46 research outputs found
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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
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
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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
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
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FDG-PET Imaging Reveals Local Brain Glucose Utilization Is Altered by Class I Histone Deacetylase Inhibitors
The purpose of this work – the first of its kind – was to evaluate the impact of chronic selective histone deacetylase (HDAC) inhibitor treatment on brain activity using uptake of the radioligand 18F-fluorodeoxyglucose and positron emission tomography (18FDG-PET). HDAC dysfunction and other epigenetic mechanisms are implicated in diverse CNS disorders and animal research suggests HDAC inhibition may provide a lead toward developing improved treatment. To begin to better understand the role of the class I HDAC subtypes HDAC 1, 2 and 3 in modulating brain activity, we utilized two benzamide inhibitors from the literature, compound 60 (Cpd-60) and CI-994 which selectively inhibit HDAC 1 and 2 or HDACs 1, 2 and 3, respectively. One day after the seventh treatment with Cpd-60 (22.5 mg/kg) or CI-994 (5 mg/kg), 18FDG-PET experiments (n = 11–12 rats per treatment group) revealed significant, local changes in brain glucose utilization. These 2–17% changes were represented by increases and decreases in glucose uptake. The pattern of changes was similar but distinct between Cpd-60 and CI-994, supporting that 18FDG-PET is a useful tool to examine the relationship between HDAC subtype activity and brain activity. Further work using additional selective HDAC inhibitors will be needed to clarify these effects as well as to understand how brain activity changes influence behavioral response.Chemistry and Chemical Biolog