Niek G.J. Leus

Histone deacetylase 3 (HDAC 3) as emerging drug target in NF-κB-mediated inflammation.

Activation of inflammatory gene expression is regulated, among other factors, by post-translational modifications of histone proteins. The most investigated type of histone modifications is lysine acetylations. Histone deacetylases (HDACs) remove acetylations from lysines, thereby influencing (inflammatory) gene expression. Intriguingly, apart from histones, HDACs also target non-histone proteins. The nuclear factor κB (NF-κB) pathway is an important regulator in the expression of numerous inflammatory genes, and acetylation plays a crucial role in regulating its responses. Several studies have shed more light on the role of HDAC 1-3 in inflammation with a particular pro-inflammatory role for HDAC 3. Nevertheless, the HDAC-NF-κB interactions in inflammatory signalling have not been fully understood. An important challenge in targeting the regulatory role of HDACs in the NF-κB pathway is the development of highly potent small molecules that selectively target HDAC iso-enzymes. This review focuses on the role of HDAC 3 in (NF-κB-mediated) inflammation and NF-κB lysine acetylation. In addition, we address the application of frequently used small molecule HDAC inhibitors as an approach to attenuate inflammatory responses, and their potential as novel therapeutics. Finally, recent progress and future directions in medicinal chemistry efforts aimed at HDAC 3-selective inhibitors are discussed.

HDAC 3-selective inhibitor RGFP966 demonstrates anti-inflammatory properties in RAW 264.7 macrophages and mouse precision-cut lung slices by attenuating NF-κB p65 transcriptional activity.

Graphical abstract

Rational Development of a Potent 15-Lipoxygenase-1 Inhibitor with in Vitro and ex Vivo Anti-inflammatory Properties

Human 15-lipoxygenase-1 (h-15-LOX-1) is a mammalian lipoxygenase and plays an important role in several inflammatory lung diseases such as asthma, COPD, and chronic bronchitis. Novel potent inhibitors of h-15-LOX-1 are required to explore the role of this enzyme further and to enable drug discovery efforts. In this study, we applied an approach in which we screened a fragment collection that is focused on a diverse substitution pattern of nitrogen-containing heterocycles such as indoles, quinolones, pyrazoles, and others. We denoted this approach substitution-oriented fragment screening (SOS) because it focuses on the identification of novel substitution patterns rather than on novel scaffolds. This approach enabled the identification of hits with good potency and clear structure-activity relationships (SAR) for h-1-5-LOX-1 inhibition. Molecular modeling enabled the rationalization of the observed SAR and supported structure-based design for further optimization to obtain inhibitor 14 d that binds with a Ki of 36 nM to the enzyme. In vitro and ex vivo biological evaluations of our best inhibitor demonstrate a significant increase of interleukin-10 (IL-10) gene expression, which indicates its anti-inflammatory properties.

The histone acetyltransferase p300 inhibitor C646 reduces pro-inflammatory gene expression and inhibits histone deacetylases

Lysine acetylations are reversible posttranslational modifications of histone and non-histone proteins that play important regulatory roles in signal transduction cascades and gene expression. Lysine acetylations are regulated by histone acetyltransferases as writers and histone deacetylases as erasers. Because of their role in signal transduction cascades, these enzymes are important players in inflammation. Therefore, histone acetyltransferase inhibitors could reduce inflammatory responses. Among the few histone acetyltransferase inhibitors described, C646 is one of the most potent (Ki of 0.4μM for histone acetyltransferase p300). C646 was described to affect the NF-κB pathway; an important pathway in inflammatory responses, which is regulated by acetylation. This pathway has been implicated in asthma and COPD. Therefore, we hypothesized that via regulation of the NF-κB signaling pathway, C646 can inhibit pro-inflammatory gene expression, and have potential for the treatment of inflammatory lung diseases. In line with this, we demonstrate here that C646 reduces pro-inflammatory gene expression in RAW264.7 murine macrophages and murine precision-cut lung slices. To unravel its effects on cellular substrates we applied mass spectrometry and found, counterintuitively, a slight increase in acetylation of histone H3. Based on this finding, and structural features of C646, we presumed inhibitory activity of C646 on histone deacetylases, and indeed found inhibition of histone deacetylases from 7μM and higher concentrations. This indicates that C646 has potential for further development towards applications in the treatment of inflammation, however, its newly discovered lack of selectivity at higher concentrations needs to be taken into account.

HDAC1-3 inhibitor MS-275 enhances IL10 expression in RAW264.7 macrophages and reduces cigarette smoke-induced airway inflammation in mice

Chronic obstructive pulmonary disease (COPD) constitutes a major health burden. Studying underlying molecular mechanisms could lead to new therapeutic targets. Macrophages are orchestrators of COPD, by releasing pro-inflammatory cytokines. This process relies on transcription factors such as NF-κB, among others. NF-κB is regulated by lysine acetylation; a post-translational modification installed by histone acetyltransferases and removed by histone deacetylases (HDACs). We hypothesized that small molecule HDAC inhibitors (HDACi) targeting class I HDACs members that can regulate NF-κB could attenuate inflammatory responses in COPD via modulation of the NF-κB signaling output. MS-275 is an isoform-selective inhibitor of HDAC1-3. In precision-cut lung slices and RAW264.7 macrophages, MS-275 upregulated the expression of both pro- and anti-inflammatory genes, implying mixed effects. Interestingly, anti-inflammatory IL10 expression was upregulated in these model systems. In the macrophages, this was associated with increased NF-κB activity, acetylation, nuclear translocation, and binding to the IL10 promoter. Importantly, in an in vivo model of cigarette smoke-exposed C57Bl/6 mice, MS-275 robustly attenuated inflammatory expression of KC and neutrophil influx in the lungs. This study highlights for the first time the potential of isoform-selective HDACi for the treatment of inflammatory lung diseases like COPD.

The relevance of Ki calculation for bi-substrate enzymes illustrated by kinetic evaluation of a novel lysine (K) acetyltransferase 8 inhibitor

Histone acetyltransferases (HATs) are important mediators of epigenetic post-translational modifications of histones that play important roles in health and disease. A disturbance of these modifications can result in disease states, such as cancer or inflammatory diseases. Inhibitors of HATs (HATi) such as lysine (K) acetyltransferase 8 (KAT8), could be used to study the epigenetic processes in diseases related to these enzymes or to investigate HATs as therapeutic targets. However, the development of HATi is challenged by the difficulties in kinetic characterization of HAT enzymes and their inhibitors to enable calculation of a reproducible inhibitory potency. In this study, a fragment screening approach was used, enabling identification of 4-amino-1-naphthol, which potently inhibited KAT8. The inhibitor was investigated for enzyme inhibition using kinetic and calorimetric binding studies. This allowed for calculation of the Ki values for both the free enzyme as well as the acetylated intermediate. Importantly, it revealed a striking difference in binding affinity between the acetylated enzyme and the free enzyme, which could not be revealed by the IC50 value. This shows that kinetic characterization of inhibitors and calculation of Ki values is crucial for determining the binding constants of HAT inhibitors. We anticipate that more comprehensive characterization of enzyme inhibition, as described here, is needed to advance the field of HAT inhibitors.

Activity-Based Probes for 15-Lipoxygenase-1

Human 15-lipoxygenase-1 (15-LOX-1) plays an important role in several inflammatory lung diseases, such as asthma, COPD, and chronic bronchitis, as well as various CNS diseases, such as Alzheimers disease, Parkinsons disease, and stroke. Activity-based probes of 15-LOX-1 are required to explore the role of this enzyme further and to enable drug discovery. In this study, we developed a 15-LOX-1 activity-based probe for the efficient activity-based labeling of recombinant 15-LOX-1. 15-LOX-1-dependent labeling in cell lysates and tissue samples was also possible. To mimic the natural substrate of the enzyme, we designed activity-based probes that covalently bind to the active enzyme and include a terminal alkene as a chemical reporter for the bioorthogonal linkage of a detectable functionality through an oxidative Heck reaction. The activity-based labeling of 15-LOX-1 should enable the investigation and identification of this enzyme in complex biological samples, thus opening up completely new opportunities for drug discovery.

Chemical epigenetics to assess the role of HDAC1-3 inhibition in macrophage pro-inflammatory gene expression

Histone deacetylases (HDACs) have been used as pharmacological targets for the treatment of various diseases. Some non-selective HDAC inhibitors (HDACi) have been clinically-used as therapeutic agents for treatment of hematological cancers but their cytotoxic side effects are an important downside. The discovery of more selective inhibitors has certified the involvement of individual HDACs in pathological processes but the elucidation of the role of specific family members in inflammatory responses still remains a challenge. Here, we report the development of closely related, structural analogues of the clinically-used HDACi Entinostat via a chemical epigenetic approach. Three compounds were designed and synthesized in which the cap moiety of Entinostat was replaced by an azobenzene group that is either para, meta or ortho substituted. The compounds were then evaluated for selectivity towards HDACs 1–3 and their effect on pro-inflammatory gene expression in macrophages. One analogue, compound 4, lacked selectivity and demonstrated inhibition of NF-κB reporter gene activity and pro-inflammatory gene expression in RAW264.7 macrophages, thus indicating that there is a delicate balance between the selectivity of HDACi over specific family members and their pro- or anti-inflammatory effects.

Small Molecule Inhibitors of Histone Deacetylases and Acetyltransferases as Potential Therapeutics in Oncology

Abstract Uncontrolled cell proliferation and resistance to apoptosis in cancer are, among others, regulated by posttranslational modifications of histone proteins. The most investigated type of histone modification is lysine acetylation. Histone acetyltransferases (HATs), acetylate histone lysine residues, and histone deacetylases (HDACs) remove acetyl residues from lysines, thereby influencing gene expression. Whereas HAT inhibitors are relatively undeveloped, four HDAC inhibitors (HDACi) reached the clinic for treatment of hematological cancers. HDACi induce apoptosis via inhibition of histone deacetylation and concomitant transcription of aberrantly silenced genes. Nevertheless, other mechanisms have also been described. Intriguingly, HDACs also target nonhistone proteins such as the nuclear factor κB (NF-κB) pathway, which is an important regulator of gene transcription for which a role in cancer has been implicated. Despite the studies that shed more light on the role of HDAC isoenzymes in the NF-κB pathway the challenge remains to develop isoenzyme-selective HDACi to target this pathway.