Yan-Ling Zhang

An aldol-based build/couple/pair strategy for the synthesis of medium- and large-sized rings: discovery of macrocyclic histone deacetylase inhibitors.

An aldol-based build/couple/pair (B/C/P) strategy was applied to generate a collection of stereochemically and skeletally diverse small molecules. In the build phase, a series of asymmetric syn- and anti-aldol reactions were performed to produce four stereoisomers of a Boc-protected γ-amino acid. In addition, both stereoisomers of O-PMB-protected alaninol were generated to provide a chiral amine coupling partner. In the couple step, eight stereoisomeric amides were synthesized by coupling the chiral acid and amine building blocks. The amides were subsequently reduced to generate the corresponding secondary amines. In the pair phase, three different reactions were employed to enable intramolecular ring-forming processes: nucleophilic aromatic substitution (S(N)Ar), Huisgen [3+2] cycloaddition, and ring-closing metathesis (RCM). Despite some stereochemical dependencies, the ring-forming reactions were optimized to proceed with good to excellent yields, providing a variety of skeletons ranging in size from 8- to 14-membered rings. Scaffolds resulting from the RCM pairing reaction were diversified on the solid phase to yield a 14 400-membered library of macrolactams. Screening of this library led to the discovery of a novel class of histone deacetylase inhibitors, which display mixed enzyme inhibition, and led to increased levels of acetylation in a primary mouse neuron culture. The development of stereo-structure/activity relationships was made possible by screening all 16 stereoisomers of the macrolactams produced through the aldol-based B/C/P strategy.

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.

Potent and selective inhibition of histone deacetylase 6 (HDAC6) does not require a surface-binding motif.

Hydroxamic acids were designed, synthesized, and evaluated for their ability to selectively inhibit human histone deacetylase 6 (HDAC6). Several inhibitors, including compound 14 (BRD9757), exhibited excellent potency and selectivity despite the absence of a surface-binding motif. The binding of these highly efficient ligands for HDAC6 is rationalized via structure-activity relationships. These results demonstrate that high selectivity and potent inhibition of HDAC6 can be achieved through careful choice of linker element only.

Discovery of the First Histone Deacetylase 6/8 Dual Inhibitors

We disclose the first small molecule histone deacetylase (HDAC) inhibitor (3, BRD73954) capable of potently and selectively inhibiting both HDAC6 and HDAC8 despite the fact that these isoforms belong to distinct phylogenetic classes within the HDAC family of enzymes. Our data demonstrate that meta substituents of phenyl hydroxamic acids are readily accommodated upon binding to HDAC6 and, furthermore, are necessary for the potent inhibition of HDAC8.

Class I HDAC imaging using [3H]CI-994 autoradiography

[3H]CI-994, a radioactive isotopologue of the benzamide CI-994, a class I histone deacetylase inhibitor (HDACi), was evaluated as an autoradiography probe for ex vivo labeling and localizing of class I HDAC (isoforms 1–3) in the rodent brain. After protocol optimization, up to 80% of total binding was attributed to specific binding. Notably, like other benzamide HDACi, [3H]CI-994 exhibits slow binding kinetics when measured in vitro with isolated enzymes and ex vivo when used for autoradiographic mapping of HDAC1–3 density. The regional distribution and density of HDAC1–3 was determined through a series of saturation and kinetics experiments. The binding properties of [3H]CI-994 to HDAC1–3 were characterized and the data were used to determine the regional Bmax of the target proteins. Kd values, determined from slice autoradiography, were between 9.17 and 15.6 nM. The HDAC1–3 density (Bmax), averaged over whole brain sections, was of 12.9 picomol · mg−1 protein. The highest HDAC1–3 density was found in the cerebellum, followed by hippocampus and cortex. Moderate to low receptor density was found in striatum, hypothalamus and thalamus. These data were correlated with semi-quantitative measures of each HDAC isoform using western blot analysis and it was determined that autoradiographic images most likely represent the sum of HDAC1, HDAC2, and HDAC3 protein density. In competition experiments, [3H]CI-994 binding can be dose-dependently blocked with other HDAC inhibitors, including suberoylanilide hydroxamic acid (SAHA). In summary, we have developed the first known autoradiography tool for imaging class I HDAC enzymes. Although validated in the CNS, [3H]CI-994 will be applicable and beneficial to other target tissues and can be used to evaluate HDAC inhibition in tissues for novel therapies being developed. [3H]CI-994 is now an enabling imaging tool to study the relationship between diseases and epigenetic regulation.

An Isochemogenic Set of Inhibitors To Define the Therapeutic Potential of Histone Deacetylases in β-Cell Protection

Modulation of histone deacetylase (HDAC) activity has been implicated as a potential therapeutic strategy for multiple diseases. However, it has been difficult to dissect the role of individual HDACs due to a lack of selective small-molecule inhibitors. Here, we report the synthesis of a series of highly potent and isoform-selective class I HDAC inhibitors, rationally designed by exploiting minimal structural changes to the clinically experienced HDAC inhibitor CI-994. We used this toolkit of isochemogenic or chemically matched inhibitors to probe the role of class I HDACs in β-cell pathobiology and demonstrate for the first time that selective inhibition of an individual HDAC isoform retains beneficial biological activity and mitigates mechanism-based toxicities. The highly selective HDAC3 inhibitor BRD3308 suppressed pancreatic β-cell apoptosis induced by inflammatory cytokines, as expected, or now glucolipotoxic stress, and increased functional insulin release. In addition, BRD3308 had no effect on human megakaryocyte differentiation, while inhibitors of HDAC1 and 2 were toxic. Our findings demonstrate that the selective inhibition of HDAC3 represents a potential path forward as a therapy to protect pancreatic β-cells from inflammatory cytokines and nutrient overload in diabetes.

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 (18)F-fluorodeoxyglucose and positron emission tomography ((18)FDG-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), (18)FDG-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 (18)FDG-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.

Inhibitors of Glycogen Synthase Kinase 3 with Exquisite Kinome-Wide Selectivity and Their Functional Effects.

The mood stabilizer lithium, the first-line treatment for bipolar disorder, is hypothesized to exert its effects through direct inhibition of glycogen synthase kinase 3 (GSK3) and indirectly by increasing GSK3s inhibitory serine phosphorylation. GSK3 comprises two highly similar paralogs, GSK3α and GSK3β, which are key regulatory kinases in the canonical Wnt pathway. GSK3 stands as a nodal target within this pathway and is an attractive therapeutic target for multiple indications. Despite being an active field of research for the past 20 years, many GSK3 inhibitors demonstrate either poor to moderate selectivity versus the broader human kinome or physicochemical properties unsuitable for use in in vitro systems or in vivo models. A nonconventional analysis of data from a GSK3β inhibitor high-throughput screening campaign, which excluded known GSK3 inhibitor chemotypes, led to the discovery of a novel pyrazolo-tetrahydroquinolinone scaffold with unparalleled kinome-wide selectivity for the GSK3 kinases. Taking advantage of an uncommon tridentate interaction with the hinge region of GSK3, we developed highly selective and potent GSK3 inhibitors, BRD1652 and BRD0209, which demonstrated in vivo efficacy in a dopaminergic signaling paradigm modeling mood-related disorders. These new chemical probes open the way for exclusive analyses of the function of GSK3 kinases in multiple signaling pathways involved in many prevalent disorders.

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-34051s 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.

PET Imaging Demonstrates Histone Deacetylase Target Engagement and Clarifies Brain Penetrance of Known and Novel Small Molecule Inhibitors in Rat

Histone deacetylase (HDAC) enzymes have been demonstrated as critical components in maintaining chromatin homeostasis, CNS development, and normal brain function. Evidence in mouse models links HDAC expression to learning, memory, and mood-related behaviors; small molecule HDAC inhibitor tool compounds have been used to demonstrate the importance of specific HDAC subtypes in modulating CNS-disease-related behaviors in rodents. So far, no direct evidence exists to understand the quantitative changes in HDAC target engagement that are necessary to alter biochemistry and behavior in a living animal. Understanding the relationship between target engagement and in vivo effect is essential in refining new ways to alleviate disease. We describe here, using positron emission tomography (PET) imaging of rat brain, the in vivo target engagement of a subset of class I/IIb HDAC enzymes implicated in CNS-disease (HDAC subtypes 1, 2, 3, and 6). We found marked differences in the brain penetrance of tool compounds from the hydroxamate and benzamide HDAC inhibitor classes and resolved a novel, highly brain penetrant benzamide, CN147, chronic treatment with which resulted in an antidepressant-like effect in a rat behavioral test. Our work highlights a new translational path for understanding the molecular and behavioral consequences of HDAC target engagement.