and discriminately by exploiting unique structural features such as the redox switch and the binding partner to modulate biological function despite these proteins previously being thought to be undruggable.

Exifone Is a Potent HDAC1 Activator with Neuroprotective Activity in Human Neuronal Models of Neurodegeneration.

Genomic instability caused by a deficiency in the DNA damage response and repair has been linked to age-related cognitive decline and neurodegenerative diseases. Preventing genomic instability that ultimately leads to neuronal death may provide a broadly effective strategy to protect against multiple potential genotoxic stressors. Recently, the zinc-dependent class I histone deacetylase (HDAC1) has been identified as a critical factor for protecting neurons from deleterious effects of DNA damage in Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD). Translating these observations to a novel neuroprotective therapy for AD, ALS, and FTD may be advanced by the identification of small molecules capable of increasing the deacetylase activity of HDAC1 selectively over other structurally similar HDACs. Here, we demonstrate that exifone, a drug previously shown to be effective in treating cognitive deficits associated with AD and Parkinson's disease, the molecular mechanism of which has remained poorly understood, potently activates the deacetylase activity of HDAC1. We show that exifone acts as a mixed, nonessential activator of HDAC1 that is capable of binding to both free and substrate-bound enzyme, resulting in an increased relative maximal rate of HDAC1-catalyzed deacetylation. Exifone can directly bind to HDAC1 based upon biolayer interferometry assays with kinetic and selectivity profiling, suggesting that HDAC1 is preferentially targeted compared to other class I HDACs and the kinase CDK5, which have also been implicated in neurodegeneration. Consistent with a mechanism of deacetylase activation intracellularly, the treatment of human induced pluripotent stem cell (iPSC)-derived neuronal cells resulted in globally decreased histone acetylation. Moreover, exifone treatment was neuroprotective in a tauopathy patient iPSC-derived neuronal model subject to oxidative stress. Taken together, these findings reveal exifone as a potent activator of HDAC1-mediated deacetylation, thereby offering a lead for novel therapeutic development aiming to protect genomic integrity in the context of neurodegeneration and aging.

Discovery of suppressors of CRMP2 phosphorylation reveals compounds that mimic the behavioral effects of lithium on amphetamine-induced hyperlocomotion.

The effective treatment of bipolar disorder (BD) represents a significant unmet medical need. Although lithium remains a mainstay of treatment for BD, limited knowledge regarding how it modulates affective behavior has proven an obstacle to discovering more effective mood stabilizers with fewer adverse side effects. One potential mechanism of action of lithium is through inhibition of the serine/threonine protein kinase GSK3ß, however, relevant substrates whose change in phosphorylation may mediate downstream changes in neuroplasticity remain poorly understood. Here, we used human induced pluripotent stem cell (hiPSC)-derived neuronal cells and stable isotope labeling by amino acids in cell culture (SILAC) along with quantitative mass spectrometry to identify global changes in the phosphoproteome upon inhibition of GSK3α/ß with the highly selective, ATP-competitive inhibitor CHIR-99021. Comparison of phosphorylation changes to those induced by therapeutically relevant doses of lithium treatment led to the identification of collapsin response mediator protein 2 (CRMP2) as being highly sensitive to both treatments as well as an extended panel of structurally distinct GSK3α/ß inhibitors. On this basis, a high-content image-based assay in hiPSC-derived neurons was developed to screen diverse compounds, including FDA-approved drugs, for their ability to mimic lithium's suppression of CRMP2 phosphorylation without directly inhibiting GSK3ß kinase activity. Systemic administration of a subset of these CRMP2-phosphorylation suppressors were found to mimic lithium's attenuation of amphetamine-induced hyperlocomotion in mice. Taken together, these studies not only provide insights into the neural substrates regulated by lithium, but also provide novel human neuronal assays for supporting the development of mechanism-based therapeutics for BD and related neuropsychiatric disorders.

Deep Learning Benchmarks on L1000 Gene Expression Data.

Gene expression data can offer deep, physiological insights beyond the static coding of the genome alone. We believe that realizing this potential requires specialized, high-capacity machine learning methods capable of using underlying biological structure, but the development of such models is hampered by the lack of published benchmark tasks and well characterized baselines. In this work, we establish such benchmarks and baselines by profiling many classifiers against biologically motivated tasks on two curated views of a large, public gene expression dataset (the LINCS corpus) and one privately produced dataset. We provide these two curated views of the public LINCS dataset and our benchmark tasks to enable direct comparisons to future methodological work and help spur deep learning method development on this modality. In addition to profiling a battery of traditional classifiers, including linear models, random forests, decision trees, K nearest neighbor (KNN) classifiers, and feed-forward artificial neural networks (FF-ANNs), we also test a method novel to this data modality: graph convolugtional neural networks (GCNNs), which allow us to incorporate prior biological domain knowledge. We find that GCNNs can be highly performant, with large datasets, whereas FF-ANNs consistently perform well. Non-neural classifiers are dominated by linear models and KNN classifiers.

Benzothiazole-Based LRRK2 Inhibitors as Wnt Enhancers and Promoters of Oligodendrocytic Fate.

Leucine rich repeat kinase 2 (LRRK2) is an enigmatic enzyme and a relevant target for Parkinson's disease (PD). However, despite the significant amount of research done in the past decade, the precise function of LRRK2 remains largely unknown. Moreover, the therapeutic potential of its inhibitors is in its infancy with the first clinical trial having just started. In the present work, the molecular mechanism of LRRK2 in the control of neurogenesis or gliogenesis was investigated. We designed and synthesized novel benzothiazole-based LRRK2 inhibitors and showed that they can modulate the Wnt/ß-catenin signaling pathway. Furthermore, compounds 5 and 14 were able to promote neural progenitors proliferation and drive their differentiation toward neuronal and oligodendrocytic cell fates. These results suggest potential new avenues for the application of LRRK2 inhibitors in demyelinating diseases in which oligodendrocyte cell-death is one of the pathological features.

An Autophagy Modifier Screen Identifies Small Molecules Capable of Reducing Autophagosome Accumulation in a Model of CLN3-Mediated Neurodegeneration.

Alterations in the autophagosomal-lysosomal pathway are a major pathophysiological feature of CLN3 disease, which is the most common form of childhood-onset neurodegeneration. Accumulating autofluorescent lysosomal storage material in CLN3 disease, consisting of dolichols, lipids, biometals, and a protein that normally resides in the mitochondria, subunit c of the mitochondrial ATPase, provides evidence that autophagosomal-lysosomal turnover of cellular components is disrupted upon loss of CLN3 protein function. Using a murine neuronal cell model of the disease, which accurately mimics the major gene defect and the hallmark features of CLN3 disease, we conducted an unbiased search for modifiers of autophagy, extending previous work by further optimizing a GFP-LC3 based assay and performing a high-content screen on a library of ~2000 bioactive compounds. Here we corroborate our earlier screening results and identify expanded, independent sets of autophagy modifiers that increase or decrease the accumulation of autophagosomes in the CLN3 disease cells, highlighting several pathways of interest, including the regulation of calcium signaling, microtubule dynamics, and the mevalonate pathway. Follow-up analysis on fluspirilene, nicardipine, and verapamil, in particular, confirmed activity in reducing GFP-LC3 vesicle burden, while also demonstrating activity in normalizing lysosomal positioning and, for verapamil, in promoting storage material clearance in CLN3 disease neuronal cells. This study demonstrates the potential for cell-based screening studies to identify candidate molecules and pathways for further work to understand CLN3 disease pathogenesis and in drug development efforts.

Structural Basis for Achieving GSK-3ß Inhibition with High Potency, Selectivity, and Brain Exposure for Positron Emission Tomography Imaging and Drug Discovery.

Using structure-guided design, several cell based assays, and microdosed positron emission tomography (PET) imaging, we identified a series of highly potent, selective, and brain-penetrant oxazole-4-carboxamide-based inhibitors of glycogen synthase kinase-3 (GSK-3). An isotopologue of our first-generation lead, [3H]PF-367, demonstrates selective and specific target engagement in vitro, irrespective of the activation state. We discovered substantial ubiquitous GSK-3-specific radioligand binding in Tg2576 Alzheimer's disease (AD), suggesting application for these compounds in AD diagnosis and identified [11C]OCM-44 as our lead GSK-3 radiotracer, with optimized brain uptake by PET imaging in nonhuman primates. GSK-3ß-isozyme selectivity was assessed to reveal OCM-51, the most potent (IC50 = 0.030 nM) and selective (>10-fold GSK-3ß/GSK-3α) GSK-3ß inhibitor known to date. Inhibition of CRMP2T514 and tau phosphorylation, as well as favorable therapeutic window against WNT/ß-catenin signaling activation, was observed in cells.

Activation of WNT and CREB signaling pathways in human neuronal cells in response to the Omega-3 fatty acid docosahexaenoic acid (DHA).

A subset of individuals with major depressive disorder (MDD) elects treatment with complementary and alternative medicines (CAMs), including the omega-3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Previous studies in rodents suggest that DHA modulates neurodevelopmental processes, including adult neurogenesis and neuroplasticity, but the molecular and cellular mechanisms of DHA's potential therapeutic effect in the context of human neurobiology have not been well established. Here we sought to address this knowledge gap by investigating the effects of DHA using human iPSC-derived neural progenitor cells (NPCs) and post-mitotic neurons using pathway-selective reporter genes, multiplexed mRNA expression profiling, and a panel of metabolism-based viability assays. Finally, real-time, live-cell imaging was employed to monitor neurite outgrowth upon DHA treatment. Overall, these studies showed that DHA treatment (0-50 µM) significantly upregulated both WNT and CREB signaling pathways in human neuronal cells in a dose-dependent manner with 2- to 3-fold increases in pathway activation. Additionally, we observed that DHA treatment enhanced survival of iPSC-derived NPCs and differentiation of post-mitotic neurons with live-cell imaging, revealing increased neurite outgrowth with DHA treatment within 24 h. Taken together, this study provides evidence that DHA treatment activates critical pathways regulating neuroplasticity, which may contribute to enhanced neuronal cell viability and neuronal connectivity. The extent to which these pathways represent molecular mechanisms underlying the potential beneficial effects of omega-3 fatty acids in MDD and other brain disorders merits further investigation.

High-throughput brain activity mapping and machine learning as a foundation for systems neuropharmacology.

Technologies for mapping the spatial and temporal patterns of neural activity have advanced our understanding of brain function in both health and disease. An important application of these technologies is the discovery of next-generation neurotherapeutics for neurological and psychiatric disorders. Here, we describe an in vivo drug screening strategy that combines high-throughput technology to generate large-scale brain activity maps (BAMs) with machine learning for predictive analysis. This platform enables evaluation of compounds' mechanisms of action and potential therapeutic uses based on information-rich BAMs derived from drug-treated zebrafish larvae. From a screen of clinically used drugs, we found intrinsically coherent drug clusters that are associated with known therapeutic categories. Using BAM-based clusters as a functional classifier, we identify anti-seizure-like drug leads from non-clinical compounds and validate their therapeutic effects in the pentylenetetrazole zebrafish seizure model. Collectively, this study provides a framework to advance the field of systems neuropharmacology.

Disruption of the psychiatric risk gene Ankyrin 3 enhances microtubule dynamics through GSK3/CRMP2 signaling.

The ankyrin 3 gene (ANK3) is a well-established risk gene for psychiatric illness, but the mechanisms underlying its pathophysiology remain elusive. We examined the molecular effects of disrupting brain-specific Ank3 isoforms in mouse and neuronal model systems. RNA sequencing of hippocampus from Ank3+/- and Ank3+/+ mice identified altered expression of 282 genes that were enriched for microtubule-related functions. Results were supported by increased expression of microtubule end-binding protein 3 (EB3), an indicator of microtubule dynamics, in Ank3+/- mouse hippocampus. Live-cell imaging of EB3 movement in primary neurons from Ank3+/- mice revealed impaired elongation of microtubules. Using a CRISPR-dCas9-KRAB transcriptional repressor in mouse neuro-2a cells, we determined that repression of brain-specific Ank3 increased EB3 expression, decreased tubulin acetylation, and increased the soluble:polymerized tubulin ratio, indicating enhanced microtubule dynamics. These changes were rescued by inhibition of glycogen synthase kinase 3 (GSK3) with lithium or CHIR99021, a highly selective GSK3 inhibitor. Brain-specific Ank3 repression in neuro-2a cells increased GSK3 activity (reduced inhibitory phosphorylation) and elevated collapsin response mediator protein 2 (CRMP2) phosphorylation, a known GSK3 substrate and microtubule-binding protein. Pharmacological inhibition of CRMP2 activity attenuated the rescue of EB3 expression and tubulin polymerization in Ank3-repressed cells by lithium or CHIR99021, suggesting microtubule instability induced by Ank3 repression is dependent on CRMP2 activity. Taken together, our data indicate that ANK3 functions in neuronal microtubule dynamics through GSK3 and its downstream substrate CRMP2. These findings reveal cellular and molecular mechanisms underlying brain-specific ANK3 disruption that may be related to its role in psychiatric illness.

Traditional and systems biology based drug discovery for the rare tumor syndrome neurofibromatosis type 2.

Neurofibromatosis 2 (NF2) is a rare tumor suppressor syndrome that manifests with multiple schwannomas and meningiomas. There are no effective drug therapies for these benign tumors and conventional therapies have limited efficacy. Various model systems have been created and several drug targets have been implicated in NF2-driven tumorigenesis based on known effects of the absence of merlin, the product of the NF2 gene. We tested priority compounds based on known biology with traditional dose-concentration studies in meningioma and schwann cell systems. Concurrently, we studied functional kinome and gene expression in these cells pre- and post-treatment to determine merlin deficient molecular phenotypes. Cell viability results showed that three agents (GSK2126458, Panobinostat, CUDC-907) had the greatest activity across schwannoma and meningioma cell systems, but merlin status did not significantly influence response. In vivo, drug effect was tumor specific with meningioma, but not schwannoma, showing response to GSK2126458 and Panobinostat. In culture, changes in both the transcriptome and kinome in response to treatment clustered predominantly based on tumor type. However, there were differences in both gene expression and functional kinome at baseline between meningioma and schwannoma cell systems that may form the basis for future selective therapies. This work has created an openly accessible resource (www.synapse.org/SynodosNF2) of fully characterized isogenic schwannoma and meningioma cell systems as well as a rich data source of kinome and transcriptome data from these assay systems before and after treatment that enables single and combination drug discovery based on molecular phenotype.

Class I Histone Deacetylase Inhibition by Tianeptinaline Modulates Neuroplasticity and Enhances Memory.

Through epigenetic and other regulatory functions, the histone deacetylase (HDAC) family of enzymes has emerged as a promising therapeutic target for central nervous system and other disorders. Here we report on the synthesis and functional characterization of new HDAC inhibitors based structurally on tianeptine, a drug used primarily to treat major depressive disorder (MDD) that has a poorly understood mechanism of action. Since the chemical structure of tianeptine resembles certain HDAC inhibitors, we profiled the in vitro HDAC inhibitory activity of tianeptine and demonstrated its ability to inhibit the lysine deacetylase activity of a subset of class I HDACs. Consistent with a model of active site Zn2+ chelation by the carboxylic acid present in tianeptine, newly synthesized analogues containing either a hydroxamic acid or ortho-aminoanilide exhibited increased potency and selectivity among the HDAC family. This in vitro potency translated to improved efficacy in a panel of high-content imaging assays designed to assess HDAC target engagement and functional effects on critical pathways involved in neuroplasticity in both primary mouse neurons and, for the first time, human neurons differentiated from pluripotent stem cells. Most notably, tianeptinaline, a class I HDAC-selective analogue of tianeptine, but not tianeptine itself, increased histone acetylation, and enhanced CREB-mediated transcription and the expression of Arc (activity-regulated cytoskeleton-associated protein). Systemic in vivo administration of tianeptinaline to mice confirmed its brain penetration and was found to enhance contextual fear conditioning, a behavioral test of hippocampal-dependent memory. Tianeptinaline and its derivatives provide new pharmacological tools to dissect chromatin-mediated neuroplasticity underlying memory and other epigenetically related processes implicated in health and disease.

A Next Generation Connectivity Map: L1000 Platform and the First 1,000,000 Profiles.

We previously piloted the concept of a Connectivity Map (CMap), whereby genes, drugs, and disease states are connected by virtue of common gene-expression signatures. Here, we report more than a 1,000-fold scale-up of the CMap as part of the NIH LINCS Consortium, made possible by a new, low-cost, high-throughput reduced representation expression profiling method that we term L1000. We show that L1000 is highly reproducible, comparable to RNA sequencing, and suitable for computational inference of the expression levels of 81% of non-measured transcripts. We further show that the expanded CMap can be used to discover mechanism of action of small molecules, functionally annotate genetic variants of disease genes, and inform clinical trials. The 1.3 million L1000 profiles described here, as well as tools for their analysis, are available at https://clue.io.

Chemogenomic analysis reveals key role for lysine acetylation in regulating Arc stability.

The role of Arc in synaptic plasticity and memory consolidation has been investigated for many years with recent evidence that defects in the expression or activity of this immediate-early gene may also contribute to the pathophysiology of brain disorders including schizophrenia and fragile X syndrome. These results bring forward the concept that reversing Arc abnormalities could provide an avenue to improve cognitive or neurological impairments in different disease contexts, but how to achieve this therapeutic objective has remained elusive. Here, we present results from a chemogenomic screen that probed a mechanistically diverse library of small molecules for modulators of BDNF-induced Arc expression in primary cortical neurons. This effort identified compounds with a range of influences on Arc, including promoting its acetylation-a previously uncharacterized post-translational modification of this protein. Together, our data provide insights into the control of Arc that could be targeted to harness neuroplasticity for clinical applications.

HDAC6 Brain Mapping with [18F]Bavarostat Enabled by a Ru-Mediated Deoxyfluorination.

Histone deacetylase 6 (HDAC6) function and dysregulation have been implicated in the etiology of certain cancers and more recently in central nervous system (CNS) disorders including Rett syndrome, Alzheimer's and Parkinson's diseases, and major depressive disorder. HDAC6-selective inhibitors have therapeutic potential, but in the CNS drug space the development of highly brain penetrant HDAC inhibitors has been a persistent challenge. Moreover, no tool exists to directly characterize HDAC6 and its related biology in the living human brain. Here, we report a highly brain penetrant HDAC6 inhibitor, Bavarostat, that exhibits excellent HDAC6 selectivity (>80-fold over all other Zn-containing HDAC paralogues), modulates tubulin acetylation selectively over histone acetylation, and has excellent brain penetrance. We further demonstrate that Bavarostat can be radiolabeled with 18F by deoxyfluorination through in situ formation of a ruthenium π-complex of the corresponding phenol precursor: the only method currently suitable for synthesis of [18F]Bavarostat. Finally, by using [18F]Bavarostat in a series of rodent and nonhuman primate imaging experiments, we demonstrate its utility for mapping HDAC6 in the living brain, which sets the stage for first-in-human neurochemical imaging of this important target.

WNT/ß-Catenin Pathway and Epigenetic Mechanisms Regulate the Pitt-Hopkins Syndrome and Schizophrenia Risk Gene TCF4.

Genetic variation within the transcription factor TCF4 locus can cause the intellectual disability and developmental disorder Pitt-Hopkins syndrome (PTHS), whereas single-nucleotide polymorphisms within noncoding regions are associated with schizophrenia. These genetic findings position TCF4 as a link between transcription and cognition; however, the neurobiology of TCF4 remains poorly understood. Here, we quantitated multiple distinct TCF4 transcript levels in human induced pluripotent stem cell-derived neural progenitors and differentiated neurons, and PTHS patient fibroblasts. We identify two classes of pharmacological treatments that regulate TCF4 expression: WNT pathway activation and inhibition of class I histone deacetylases. In PTHS fibroblasts, both of these perturbations upregulate a subset of TCF4 transcripts. Finally, using chromatin immunoprecipitation sequencing in conjunction with genome-wide transcriptome analysis, we identified TCF4 target genes that may mediate the effect of TCF4 loss on neuroplasticity. Our studies identify new pharmacological assays, tools, and targets for the development of therapeutics for cognitive disorders.

Selectivity and Kinetic Requirements of HDAC Inhibitors as Progranulin Enhancers for Treating Frontotemporal Dementia.

Frontotemporal dementia (FTD) arises from neurodegeneration in the frontal, insular, and anterior temporal lobes. Autosomal dominant causes of FTD include heterozygous mutations in the GRN gene causing haploinsufficiency of progranulin (PGRN) protein. Recently, histone deacetylase (HDAC) inhibitors have been identified as enhancers of PGRN expression, although the mechanisms through which GRN is epigenetically regulated remain poorly understood. Using a chemogenomic toolkit, including optoepigenetic probes, we show that inhibition of class I HDACs is sufficient to upregulate PGRN in human neurons, and only inhibitors with apparent fast binding to their target HDAC complexes are capable of enhancing PGRN expression. Moreover, we identify regions in the GRN promoter in which elevated H3K27 acetylation and transcription factor EB (TFEB) occupancy correlate with HDAC-inhibitor-mediated upregulation of PGRN. These findings have implications for epigenetic and cis-regulatory mechanisms controlling human GRN expression and may advance translational efforts to develop targeted therapeutics for treating PGRN-deficient FTD.

Probing the lithium-response pathway in hiPSCs implicates the phosphoregulatory set-point for a cytoskeletal modulator in bipolar pathogenesis.

The molecular pathogenesis of bipolar disorder (BPD) is poorly understood. Using human-induced pluripotent stem cells (hiPSCs) to unravel such mechanisms in polygenic diseases is generally challenging. However, hiPSCs from BPD patients responsive to lithium offered unique opportunities to discern lithium's target and hence gain molecular insight into BPD. By profiling the proteomics of BDP-hiPSC-derived neurons, we found that lithium alters the phosphorylation state of collapsin response mediator protein-2 (CRMP2). Active nonphosphorylated CRMP2, which binds cytoskeleton, is present throughout the neuron; inactive phosphorylated CRMP2, which dissociates from cytoskeleton, exits dendritic spines. CRMP2 elimination yields aberrant dendritogenesis with diminished spine density and lost lithium responsiveness (LiR). The "set-point" for the ratio of pCRMP2:CRMP2 is elevated uniquely in hiPSC-derived neurons from LiR BPD patients, but not with other psychiatric (including lithium-nonresponsive BPD) and neurological disorders. Lithium (and other pathway modulators) lowers pCRMP2, increasing spine area and density. Human BPD brains show similarly elevated ratios and diminished spine densities; lithium therapy normalizes the ratios and spines. Consistent with such "spine-opathies," human LiR BPD neurons with abnormal ratios evince abnormally steep slopes for calcium flux; lithium normalizes both. Behaviorally, transgenic mice that reproduce lithium's postulated site-of-action in dephosphorylating CRMP2 emulate LiR in BPD. These data suggest that the "lithium response pathway" in BPD governs CRMP2's phosphorylation, which regulates cytoskeletal organization, particularly in spines, modulating neural networks. Aberrations in the posttranslational regulation of this developmentally critical molecule may underlie LiR BPD pathogenesis. Instructively, examining the proteomic profile in hiPSCs of a functional agent-even one whose mechanism-of-action is unknown-might reveal otherwise inscrutable intracellular pathogenic pathways.

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.

Dissecting structure-activity-relationships of crebinostat: Brain penetrant HDAC inhibitors for neuroepigenetic regulation.

Targeting chromatin-mediated epigenetic regulation has emerged as a potential avenue for developing novel therapeutics for a wide range of central nervous system disorders, including cognitive disorders and depression. Histone deacetylase (HDAC) inhibitors have been pursued as cognitive enhancers that impact the regulation of gene expression and other mechanisms integral to neuroplasticity. Through systematic modification of the structure of crebinostat, a previously discovered cognitive enhancer that affects genes critical to memory and enhances synaptogenesis, combined with biochemical and neuronal cell-based screening, we identified a novel hydroxamate-based HDAC inhibitor, here named neurinostat, with increased potency compared to crebinostat in inducing neuronal histone acetylation. In addition, neurinostat was found to have a pharmacokinetic profile in mouse brain modestly improved over that of crebinostat. This discovery of neurinostat and demonstration of its effects on neuronal HDACs adds to the available pharmacological toolkit for dissecting the molecular and cellular mechanisms of neuroepigenetic regulation in health and disease.