Design, Synthesis, and Evaluation of Hydroxamic Acid-Based Molecular Probes for In Vivo Imaging of Histone Deacetylase (HDAC) in Brain

Hydroxamic acid-based histone deacetylase inhibitors (HDACis) are a class of molecules with therapeutic potential currently reflected in the use of suberoylanilide hydroxamic acid (SAHA; Vorinostat) to treat cutaneous T-cell lymphomas (CTCL). HDACis may have utility beyond cancer therapy, as preclinical studies have ascribed HDAC inhibition as beneficial in areas such as heart disease, diabetes, depression, neurodegeneration, and other disorders of the central nervous system (CNS). However, little is known about the pharmacokinetics (PK) of hydroxamates, particularly with respect to CNS-penetration, distribution, and retention. To explore the rodent and non-human primate (NHP) brain permeability of hydroxamic acid-based HDAC inhibitors using positron emission tomography (PET), we modified the structures of belinostat (PXD101) and panobinostat (LBH-589) to incorporate carbon-11. We also labeled PCI 34051 through carbon isotope substitution. After characterizing the in vitro affinity and efficacy of these compounds across nine recombinant HDAC isoforms spanning Class I and Class II family members, we determined the brain uptake of each inhibitor. Each labeled compound has low uptake in brain tissue when administered intravenously to rodents and NHPs. In rodent studies, we observed that brain accumulation of the radiotracers were unaffected by the pre-administration of unlabeled inhibitors. Knowing that CNS-penetration may be desirable for both imaging applications and therapy, we explored whether a liquid chromatography, tandem mass spectrometry (LC-MS-MS) method to predict brain penetrance would be an appropriate method to pre-screen compounds (hydroxamic acid-based HDACi) prior to PET radiolabeling. LC-MS-MS data were indeed useful in identifying additional lead molecules to explore as PET imaging agents to visualize HDAC enzymes in vivo. However, HDACi brain penetrance predicted by LC-MS-MS did not strongly correlate with PET imaging results. This underscores the importance of in vivo PET imaging tools in characterizing putative CNS drug lead compounds and the continued need to discover effect PET tracers for neuroepigenetic imaging.Chemistry and Chemical Biolog

Selective HDAC Inhibition for the Disruption of Latent HIV-1 Infection

Selective histone deacetylase (HDAC) inhibitors have emerged as a potential anti-latency therapy for persistent human immunodeficiency virus type 1 (HIV-1) infection. We utilized a combination of small molecule inhibitors and short hairpin (sh)RNA-mediated gene knockdown strategies to delineate the key HDAC(s) to be targeted for selective induction of latent HIV-1 expression. Individual depletion of HDAC3 significantly induced expression from the HIV-1 promoter in the 2D10 latency cell line model. However, depletion of HDAC1 or −2 alone or in combination did not significantly induce HIV-1 expression. Co-depletion of HDAC2 and −3 resulted in a significant increase in expression from the HIV-1 promoter. Furthermore, concurrent knockdown of HDAC1, −2, and −3 resulted in a significant increase in expression from the HIV-1 promoter. Using small molecule HDAC inhibitors of differing selectivity to ablate the residual HDAC activity that remained after (sh)RNA depletion, the effect of depletion of HDAC3 was further enhanced. Enzymatic inhibition of HDAC3 with the selective small-molecule inhibitor BRD3308 activated HIV-1 transcription in the 2D10 cell line. Furthermore, ex vivo exposure to BRD3308 induced outgrowth of HIV-1 from resting CD4+ T cells isolated from antiretroviral-treated, aviremic HIV+ patients. Taken together these findings suggest that HDAC3 is an essential target to disrupt HIV-1 latency, and inhibition of HDAC2 may also contribute to the effort to purge and eradicate latent HIV-1 infection

Original Article Design, synthesis, and evaluation of hydroxamic acid-based molecular probes for in vivo imaging of histone deacetylase (HDAC) in brain

Abstract: Hydroxamic acid-based histone deacetylase inhibitors (HDACis) are a class of molecules with therapeutic potential currently reflected in the use of suberoylanilide hydroxamic acid (SAHA; Vorinostat) to treat cutaneous T-cell lymphomas (CTCL). HDACis may have utility beyond cancer therapy, as preclinical studies have ascribed HDAC inhibition as beneficial in areas such as heart disease, diabetes, depression, neurodegeneration, and other disorders of the central nervous system (CNS). However, little is known about the pharmacokinetics (PK) of hydroxamates, particularly with respect to CNS-penetration, distribution, and retention. To explore the rodent and non-human primate (NHP) brain permeability of hydroxamic acid-based HDAC inhibitors using positron emission tomography (PET), we modified the structures of belinostat (PXD101) and panobinostat (LBH-589) to incorporate carbon-11. We also labeled PCI 34051 through carbon isotope substitution. After characterizing the in vitro affinity and efficacy of these compounds across nine recombinant HDAC isoforms spanning Class I and Class II family members, we determined the brain uptake of each inhibitor. Each labeled compound has low uptake in brain tissue when administered intravenously to rodents and NHPs. In rodent studies, we observed that brain accumulation of the radiotracers were unaffected by the pre-administration of unlabeled inhibitors. Knowing that CNS-penetration may be desirable for both imaging applications and therapy, we explored whether a liquid chromatography, tandem mass spectrometry (LC-MS-MS) method to predict brain penetrance would be an appropriate method to pre-screen compounds (hydroxamic acid-based HDACi) prior to PET radiolabeling. LC-MS-MS data were indeed useful in identifying additional lead molecules to explore as PET imaging agents to visualize HDAC enzymes in vivo. However, HDACi brain penetrance predicted by LC-MS-MS did not strongly correlate with PET imaging results. This underscores the importance of in vivo PET imaging tools in characterizing putative CNS drug lead compounds and the continued need to discover effect PET tracers for neuroepigenetic imaging

A Selective HDAC 1/2 Inhibitor Modulates Chromatin and Gene Expression in Brain and Alters Mouse Behavior in Two Mood-Related Tests

Psychiatric diseases, including schizophrenia, bipolar disorder and major depression, are projected to lead global disease burden within the next decade. Pharmacotherapy, the primary – albeit often ineffective – treatment method, has remained largely unchanged over the past 50 years, highlighting the need for novel target discovery and improved mechanism-based treatments. Here, we examined in wild type mice the impact of chronic, systemic treatment with Compound 60 (Cpd-60), a slow-binding, benzamide-based inhibitor of the class I histone deacetylase (HDAC) family members, HDAC1 and HDAC2, in mood-related behavioral assays responsive to clinically effective drugs. Cpd-60 treatment for one week was associated with attenuated locomotor activity following acute amphetamine challenge. Further, treated mice demonstrated decreased immobility in the forced swim test. These changes are consistent with established effects of clinical mood stabilizers and antidepressants, respectively. Whole-genome expression profiling of specific brain regions (prefrontal cortex, nucleus accumbens, hippocampus) from mice treated with Cpd-60 identified gene expression changes, including a small subset of transcripts that significantly overlapped those previously reported in lithium-treated mice. HDAC inhibition in brain was confirmed by increased histone acetylation both globally and, using chromatin immunoprecipitation, at the promoter regions of upregulated transcripts, a finding consistent with in vivo engagement of HDAC targets. In contrast, treatment with suberoylanilide hydroxamic acid (SAHA), a non-selective fast-binding, hydroxamic acid HDAC 1/2/3/6 inhibitor, was sufficient to increase histone acetylation in brain, but did not alter mood-related behaviors and had dissimilar transcriptional regulatory effects compared to Cpd-60. These results provide evidence that selective inhibition of HDAC1 and HDAC2 in brain may provide an epigenetic-based target for developing improved treatments for mood disorders and other brain disorders with altered chromatin-mediated neuroplasticity.Stanley Medical Research InstituteNational Institutes of Health (U.S.) (R01DA028301)National Institutes of Health (U.S.) (R01DA030321

Identification of Antifungal Compounds Active against Candida albicans Using an Improved High-Throughput Caenorhabditis elegans Assay

Candida albicans, the most common human pathogenic fungus, can establish a persistent lethal infection in the intestine of the microscopic nematode Caenorhabditis elegans. The C. elegans–C. albicans infection model was previously adapted to screen for antifungal compounds. Modifications to this screen have been made to facilitate a high-throughput assay including co-inoculation of nematodes with C. albicans and instrumentation allowing precise dispensing of worms into assay wells, eliminating two labor-intensive steps. This high-throughput method was utilized to screen a library of 3,228 compounds represented by 1,948 bioactive compounds and 1,280 small molecules derived via diversity-oriented synthesis. Nineteen compounds were identified that conferred an increase in C. elegans survival, including most known antifungal compounds within the chemical library. In addition to seven clinically used antifungal compounds, twelve compounds were identified which are not primarily used as antifungal agents, including three immunosuppressive drugs. This assay also allowed the assessment of the relative minimal inhibitory concentration, the effective concentration in vivo, and the toxicity of the compound in a single assay

Studies directed towards the total synthesis of spongistatin 1 and the cyclitols.

My research has focused on the total synthesis of spongistatin 1, a highly cytotoxic antineoplastic agent isolated from a variety of marine sponges, as well as the total synthesis of several cyclitols. Our synthetic approach within each project relied on a divergent strategy taking advantage of the structural homology between distinct fragments of the natural product in the case of spongistatin 1 and between different members of the conduritol and inositol family in the case of the cyclitols. Within the spongistatin project, we have synthesized both the A--B and C--D spiroketals in spongistatin 1 using a kinetically controlled intramolecular iodospiroketalization reaction of a suitably functionalized glycal precursor. We achieved good stereochemical control in the synthesis of both of the spiroketal centers using this methodology. Utilizing a common late stage intermediate we were able to complete the synthesis of both spiroketals (constituting a 24 carbon segment of the natural product) in 32 total synthetic steps. Our approach to the cyclitols relied on selective Peterson elimination reactions and Fleming-Tamao oxidation of beta-hydroxy cyclohexylsilanes. The beta-hydroxy cyclohexylsilanes were accessed via the dihydroxylation of cyclohexenylsilanes which were derived via the ring closing metathesis of an acyclic dienylsilane which was prepared via the silylallylboration of a tartrate derived aldehyde with [(E)-gamma-(dimethylphenylsilyl)allyl]diisopinocampheylborane. These studies culminated in the total synthesis of conduritol B, C, D and F and D-(+)-chiro and neo-inositol.Ph.D.Organic chemistryPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/123606/2/3096111.pd

Hydroxamate-Based Histone Deacetylase Inhibitors Can Protect Neurons from Oxidative Stress via a Histone Deacetylase-Independent Catalase-Like Mechanism

Histone deacetylase (HDAC) inhibitors have shown enormous promise for treating various disease states, presumably due to their ability to modulate acetylation of histone and non-histone proteins. Many of these inhibitors contain functional groups capable of strongly chelating metal ions. We demonstrate that several members of one such class of compounds, the hydroxamate-based HDAC inhibitors, can protect neurons from oxidative stress via an HDAC-independent mechanism. This previously unappreciated antioxidant mechanism involves the in situ formation of hydroxamate-iron complexes that catalyze the decomposition of hydrogen peroxide in a manner reminiscent of catalase. We demonstrate that while many hydroxamate-containing HDAC inhibitors display a propensity for binding iron, only a subset form active catalase mimetics capable of protecting neurons from exogenous H2O2. In addition to their impact on stroke and neurodegenerative disease research, these results highlight the possibility that HDAC-independent factors might play a role in the therapeutic effects of hydroxamate-based HDAC inhibitors

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

Selective inhibition of histone deacetylase 3 (HDAC3) prevents glucolipotoxicity-induced β-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 β-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. β-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