Discovery of 1-Benzhydryl-Piperazine-Based HDAC Inhibitors with Anti-Breast Cancer Activity: Synthesis, Molecular Modeling, In Vitro and In Vivo Biological Evaluation

Abstract Isoform-selective histone deacetylase (HDAC) inhibition is promoted as a rational strategy to develop safer anti-cancer drugs compared to non-selective HDAC inhibitors. Despite this presumed benefit, considerably more non-selective HDAC inhibitors have undergone clinical trials. In this report, we detail the design and discovery of potent HDAC inhibitors, with 1-benzhydryl piperazine as a surface recognition group, that differ in hydrocarbon linker. In vitro HDAC screening identified two selective HDAC6 inhibitors with nanomolar IC50 values, as well as two non-selective nanomolar HDAC inhibitors. Structure-based molecular modeling was employed to study the influence of linker chemistry of synthesized inhibitors on HDAC6 potency. The breast cancer cell lines (MDA-MB-231 and MCF-7) were used to evaluate compound-mediated in vitro anti-cancer, anti-migratory, and anti-invasive activities. Experiments on the zebrafish MDA-MB-231 xenograft model revealed that a novel non-selective HDAC inhibitor with a seven-carbon-atom linker exhibits potent anti-tumor, anti-metastatic, and anti-angiogenic effects when tested at low micromolar concentrations

AKT activation controls cell survival in response to HDAC6 inhibition.

HDAC6 is emerging as an important therapeutic target for cancer. We investigated mechanisms responsible for survival of tumor cells treated with a HDAC6 inhibitor. Expression of the 20 000 genes examined did not change following HDAC6 treatment in vivo. We found that HDAC6 inhibition led to an increase of AKT activation (P-AKT) in vitro, and genetic knockdown of HDAC6 phenocopied drug-induced AKT activation. The activation of AKT was not observed in PTEN null cells; otherwise, PTEN/PIK3CA expression per se did not predict HDAC6 inhibitor sensitivity. Interestingly, HDAC6 inhibitor treatment led to inactivating phosphorylation of PTEN (P-PTEN Ser380), which likely led to the increased P-AKT in cells that express PTEN. Synergy was observed with phosphatidylinositol 3-kinases (PI3K) inhibitor treatment in vitro, accompanied by increased caspase 3/7 activity. Furthermore, combination of HDAC6 inhibitor with a PI3K inhibitor caused substantial tumor growth inhibition in vivo compared with either treatment alone, also detectable by Ki-67 immunostaining and (18)F-FLT positron emission tomography (PET). In aggregate AKT activation appears to be a key survival mechanism for HDAC6 inhibitor treatment. Our findings indicate that dual inhibition of HDAC6 and P-AKT may be necessary to substantially inhibit growth of solid tumors

The therapeutic strategy of HDAC6 inhibitors in lymphoproliferative disease

Histone deacetylases (HDACs) are master regulators of chromatin remodeling, acting as epigenetic regulators of gene expression. In the last decade, inhibition of HDACs has become a target for specific epigenetic modifications related to cancer development. Overexpression of HDAC has been observed in several hematologic malignancies. Therefore, the observation that HDACs might play a role in various hematologic malignancies has brought to the development of HDAC inhibitors as potential antitumor agents. Recently, the class IIb, HDAC6, has emerged as one potential selective HDACi. This isoenzyme represents an important pharmacological target for selective inhibition. Its selectivity may reduce the toxicity related to the off-target effects of pan-HDAC inhibitors. HDAC6 has also been studied in cancer especially for its ability to coordinate a variety of cellular processes that are important for cancer pathogenesis. HDAC6 has been reported to be overexpressed in lymphoid cells and its inhibition has demonstrated activity in preclinical and clinical study of lymphoproliferative disease. Various studies of HDAC6 inhibitors alone and in combination with other agents provide strong scientific rationale for the evaluation of these new agents in the clinical setting of hematological malignancies. In this review, we describe the HDACs, their inhibitors, and the recent advances of HDAC6 inhibitors, their mechanisms of action and role in lymphoproliferative disorders

A Therapeutic Perspective of HDAC8 in Different Diseases: An Overview of Selective Inhibitors

Histone deacetylases (HDACs) are epigenetic enzymes which participate in transcriptional repression and chromatin condensation mechanisms by removing the acetyl moiety from acetylated ε-amino group of histone lysines and other non-histone proteins. In recent years, HDAC8, a class I HDAC, has emerged as a promising target for different disorders, including X-linked intellectual disability, fibrotic diseases, cancer, and various neuropathological conditions. Selective HDAC8 targeting is required to limit side effects deriving from the treatment with pan-HDAC inhibitors (HDACis); thus, many endeavours have focused on the development of selective HDAC8is. In addition, polypharmacological approaches have been explored to achieve a synergistic action on multi-factorial diseases or to enhance the drug efficacy. In this frame, proteolysis-targeting chimeras (PROTACs) might be regarded as a dual-targeting approach for attaining HDAC8 proteasomal degradation. This review highlights the most relevant and recent advances relative to HDAC8 validation in various diseases, providing a snapshot of the current selective HDAC8is, with a focus on polyfunctional modulators

Development of HDAC Inhibitors Exhibiting Therapeutic Potential in T-Cell Prolymphocytic Leukemia

Epigenetic targeting has emerged as an efficacious therapy for hematological cancers. The rare and incurable T-cell prolymphocytic leukemia (T-PLL) is known for its aggressive clinical course. Current epigenetic agents such as histone deacetylase (HDAC) inhibitors are increasingly used for targeted therapy. Through a structure-activity relationship (SAR) study, we developed an HDAC6 inhibitor KT-531, which exhibited higher potency in T-PLL compared to other hematological cancers. KT-531 displayed strong HDAC6 inhibitory potency and selectivity, on-target biological activity, and a safe therapeutic window in nontransformed cell lines. In primary T-PLL patient cells, where HDAC6 was found to be overexpressed, KT-531 exhibited strong biological responses, and safety in healthy donor samples. Notably, combination studies in T-PLL patient samples demonstrated KT-531 synergizes with approved cancer drugs, bendamustine, idasanutlin, and venetoclax. Our work suggests HDAC inhibition in T-PLL could afford sufficient therapeutic windows to achieve durable remission either as standalone or in combination with targeted drugs.Peer reviewe

Novel chemical tools to target two-pore channel 2, P-glycoprotein and histone deacetylase 6 in cancer

The identification of previously unknown targets as well as the development of efficacious inhibitors for known targets are key factors to make more patients benefit from tumor therapy. For instance, the role of two-pore channel 2 (TPC2), one of the few cation channels localized on endolysosomal membranes, in cancer remains poorly understood. Here, we report that TPC2 knockout reduces proliferation of liver cancer cells in vitro, affects their energy metabolism and successfully abrogates tumor growth in vivo. Concurrently, we have identified novel, simplified analogues of the alkaloid tetrandrine (SG-005 and SG-094) as potent TPC2 inhibitors by screening a library of benzyltetrahydroisoquinoline derivatives using cell proliferation assays, endolysosomal patch clamp and calcium imaging. Removal of dispensable substructures of the lead molecule tetrandrine increases antiproliferative properties against several cancer cell lines and impairs proangiogenic signaling of endothelial cells to a greater extent than tetrandrine. Simultaneously, toxic effects on non-cancerous cells are reduced, allowing in vivo administration and revealing the first TPC2 inhibitor with antitumor efficacy in mice (SG-094). Hence, our study unveils TPC2 as valid target for cancer therapy and provides novel, easily accessible tetrandrine analogues as promising option for effective pharmacological interference. Furthermore, in-depth studies were conducted to investigate a postulated mechanism of metabolic toxification of tetrandrine. A combined medicinal chemistry and cell biology approach showed that a reduction of the toxicity of tetrandrine cannot be achieved by replacing or eliminating the hypothesized metabolically instable functional group, clearly indicating that the proposed pathway is not the primary cause for the in vitro toxicity of tetrandrine and related alkaloids. Moreover, we have uncovered that the simplified tetrandrine congeners SG-005 and SG-094 additionally inhibit P-glycoprotein (P-gp), a drug efflux pump associated with multidrug resistance and treatment failure in tumor therapy. Since no approved molecules targeting P-gp are currently available, SG-005 and SG-094 might represent promising candidates to treat drug-resistant cancers owing to their favorable drug-like properties. Generally, the dual mode of action of isoquinoline-based TPC2/P-gp antagonists is mentioned here for the first time. Based on this, the known third-generation P-gp inhibitor elacridar was exemplarily studied for its potential to block TPC2, revealing another potent TPC2 blocker and thereby challenging the assumption of elacridar specifically acting on efflux pumps. Hence, on the one hand, a new lead structure (elacridar) for the development of prospective TPC2 blockers is provided. On the other hand, hints for common structural motifs on TPC2 and P-gp are given, which can facilitate the search for additional TPC2 antagonists. We further uncovered that TPC2 and P-gp do not only share mutual small molecule inhibitors, but also seem to be functionally connected. This is reflected by the higher sensitivity of TPC2-deficient, drug-resistant leukemia cells to vincristine, opening the stage for further studying the implication of TPC2 in processes related to (P-gp-mediated) chemoresistance. Summarizing, this work clearly illustrates that the endolysosomal cation channel TPC2 is a suitable target for tumor therapy. Additionally, synthetically accessible, potent TPC2 blockers were developed as promising preclinical candidates, making TPC2 a druggable protein target. Further, an implication of TPC2 and blockers of this channel in chemoresistance was uncovered, both by TPC2 promoting chemoresistance as well as by the dual action of isoquinolines on TPC2 and the drug efflux pump P-gp. Histone deacetylase 6 (HDAC6) is another protein that has gained attention as target for tumor therapy. HDAC6 is primarily located to the cytoplasm, where it deacetylates several non-histone proteins and thereby alters critical cancer-related pathways. Selective targeting of HDAC6 is aimed to reduce the toxicity associated with pan-HDAC inhibition and, along this line, we have developed and characterized potent and selective HDAC6 inhibitors (KV-46, KV-70, KV-181) with a phenothiazine system as cap group and a benzhydroxamic acid moiety as zinc-binding group. In accordance with effects of specific HDAC6 inhibition, KV-46, KV-70 and KV-181 are relatively non-toxic to healthy liver cells and moderately effective at reducing cancer cell proliferation and inducing apoptosis. Further, KV-46, KV-70 and KV-181 exposure increases the expression of critical protein markers of the unfolded protein response and the immune response, suggesting a potential benefit of combining HDAC6 inhibitors with proteasome inhibitors or immunomodulatory agents

Design, Synthesis And Biological Evaluation Of Histone Deacetylase (hdac) Inhibitors: Saha (vorinostat) Analogs And Biaryl Indolyl Benzamide Inhibitors Display Isoform Selectivity

HDAC proteins have emerged as interesting targets for anti-cancer drugs due to their involvement in cancers, as well as several other diseases. Several HDAC inhibitors have been approved by the FDA as anti-cancer drugs, including SAHA (suberoylanilide hydroxamic acid, Vorinostat). Unfortunately, SAHA inhibits most HDAC isoforms, which limit its use as a pharmacological tool and may lead to side effects in the clinic. In this work we were interested in developing isoform selective HDAC inhibitors, which may decrease or eliminate the side effects associated with non-selective inhibitors treatment. In addition, isoform selective HDAC inhibitors can be used as biological tools to help understand the HDAC-related cancer biology. Our strategy was based on synthesis and screening of several derivatives of the non-selective FDA approved drug SAHA substituted at different positions of the linker region. Several SAHA analogs modified at the C4 and C5 positions of the linker were synthesized. The new C4- and C5-modified SAHA libraries, along with the previously synthesized C2-modified SAHA analogs were screened in vitro and in cellulo for HDAC isoform selectivity. Interestingly, several analogs exhibited dual HDAC6/HDAC8 selectivity. Enantioselective syntheses of the pure enantiomers of some of the interesting analogs were performed and the enantiomers were screened in vitro. Among the most interesting analogs, (R)-C4-benzyl SAHA displayed 520- to 1300-fold selectivity for HDAC6 and HDAC8 over HDAC1, 2, and 3, with IC50 values of 48 and 27 nM with HDAC6 and 8, respectively. Docking studies were performed to provide structural rationale for the observed selectivity of the new analogs. In addition, rational design, synthesis, and screening of several other biaryl indolyl benzamide HDAC inhibitors is discussed, and some showed modest HDAC1 selectivity. The new biaryl indolyl benzamides can be useful to further develop HDAC1 selective inhibitors. The dual HDAC6/8 selective inhibitors can be used as lead compounds and as a chemical tool to study HDAC related cancer biology. The observed enhancement of selectivity upon modifying the linker region of the non-selective inhibitor SAHA shows that modifying current drugs, like SAHA, could lead to substantial improvement in its pharmacodynamic properties

Novel chemical tools to target two-pore channel 2, P-glycoprotein and histone deacetylase 6 in cancer

The identification of previously unknown targets as well as the development of efficacious inhibitors for known targets are key factors to make more patients benefit from tumor therapy. For instance, the role of two-pore channel 2 (TPC2), one of the few cation channels localized on endolysosomal membranes, in cancer remains poorly understood. Here, we report that TPC2 knockout reduces proliferation of liver cancer cells in vitro, affects their energy metabolism and successfully abrogates tumor growth in vivo. Concurrently, we have identified novel, simplified analogues of the alkaloid tetrandrine (SG-005 and SG-094) as potent TPC2 inhibitors by screening a library of benzyltetrahydroisoquinoline derivatives using cell proliferation assays, endolysosomal patch clamp and calcium imaging. Removal of dispensable substructures of the lead molecule tetrandrine increases antiproliferative properties against several cancer cell lines and impairs proangiogenic signaling of endothelial cells to a greater extent than tetrandrine. Simultaneously, toxic effects on non-cancerous cells are reduced, allowing in vivo administration and revealing the first TPC2 inhibitor with antitumor efficacy in mice (SG-094). Hence, our study unveils TPC2 as valid target for cancer therapy and provides novel, easily accessible tetrandrine analogues as promising option for effective pharmacological interference. Furthermore, in-depth studies were conducted to investigate a postulated mechanism of metabolic toxification of tetrandrine. A combined medicinal chemistry and cell biology approach showed that a reduction of the toxicity of tetrandrine cannot be achieved by replacing or eliminating the hypothesized metabolically instable functional group, clearly indicating that the proposed pathway is not the primary cause for the in vitro toxicity of tetrandrine and related alkaloids. Moreover, we have uncovered that the simplified tetrandrine congeners SG-005 and SG-094 additionally inhibit P-glycoprotein (P-gp), a drug efflux pump associated with multidrug resistance and treatment failure in tumor therapy. Since no approved molecules targeting P-gp are currently available, SG-005 and SG-094 might represent promising candidates to treat drug-resistant cancers owing to their favorable drug-like properties. Generally, the dual mode of action of isoquinoline-based TPC2/P-gp antagonists is mentioned here for the first time. Based on this, the known third-generation P-gp inhibitor elacridar was exemplarily studied for its potential to block TPC2, revealing another potent TPC2 blocker and thereby challenging the assumption of elacridar specifically acting on efflux pumps. Hence, on the one hand, a new lead structure (elacridar) for the development of prospective TPC2 blockers is provided. On the other hand, hints for common structural motifs on TPC2 and P-gp are given, which can facilitate the search for additional TPC2 antagonists. We further uncovered that TPC2 and P-gp do not only share mutual small molecule inhibitors, but also seem to be functionally connected. This is reflected by the higher sensitivity of TPC2-deficient, drug-resistant leukemia cells to vincristine, opening the stage for further studying the implication of TPC2 in processes related to (P-gp-mediated) chemoresistance. Summarizing, this work clearly illustrates that the endolysosomal cation channel TPC2 is a suitable target for tumor therapy. Additionally, synthetically accessible, potent TPC2 blockers were developed as promising preclinical candidates, making TPC2 a druggable protein target. Further, an implication of TPC2 and blockers of this channel in chemoresistance was uncovered, both by TPC2 promoting chemoresistance as well as by the dual action of isoquinolines on TPC2 and the drug efflux pump P-gp. Histone deacetylase 6 (HDAC6) is another protein that has gained attention as target for tumor therapy. HDAC6 is primarily located to the cytoplasm, where it deacetylates several non-histone proteins and thereby alters critical cancer-related pathways. Selective targeting of HDAC6 is aimed to reduce the toxicity associated with pan-HDAC inhibition and, along this line, we have developed and characterized potent and selective HDAC6 inhibitors (KV-46, KV-70, KV-181) with a phenothiazine system as cap group and a benzhydroxamic acid moiety as zinc-binding group. In accordance with effects of specific HDAC6 inhibition, KV-46, KV-70 and KV-181 are relatively non-toxic to healthy liver cells and moderately effective at reducing cancer cell proliferation and inducing apoptosis. Further, KV-46, KV-70 and KV-181 exposure increases the expression of critical protein markers of the unfolded protein response and the immune response, suggesting a potential benefit of combining HDAC6 inhibitors with proteasome inhibitors or immunomodulatory agents

Synthesis and analysis of pseudo-octahedral metal complexes as anticancer agents

Chemotherapy is one of the most predominantly used treatments for cancer. Identifying desirable features required of anticancer agents has rapidly developed alongside our growing understanding of the disease. Metal complexes are of interest as drug candidates as they provide advantages over purely organic systems via modular synthesis, variable geometries and oxidation states. Currently, platinum drugs such as cisplatin remain the most common metal-based treatments, despite their known toxicity towards healthy cells. Viable complexes are therefore sought as alternatives that overcome the limitations of platinum drugs. The synthesis and analysis of three piano-stool metal complex series are described, each investigating a distinct mechanism of action. Firstly, pyridylphosphinate complexes were employed as potential DNA-binding agents. The complexes possess a labile monodentate halide ligand, which exchanges intracellularly, forming the aqua species, enabling the binding of DNA. The metal, arene and ligand substituents were varied to tune the properties of the complexes. Of the resulting systems, few displayed notable cytotoxicity, and these complexes were pursued no further. The bulk of this thesis focusses on HDAC enzymes as a discrete target for anticancer therapy. New complexes were developed incorporating arene-metal motifs along with ligands designed to trigger HDAC inhibition. The biological properties of this series were explored and revealed moderate to good cytotoxicity, as well as HDAC inhibition to be the likely mechanism of action. Structural modifications of the parent complexes were devised to optimise selectivity between HDAC enzyme isoforms. Relative to the ligand alone, an increase in specificity of the metal-ligand complexes was observed, successfully demonstrating the benefits of incorporating a metal-arene motif. Biological assays including cellular uptake, catalytic domain selectivity and uptake mechanism are reported to examine the varied behaviour of these complexes. Hypoxic activation was the final mechanism of action studied. Two complexes were synthesised possessing a reducible protecting group, to produce the active species selectively under conditions of hypoxia. Such activity was utilised to minimise off-target binding and enhance tumour region selectivity