Harnessing the Role of HDAC6 in Idiopathic Pulmonary Fibrosis: Design, Synthesis, Structural Analysis, and Biological Evaluation of Potent Inhibitors.

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease characterized by a progressive-fibrosing phenotype. IPF has been associated with aberrant HDAC activities confirmed by our immunohistochemistry studies on HDAC6 overexpression in IPF lung tissues. We herein developed a series of novel hHDAC6 inhibitors, having low inhibitory potency over hHDAC1 and hHDAC8, as potential pharmacological tools for IPF treatment. Their inhibitory potency was combined with low in vitro and in vivo toxicity. Structural analysis of 6h and structure-activity relationship studies contributed to the optimization of the binding mode of the new molecules. The best-performing analogues were tested for their efficacy in inhibiting fibrotic sphere formation and cell viability, proving their capability in reverting the IPF phenotype. The efficacy of analogue 6h was also determined in a validated human lung model of TGF-ß1-dependent fibrogenesis. The results highlighted in this manuscript may pave the way for the identification of first-in-class molecules for the treatment of IPF.

Structural analysis of histone deacetylase 8 mutants associated with Cornelia de Lange Syndrome spectrum disorders.

Cornelia de Lange Syndrome (CdLS) and associated spectrum disorders are characterized by one or more congenital anomalies including distinctive facial features, upper limb abnormalities, intellectual disability, and other symptoms. The molecular genetic basis of CdLS is linked to defects in cohesin, a protein complex that functions in sister chromatid cohesion, chromatin organization, and transcriptional regulation. Histone deacetylase 8 (HDAC8) plays an important role in cohesin function by catalyzing the deacetylation of SMC3, which is required for efficient recycling of the cohesin complex. Missense mutations in HDAC8 have been identified in children diagnosed with CdLS spectrum disorders, and here we outline structure-function relationships for four of these mutations. Specifically, we report the 1.50 Å-resolution structure of the I45T HDAC8-suberoylanilide hydroxamic acid complex, the 1.84 Å-resolution structure of E66D/Y306F HDAC8 complexed with a peptide assay substrate, and the 2.40 Å-resolution structure of G320R HDAC8 complexed with the inhibitor M344. Additionally, we present a computationally generated model of D176G HDAC8. These structures illuminate new structure-function relationships for HDAC8 and highlight the importance of long-range interactions in the protein scaffold that can influence catalytic function.

Spiroindoline-Capped Selective HDAC6 Inhibitors: Design, Synthesis, Structural Analysis, and Biological Evaluation.

Histone deacetylase inhibitors (HDACi) have emerged as promising therapeutics for the treatment of neurodegeneration, cancer, and rare disorders. Herein, we report the development of a series of spiroindoline-based HDAC6 isoform-selective inhibitors based on the X-ray crystal studies of the hit 6a. We identified compound 6j as the most potent and selective hHDAC6 inhibitor of the series. Biological investigation of compounds 6b, 6h, and 6j demonstrated their antiproliferative activity against several cancer cell lines. Western blotting studies indicated that they were able to increase tubulin acetylation, without significant variation in histone acetylation state, and induced PARP cleavage indicating their apoptotic potential at the molecular level. 6j induced HDAC6-dependent pSTAT3 inhibition.

Binding of inhibitors to active-site mutants of CD1, the enigmatic catalytic domain of histone deacetylase 6.

The zinc hydrolase histone deacetylase 6 (HDAC6) is unique among vertebrate deacetylases in that it contains two catalytic domains, designated CD1 and CD2. Both domains are fully functional as lysine deacetylases in vitro. However, the in vivo function of only the CD2 domain is well defined, whereas that of the CD1 domain is more enigmatic. Three X-ray crystal structures of HDAC6 CD1-inhibitor complexes are now reported to broaden the understanding of affinity determinants in the active site. Notably, cocrystallization with inhibitors was facilitated by using active-site mutants of zebrafish HDAC6 CD1. The first mutant studied, H82F/F202Y HDAC6 CD1, was designed to mimic the active site of human HDAC6 CD1. The structure of its complex with trichostatin A was generally identical to that with the wild-type zebrafish enzyme. The second mutant studied, K330L HDAC6 CD1, was prepared to mimic the active site of HDAC6 CD2. It has previously been demonstrated that this substitution does not perturb inhibitor binding conformations in HDAC6 CD1; here, this mutant facilitated cocrystallization with derivatives of the cancer chemotherapy drug suberoylanilide hydroxamic acid (SAHA). These crystal structures allow the mapping of inhibitor-binding regions in the outer active-site cleft, where one HDAC isozyme typically differs from another. It is expected that these structures will help to guide the structure-based design of inhibitors with selectivity against HDAC6 CD1, which in turn will enable new chemical biology approaches to probe its cellular function.

Multicomponent Synthesis, Binding Mode, and Structure-Activity Relationship of Selective Histone Deacetylase 6 (HDAC6) Inhibitors with Bifurcated Capping Groups.

Histone deacetylase 6 (HDAC6) is an emerging target for the treatment of cancer, neurodegenerative diseases, inflammation, and other diseases. Here, we present the multicomponent synthesis and structure-activity relationship of a series of tetrazole-based HDAC6 inhibitors. We discovered the hit compound NR-160 by investigating the inhibition of recombinant HDAC enzymes and protein acetylation. A cocrystal structure of HDAC6 complexed with NR-160 disclosed that the steric complementarity of the bifurcated capping group of NR-160 to the L1 and L2 loop pockets may be responsible for its HDAC6-selective inhibition. While NR-160 displayed only low cytotoxicity as a single agent against leukemia cell lines, it augmented the apoptosis induction of the proteasome inhibitor bortezomib in combination experiments significantly. Furthermore, a combinatorial high-throughput drug screen revealed significantly enhanced cytotoxicity when NR-160 was used in combination with epirubicin and daunorubicin. The synergistic effect in combination with bortezomib and anthracyclines highlights the potential of NR-160 in combination therapies.

Design and Synthesis of Dihydroxamic Acids as HDAC6/8/10 Inhibitors.

We report the synthesis and evaluation of a class of selective multitarget agents for the inhibition of HDAC6, HDAC8, and HDAC10. The concept for this study grew out of a structural analysis of the two selective inhibitors Tubastatin A (HDAC6/10) and PCI-34051 (HDAC8), which we recognized share the same N-benzylindole core. Hybridization of the two inhibitor structures resulted in dihydroxamic acids with benzyl-indole and -indazole core motifs. These substances exhibit potent activity against HDAC6, HDAC8, and HDAC10, while retaining selectivity over HDAC1, HDAC2, and HDAC3. The best substance inhibited the viability of the SK-N-BE(2)C neuroblastoma cell line with an IC50 value similar to a combination treatment with Tubastatin A and PCI-34051. This compound class establishes a proof of concept for such hybrid molecules and could serve as a starting point for the further development of enhanced HDAC6/8/10 inhibitors.

Structural determinants of affinity and selectivity in the binding of inhibitors to histone deacetylase 6.

Histone deacetylase 6 (HDAC6) is associated with multiple neurological disorders as well as aggressive cancers, making its selective inhibition highly desirable for therapeutic purposes. The basic molecular design of an effective HDAC6 inhibitor consists of a zinc-binding group, a linker, and a capping group capable of making interactions at the mouth of the active site. To date, more than 50 high-resolution X-ray crystal structures of HDAC6-inhibitor complexes have been reported, many of which reveal intermolecular interactions that contribute to isozyme affinity and selectivity. Here, we review the key features of HDAC6 inhibitor design illuminated by these structural studies.

Exploring Structural Determinants of Inhibitor Affinity and Selectivity in Complexes with Histone Deacetylase 6.

Inhibition of histone deacetylase 6 (HDAC6) has emerged as a promising therapeutic strategy for the treatment of cancer, chemotherapy-induced peripheral neuropathy, and neurodegenerative disease. The recent X-ray crystal structure determination of HDAC6 enables an understanding of structural features directing affinity and selectivity in the active site. Here, we present the X-ray crystal structures of five HDAC6-inhibitor complexes that illuminate key molecular features of the inhibitor linker and capping groups that facilitate and differentiate binding to HDAC6. In particular, aromatic and heteroaromatic linkers nestle within an aromatic cleft defined by F583 and F643, and different aromatic linkers direct the capping group toward shallow pockets defined by the L1 loop, the L2 loop, or somewhere in between these pockets. These results expand our understanding of factors contributing to the selective inhibition of HDAC6, particularly regarding interactions that can be targeted in the region of the L2 pocket.

Structural Basis of Catalysis and Inhibition of HDAC6 CD1, the Enigmatic Catalytic Domain of Histone Deacetylase 6.

Histone deacetylase 6 (HDAC6) is emerging as a target for inhibition in therapeutic strategies aimed at treating cancer, neurodegenerative disease, and other disorders. Among the metal-dependent HDAC isozymes, HDAC6 is unique in that it contains two catalytic domains, CD1 and CD2. CD2 is a tubulin deacetylase and a tau deacetylase, and the development of HDAC6-selective inhibitors has focused exclusively on this domain. In contrast, there is a dearth of structural and functional information regarding CD1, which exhibits much narrower substrate specificity in comparison with CD2. As the first step in addressing the CD1 information gap, we now present X-ray crystal structures of seven inhibitor complexes with wild-type, Y363F, and K330L HDAC6 CD1. These structures broaden our understanding of molecular features that are important for catalysis and inhibitor binding. The active site of HDAC6 CD1 is wider than that of CD2, which is unexpected in view of the narrow substrate specificity of CD1. Amino acid substitutions between HDAC6 CD1 and CD2, as well as conformational differences in conserved residues, define striking differences in active site contours. Catalytic activity measurements with HDAC6 CD1 confirm the preference for peptide substrates containing C-terminal acetyllysine residues. However, these measurements also show that CD1 exhibits weak activity for peptide substrates bearing certain small amino acids on the carboxyl side of the scissile acetyllysine residue. Taken together, these results establish a foundation for understanding the structural basis of HDAC6 CD1 catalysis and inhibition, pointing to possible avenues for the development of HDAC6 CD1-selective inhibitors.

Methods for the expression, purification, and crystallization of histone deacetylase 6-inhibitor complexes.

Histone deacetylase (HDAC) isozymes modulate numerous regulatory signals and pathways in biological systems, hence serving as targets for drug design. For example, HDAC6 is the cytosolic tubulin deacetylase and its inhibition compromises microtubule dynamics, leading to cancer cell cycle arrest and apoptosis. The design of inhibitors that selectively target HDAC6 is desirable to avoid side effects resulting from the inhibition of off-target HDACs. High resolution X-ray crystal structures of HDAC6 have accelerated structure-based approaches to drug design targeting HDAC6. Crystal structure analysis reveals that the tubulin deacetylase domain of human HDAC6 (catalytic domain 2, also known as CD2) is very similar to that of HDAC6 CD2 from Danio rerio (zebrafish, designated zCD2). Thus, zCD2 is a valid surrogate of human HDAC6 CD2, the actual drug target; moreover, zCD2 is much more easily prepared and crystallized. A plasmid containing the zCD2 construct for heterologous expression in Escherichia coli is available through Addgene (#122031). In this chapter, we review the preparation, purification, and crystallization of zCD2-inhibitor complexes. These methods enable the rapid acquisition of structural data regarding optimal zinc-binding groups, capping groups, and linkers in the discovery of new and selective HDAC6 inhibitors.

Structure and Function of the Acetylpolyamine Amidohydrolase from the Deep Earth Halophile Marinobacter subterrani.

Polyamines are small organic cations that are essential for cellular function in all kingdoms of life. Polyamine metabolism is regulated by enzyme-catalyzed acetylation-deacetylation cycles in a fashion similar to the epigenetic regulation of histone function in eukaryotes. Bacterial polyamine deacetylases are particularly intriguing, because these enzymes share the fold and function of eukaryotic histone deacetylases. Recently, acetylpolyamine amidohydrolase from the deep earth halophile Marinobacter subterrani (msAPAH) was described. This Zn2+-dependent deacetylase shares 53% amino acid sequence identity with the acetylpolyamine amidohydrolase from Mycoplana ramosa (mrAPAH) and 22% amino acid sequence identity with the catalytic domain of histone deacetylase 10 from Danio rerio (zebrafish; zHDAC10), the eukaryotic polyamine deacetylase. The X-ray crystal structure of msAPAH, determined in complexes with seven different inhibitors as well as the acetate coproduct, shows how the chemical strategy of Zn2+-dependent amide hydrolysis and the catalytic specificity for cationic polyamine substrates is conserved in a subterranean halophile. Structural comparisons with mrAPAH reveal that an array of aspartate and glutamate residues unique to msAPAH enable the binding of one or more Mg2+ ions in the active site and elsewhere on the protein surface. Notwithstanding these differences, activity assays with a panel of acetylpolyamine and acetyllysine substrates confirm that msAPAH is a broad-specificity polyamine deacetylase, much like mrAPAH. The broad substrate specificity contrasts with the narrow substrate specificity of zHDAC10, which is highly specific for N8-acetylspermidine hydrolysis. Notably, quaternary structural features govern the substrate specificity of msAPAH and mrAPAH, whereas tertiary structural features govern the substrate specificity of zHDAC10.

Discovery of the First-in-Class Dual Histone Deacetylase-Proteasome Inhibitor.

Dual- or multitarget drugs have emerged as a promising alternative to combination therapies. Proteasome inhibitors (PIs) possess synergistic activity with histone deacetylase (HDAC) inhibitors due to the simultaneous blockage of the ubiquitin degradation and aggresome pathways. Here, we present the design, synthesis, binding modes, and anticancer properties of RTS-V5 as the first-in-class dual HDAC-proteasome ligand. The inhibition of both targets was confirmed by biochemical and cellular assays as well as X-ray crystal structures of the 20S proteasome and HDAC6 complexed with RTS-V5. Cytotoxicity assays with leukemia and multiple myeloma cell lines as well as therapy refractory primary patient-derived leukemia cells demonstrated that RTS-V5 possesses potent and selective anticancer activity. Our results will thus guide the structure-based optimization of dual HDAC-proteasome inhibitors for the treatment of hematological malignancies.

Histone Deacetylase 6-Selective Inhibitors and the Influence of Capping Groups on Hydroxamate-Zinc Denticity.

Four crystal structures are presented of histone deacetylase 6 (HDAC6) complexes with para-substituted phenylhydromaxamate inhibitors, including bulky peptoids. These structures provide insight regarding the design of capping groups that confer selectivity for binding to HDAC6, specifically with regard to interactions in a pocket formed by the L1 loop. Capping group interactions may also influence hydroxamate-Zn2+ coordination with monodentate or bidentate geometry.

Multicomponent Synthesis and Binding Mode of Imidazo[1,2- a]pyridine-Capped Selective HDAC6 Inhibitors.

The multicomponent synthesis of a mini-library of histone deacetylase inhibitors with imidazo[1,2- a]pyridine-based cap groups is presented. The biological evaluation led to the discovery of the hit compound MAIP-032 as a selective HDAC6 inhibitor with promising anticancer activity. The X-ray structure of catalytic domain 2 from Danio rerio HDAC6 complexed with MAIP-032 revealed a monodentate zinc-binding mode.