David J. Witter

Histone deacetylase inhibitors.

Histone deacetylase enzymes, which have been divided into three distinct structural classes, operate by zinc-dependent (class I/II) or NAD-dependent (class III) mechanisms. Class I/II histone deacetylase (HDAC) enzymes are an emerging therapeutic target for the treatment of cancer and other diseases.1-5 These enzymes, as part of multiprotein complexes, catalyze the removal of acetyl groups from lysine residues on proteins, including histones. HDAC inhibitors have been shown to bind directly to the HDAC active site and thereby block substrate access, causing a resultant accumulation of acetylated histones.1,3,6 These agents possess diverse biological activities and can affect differentiation, growth arrest, and/or apoptosis in transformed cell cultures. In vivo xenograft studies have further demonstrated many of these agents to be effective in the inhibition of tumor growth. A wide range of structures have been shown to inhibit the activity of class I/II HDAC enzymes, and with few exceptions, these can be divided into structural classes including small-molecule hydroxamates, carboxylates, benzamides, electrophilic ketones, and cyclic peptides. Despite the variety of structural characteristics, all of these HDAC inhibitors can be broadly characterized by a common pharmacophore that includes key elements of inhibitor-enzyme interactions. In addition to the availability of crystal structures, homology models have further aided in the identification and rational design of new HDAC inhibitors for use as chemical tools and potential therapeutics. The high level of interest in developing efficacious HDAC inhibitors and the availability of design tools have led to an expansive group of agents that target class I/II HDACs. This review encompasses the medicinal chemistry and structureactivity relationships (SAR) underlying advances in HDAC class I/II inhibitor discovery, design, and optimization.5,7,8

Exploration of the internal cavity of histone deacetylase (HDAC) with selective HDAC1/HDAC2 inhibitors (SHI-1:2)

We report herein the initial exploration of novel selective HDAC1/HDAC2 inhibitors (SHI-1:2). Optimized SHI-1:2 structures exhibit enhanced intrinsic activity against HDAC1 and HDAC2, and are greater than 100-fold selective versus other HDACs, including HDAC3. Based on the SAR of these agents and our current understanding of the HDAC active site, we postulate that the SHI-1:2 extend the existing HDAC inhibitor pharmacophore to include an internal binding domain.

Structure-dependent Impairment of Intracellular Apolipoprotein E4 Trafficking and Its Detrimental Effects Are Rescued by Small-molecule Structure Correctors

Apolipoprotein (apo) E4 is the major genetic risk factor for Alzheimer disease (AD) and likely contributes to neuropathology through various pathways. Here we report that the intracellular trafficking of apoE4 is impaired in Neuro-2a cells and primary neurons, as shown by measuring fluorescence recovery after photobleaching. In Neuro-2a cells, more apoE4 than apoE3 molecules remained immobilized in the endoplasmic reticulum (ER) and the Golgi apparatus, and the lateral motility of apoE4 was significantly lower in the Golgi apparatus (but not in the ER) than that of apoE3. Likewise, the immobile fraction was larger, and the lateral motility was lower for apoE4 than apoE3 in mouse primary hippocampal neurons. ApoE4 with the R61T mutation, which abolishes apoE4 domain interaction, was less immobilized, and its lateral motility was comparable with that of apoE3. The trafficking impairment of apoE4 was also rescued by disrupting domain interaction with the small-molecule structure correctors GIND25 and PH002. PH002 also rescued apoE4-induced impairments of neurite outgrowth in Neuro-2a cells and dendritic spine development in primary neurons. ApoE4 did not affect trafficking of amyloid precursor protein, another AD-related protein, through the secretory pathway. Thus, domain interaction renders more newly synthesized apoE4 molecules immobile and slows their trafficking along the secretory pathway. Correcting the pathological structure of apoE4 by disrupting domain interaction is a potential therapeutic approach to treat or prevent AD related to apoE4.

Design and synthesis of SH3 domain binding ligands: Modifications of the consensus sequence XPpXP

Spirolactam-based Pro-Pro mimetics incorporated in the consensus sequence XPpXP, lead to effective nonpeptide ligands of SH3 domains.

Phenylglycine and phenylalanine derivatives as potent and selective HDAC1 inhibitors (SHI-1)

An HTS screening campaign identified a series of low molecular weight phenols that showed excellent selectivity (>100-fold) for HDAC1/HDAC2 over other Class I and Class II HDACs. Evolution and optimization of this HTS hit series provided HDAC1-selective (SHI-1) compounds with excellent anti-proliferative activity and improved physical properties. Dose-dependent efficacy in a mouse HCT116 xenograft model was demonstrated with a phenylglycine SHI-1 analog.

A Divergent Approach to the Synthesis of 3-Substituted-2-pyrazolines: Suzuki Cross-Coupling of 3-Sulfonyloxy-2-pyrazolines

The efficient Suzuki cross-coupling of pyrazoline nonaflates with organoboron reagents was achieved to afford diverse 3-substituted-2-pyrazolines in excellent yield. The nonaflates displayed improved reactivity over the corresponding triflates and smoothly coupled to a variety of aryl- and heteroarylboronic acids. This process and its broad scope constitute a rapid, divergent strategy for the synthesis of elaborated 2-pyrazolines that are not readily obtained via conventional methods.

Biosynthesis of ML-236C and the hypocholesterolemic agents compactin by Penicillium aurantiogriseum and lovastatin by Aspergillus terreus: determination of the origin of carbon, hydrogen and oxygen atoms by 13C NMR spectrometry and observation of unusual labelling of acetate-derived oxygens by 18O2

Sodium [1-13C, 2-2H3]-, [2-13C, 2-2H3]- and [1-13C, 18O2]-acetate are incorporated in separate experiments into ML-236C 2 and the hypocholesterolemic agent compactin 3 by cultures of Penicillium aurantiogriseum, and the regiochemical distribution of 2H, 13C and 18O is determined by 13C NMR spectrometry. In addition, sodium [1-13C, 18O2]acetate and 18O2 are incorporated into lovastatin (mevinolin) 4 by cultures of Aspergillus terreus to re-examine the origin of oxygen atoms. The results show that the main-chain oxygen atoms of 2–4 originate from acetate, and the C-8 oxygen atom of 3 and 4 is derived from molecular oxygen. However, detailed mass spectral analysis shows that significant amounts of aerobic oxygen can be incorporated at sites normally labelled by acetate oxygen, presumably through generation of [18O]acetate by ω-oxidation of fats. On the basis of labelling results, a mechanism is proposed to account for the formation of the bicyclic ring system in these compounds.

Exploring the pharmacokinetic properties of phosphorus-containing selective HDAC 1 and 2 inhibitors (SHI-1:2).

We report the preparation and structure-activity relationships of phosphorus-containing histone deacetylase inhibitors. A strong trend between decreasing phosphorus functional group size and superior mouse pharmacokinetic properties was identified. In addition, optimized candidates showed tumor growth inhibition in xenograft studies.

Delayed and Prolonged Histone Hyperacetylation with a Selective HDAC1/HDAC2 Inhibitor.

The identification and in vitro and in vivo characterization of a potent SHI-1:2 are described. Kinetic analysis indicated that biaryl inhibitors exhibit slow binding kinetics in isolated HDAC1 and HDAC2 preparations. Delayed histone hyperacetylation and gene expression changes were also observed in cell culture, and histone acetylation was observed in vivo beyond disappearance of drug from plasma. In vivo studies further demonstrated that continuous target inhibition was well tolerated and efficacious in tumor-bearing mice, leading to tumor growth inhibition with either once-daily or intermittent administration.

Evaluation of JAK3 biology in autoimmune disease using a highly selective, irreversible JAK3 inhibitor

Reversible janus associated kinase (JAK) inhibitors such as tofacitinib and decernotinib block cytokine signaling and are efficacious in treating autoimmune diseases. However, therapeutic doses are limited due to inhibition of other JAK/signal transducer and activator of transcription pathways associated with hematopoiesis, lipid biogenesis, infection, and immune responses. A selective JAK3 inhibitor may have a better therapeutic index; however, until recently, no compounds have been described that maintain JAK3 selectivity in cells, as well as against the kinome, with good physicochemical properties to test the JAK3 hypothesis in vivo. To quantify the biochemical basis for JAK isozyme selectivity, we determined that the apparent Km value for each JAK isozyme ranged from 31.8 to 2.9 μM for JAK1 and JAK3, respectively. To confirm compound activity in cells, we developed a novel enzyme complementation assay that read activity of single JAK isozymes in a cellular context. Reversible JAK3 inhibitors cannot achieve sufficient selectivity against other isozymes in the cellular context due to inherent differences in enzyme ATP Km values. Therefore, we developed irreversible JAK3 compounds that are potent and highly selective in vitro in cells and against the kinome. Compound 2, a potent inhibitor of JAK3 (0.15 nM) was 4300-fold selective for JAK3 over JAK1 in enzyme assays, 67-fold [interleukin (IL)-2 versus IL-6] or 140-fold [IL-2 versus erythropoietin or granulocyte-macrophage colony-stimulating factor (GMCSF)] selective in cellular reporter assays and >35-fold selective in human peripheral blood mononuclear cell assays (IL-7 versus IL-6 or GMCSF). In vivo, selective JAK3 inhibition was sufficient to block the development of inflammation in a rat model of rheumatoid arthritis, while sparing hematopoiesis.