Enhancing anti-cancer capacity: Novel class I/II HDAC inhibitors modulate EMT, cell cycle, and apoptosis pathways.

Cancer has been a leading cause of death over the last few decades in western countries as well as in Taiwan. However, traditional therapies are limited by the adverse effects of chemotherapy and radiotherapy, and tumor recurrence may occur. Therefore, it is critical to develop novel therapeutic drugs. In the field of HDAC inhibitor development, apart from the hydroxamic acid moiety, 2-aminobenzamide also functions as a zinc-binding domain, which is shown in well-known HDAC inhibitors such as Entinostat and Chidamide. With recent successful experiences in synthesizing 1-(phenylsulfonyl)indole-based compounds, in this study, we further combined two features of the above chemical compounds and generated indolyl benzamides. Compounds were screened in different cancer cell lines, and enzyme activity was examined to demonstrate their potential for anti-HDAC activity. Various biological functional assays evidenced that two of these compounds could suppress cancer growth and migration capacity, through regulating epithelial-mesenchymal transition (EMT), cell cycle, and apoptosis mechanisms. Data from 3D cancer cells and the in vivo zebrafish model suggested the potential of these compounds in cancer therapy in the future.

Synthesis and evaluation of potent (iso)ellipticine-based inhibitors of MYLK4 accessed via expeditious synthesis from isoquinolin-5-ol.

The K2S2O8-mediated generation of p-iminoquinone contributed to the regioselective substitution of isoquinolin-5,8-dione. This hydroxyl group-guided substitution was also applied to selected heterocycles and addressed the regioselectivity issue of quinones. This study has provided an expeditious pathway from isoquinolin-5-ol (5) to ellipticine (1) and isoellipticine (2), which benefits the comprehensive comparison of their activity. Compounds 1 and 2 displayed marked MYLK4 inhibitory activity with IC50 values of 7.1 and 6.1 nM, respectively. In the cellular activity of AML cells (MV-4-11 and MOLM-13), compound 1 showed better AML activity than compound 2.

6-Regioisomeric 5,8-quinolinediones as potent CDC25 inhibitors against colorectal cancers.

Precise and accurate control of cell cycle progression is required to maintain cell identity and proliferation. Failing to keep it will lead to genome instability and tumorigenesis. Cell Division Cycle 25 (CDC25) phosphatases are the key to regulating the activity of the master cell cycle controller, cyclin-dependent kinases (CDKs). Dysregulation of CDC25 has been shown to associate with several human malignancies. Here, we reported a series of derivatives of the CDC25 inhibitor, NSC663284, bearing quinones as core scaffolds and morpholin alkylamino side chains. Among these derivatives, the cytotoxic activity of the 6-isomer of 5,8-quinolinedione derivatives (6b, 16b, 17b, and 18b) displayed higher potency against colorectal cancer (CRC) cells. Compound 6b possessed the most antiproliferative activity, with IC50 values of 0.59 µM (DLD1) and 0.44 µM (HCT116). The treatment of compound 6b resulted in a remarkable effect on cell cycle progression, blocking S-phase progression in DLD1 cells straight away while slowing S-phase progression and accumulated cells in the G2/M phase in HCT116 cells. Furthermore, we showed that compound 6b inhibited CDK1 dephosphorylation and H4K20 methylation in cells. The treatment with compound 6b induced DNA damage and triggered apoptosis. Our study identifies compound 6b as a potent CDC25 inhibitor that induces genome instability and kills cancer cells through an apoptotic pathway, deserving further investigation to fulfill its candidacy as an anti-CRC agent.

Definition of fatty acid transport protein-2 (FATP2) structure facilitates identification of small molecule inhibitors for the treatment of diabetic complications.

Diabetes is a major public health problem due to morbidity and mortality associated with end organ complications. Uptake of fatty acids by Fatty Acid Transport Protein-2 (FATP2) contributes to hyperglycemia, diabetic kidney and liver disease pathogenesis. Because FATP2 structure is unknown, a homology model was constructed, validated by AlphaFold2 prediction and site-directed mutagenesis, and then used to conduct a virtual drug discovery screen. In silico similarity searches to two low-micromolar IC50 FATP2 inhibitors, followed by docking and pharmacokinetics predictions, narrowed a diverse 800,000 compound library to 23 hits. These candidates were further evaluated for inhibition of FATP2-dependent fatty acid uptake and apoptosis in cells. Two compounds demonstrated nanomolar IC50, and were further characterized by molecular dynamic simulations. The results highlight the feasibility of combining a homology model with in silico and in vitro screening, to economically identify high affinity inhibitors of FATP2, as potential treatment for diabetes and its complications.

CuI-mediated synthesis of 1-aryl-5,6,7-trimethoxybenzimidazoles as potent antitubulin agents.

In situ CuI-mediated cyclization methodology helped yield benzimidazoles with different substitution manner, such as 1,2-diarylbenzimidazoles (4 and 5) and 1-arylbenzimidazoles (6-15). The result of structure-activity relationship (SAR) study confirmed the significance of the 5,6,7-trimethoxybenzimidazole moiety, and the representative derivatives (8-10) exhibited marked antiproliferative activity against A549, HCT-116, and PC-3 cells; in addition, they are able to inhibit the polymerization of tubulin. Among them, compound 10 inhibited the growth of A549, HCT-116, and PC-3 cells with a mean IC50 value of 0.07 µM, and its IC50 value of tubulin polymerization is 0.26 µM.

Correction: CuI-mediated synthesis of 1-aryl-5,6,7-trimethoxybenzimidazoles as potent antitubulin agents.

[This corrects the article DOI: 10.1039/D3RA01927F.].

In silico identification and biological evaluation of a selective MAP4K4 inhibitor against pancreatic cancer.

Inhibiting a specific target in cancer cells and reducing unwanted side effects has become a promising strategy in pancreatic cancer treatment. MAP4K4 is associated with pancreatic cancer development and correlates with poor clinical outcomes. By phosphorylating MKK4, proteins associated with cell apoptosis and survival are translated. Therefore, inhibiting MAP4K4 activity in pancreatic tumours is a new therapeutic strategy. Herein, we performed a structure-based virtual screening to identify MAP4K4 inhibitors and discovered the compound F389-0746 with a potent inhibition (IC50 120.7 nM). The results of kinase profiling revealed that F389-0746 was highly selective to MAP4K4 and less likely to cause side effects. Results of in vitro experiments showed that F389-0746 significantly suppressed cancer cell growth and viability. Results of in vivo experiments showed that F389-0746 displayed comparable tumour growth inhibition with the group treated with gemcitabine. These findings suggest that F389-0746 has promising potential to be further developed as a novel pancreatic cancer treatment.

Structure-based virtual screening and biological evaluation of novel small-molecule BTK inhibitors.

Bruton tyrosine kinase (BTK) is linked to multiple signalling pathways that regulate cellular survival, activation, and proliferation. A covalent BTK inhibitor has shown favourable outcomes for treating B cell malignant leukaemia. However, covalent inhibitors require a high reactive warhead that may contribute to unexpected toxicity, poor selectivity, or reduced effectiveness in solid tumours. Herein, we report the identification of a novel noncovalent BTK inhibitor. The binding interactions (i.e. interactions from known BTK inhibitors) for the BTK binding site were identified and incorporated into a structure-based virtual screening (SBVS). Top-rank compounds were selected and testing revealed a BTK inhibitor with >50% inhibition at 10 µM concentration. Examining analogues revealed further BTK inhibitors. When tested across solid tumour cell lines, one inhibitor showed favourable inhibitory activity, suggesting its potential for targeting BTK malignant tumours. This inhibitor could serve as a basis for developing an effective BTK inhibitor targeting solid cancers.

A novel HDAC1/2 inhibitor suppresses colorectal cancer through apoptosis induction and cell cycle regulation.

Colorectal cancer (CRC) is one of the leading causes of death around the world, and synthetic chemicals targeting specific proteins or various molecular pathways for tumor suppression, such as histone deacetylases (HADC) inhibitors, are under intensively studied. The target of HDAC involves in regulating critical cellular mechanisms and underpins the progression of anticancer therapy. However, little is known about the antitumor mechanisms of class I specific HDAC inhibitors in CRC. We structurally designed and synthesized benzamide-based compounds, examined their anticancer activity in several solid tumors, and identified compound 9 with high potential. Results from the in vitro enzyme and cell-based studies demonstrated that compound 9 as a selective HDAC1/2 inhibitor that possessed short-term and long-term suppression capacities against colorectal cancer cells. Investigation of molecular regulatory mechanisms of 9 in colorectal cancer cells by biological functional assays evidenced that treatment of compound 9 could activate apoptosis, induce cell cycle arrest, facilitate DNA damage process, and suppress cancer migration. A non-cancerous cell line and the in vivo zebrafish model were applied for safety evaluation. In summary, our results demonstrate that compound 9 is a promising lead drug worth further investigation for development of future cancer therapeutic agents.

Design, synthesis and biological evaluation of bisindole derivatives as anticancer agents against Tousled-like kinases.

This study presents the design, synthesis, and characterization of bisindole molecules as anti-cancer agents against Tousled-like kinases (TLKs). We show that compound 2 composed of an indirubin-3'-oxime group linked with a (N-methylpiperidin-2-yl)ethyl moiety possessed inhibitory activity toward both TLK1 and TLK2 in vitro and diminished the phosphorylation level of the downstream substrate anti-silencing function 1 (ASF1) in replicating cells. The treatment of compound 2 impaired DNA replication, slowed S-phase progression, and triggered DNA damage response in replicating cells. Structure optimization further discovered six derivatives exhibiting potent TLK inhibitory activity and revealed the importance of the tertiary amine-containing moiety of the side chain. Moreover, the derivatives 6, 17, 19, and 20 strongly suppressed the growth of triple-negative breast cancer MDA-MB-231 cells, non-small cell lung cancer A549 cells, and colorectal cancer HCT-116 cells, while normal lung fibroblast MRC5 and IMR90 cells showed a lower response to these compounds. Taken together, this study identifies tertiary amine-linked indirubin-3'-oximes as potent anticancer agents that inhibit TLK activity.

Effect of phenylurea hydroxamic acids on histone deacetylase and VEGFR-2.

A series of phenylurea hydroxamic acids incorporating pharmacophores of inhibitors of HDAC inhibitors and VEGFR-2 has been designed. Most of the compounds show antiproliferative activity comparable to that of Vorinostat and Sorafenib, and better EPC inhibitory activity. Enzymatic assays and Western blotting results indicated that compound 14 not only inhibits HDAC but also has slight VEGFR-2 inhibitory activity. A docking study revealed that the polar hydroxamic acid retains the interaction with HDAC through a zinc ion and also interacts with some residues of the active site of VEGFR-2. Despite 14 displaying a weaker VEGFR-2 activity, a possible route to develop potent HDAC/VEGFR-2 inhibitors is suggested.

Ring size changes in the development of class I HDAC inhibitors.

Five pathways involving different ring structures led to generation of fourteen thienylbenzamides (7-20) which display the structure-activity relationships of class I HDAC inhibitors. All the synthesised compounds inhibit HDAC1 and HDAC2 selectively over other isoforms and many inhibit DLD1 and HCT116 cells more effectively than a parent compound. Compounds 8 and 16 inhibit HCT116 cells by activation of the apoptosis pathway.

Effect of 3-subsitution of quinolinehydroxamic acids on selectivity of histone deacetylase isoforms.

A series of 3-subsituted quinolinehydroxamic acids has been synthesised and evaluated for their effect on human lung cancer cell line (A549), human colorectal cancer cell line (HCT116) and HDAC isoforms 1, 2, 6, and 8. The results indicated that substitution at C3 of quinoline is favoured for HDAC6 selectivity. Two compounds (25 and 26) were also found to be potent anti-proliferative compounds with IC50 values ranging from 1.29 to 2.13 µM against A549 and HCT116 cells. These compounds displayed remarkable selectivity for HDAC6 over other HDAC isoforms with nanomolar IC50 values. Western blot analysis revealed that compounds of this series activate apoptotic caspase pathway as indicated by cleavage of caspase 3, 8, and 9 and also increase phosphorylated H2AX thus inducing DNA double strand fragmentation in a concentration dependent manner. Flow cytometric analysis also displayed a dose dependent increase of cell population in sub G1 phase.

HDAC inhibitor protects chronic cerebral hypoperfusion and oxygen-glucose deprivation injuries via H3K14 and H4K5 acetylation-mediated BDNF expression.

Vascular dementia (VaD) is the second most common cause of dementia, but the treatment is still lacking. Although many studies have reported that histone deacetylase inhibitors (HDACis) confer protective effects against ischemic and hypoxic injuries, their role in VaD is still uncertain. Previous studies shown, one HDACi protected against cognitive decline in animals with chronic cerebral hypoperfusion (CCH). However, the underlying mechanisms remain elusive. In this study, we tested several 10,11-dihydro-5H-dibenzo[b,f]azepine hydroxamates, which act as HDACis in the CCH model (in vivo), and SH-SY5Y (neuroblastoma cells) with oxygen-glucose deprivation (OGD, in vitro). We identified a compound 13, which exhibited the best cell viability under OGD. The compound 13 could increase, in part, the protein levels of brain-derived neurotrophic factor (BDNF). It increased acetylation status on lysine 14 residue of histone 3 (H3K14) and lysine 5 of histone 4 (H4K5). We further clarified which promoters (I, II, III, IV or IX) could be affected by histone acetylation altered by compound 13. The results of chromatin immunoprecipitation and Q-PCR analysis indicate that an increase in H3K14 acetylation leads to an increase in the expression of BDNF promoter II, while an increase in H4K5 acetylation results in an increase in the activity of BDNF promoter II and III. Afterwards, these cause an increase in the expression of BDNF exon II, III and coding exon IX. In summary, the HDACi compound 13 may increase BDNF specific isoforms expression to rescue the ischemic and hypoxic injuries through changes of acetylation on histones.

Synthesis and biological evaluation of 2-quinolineacrylamides.

A series of C6-substituted N-hydroxy-2-quinolineacrylamides (3-15), with four types of bridging groups have been synthesized. Most of these compounds exhibit antiproliferative activity against A549 and HCT116 cells and Western blot analysis revealed that they are able to inhibit HDAC. Measurement of the HDAC isoform activity of ether-containing compounds showed that compound 9 has distinct HDAC6 selectivity, more than 300-fold over other isoforms. This paper describes the development of 6-aryloxy-N-hydroxy-2-quinolineacrylamides as potential HDAC6 inhibitors.

Protective effects of 10,11-dihydro-5H-dibenzo[b,f]azepine hydroxamates on vascular cognitive impairment.

A series of 10,11-dihydro-5H-dibenzo [b,f]azepine hydroxamates (4-15) were synthesized, behaving as histone deacetylase inhibitors, and examined for their influence on vascular cognitive impairment (VCI), which correlated with dementia. The results revealed that (E)-3-(4-(((3-(3-chloro-10,11-dihydro-5H-dibenzo [b,f]azepin-5-yl)propyl)amino)methyl)phenyl)-N-hydroxy-acrylamide (13) increases cerebral blood flow (CBF), attenuates cognitive impairment, and improves hippocampal atrophy in in vivo study. It is also able to increase the level of histone acetylation (H3K14 or H4K5) in the cortex and hippocampus of chronic cerebral hypoperfusion (CCH) mice; as a result, it could be a potential HDAC inhibitor for the treatment of vascular cognitive impairment.

Structure-guided design of anti-cancer ribonucleotide reductase inhibitors.

Ribonucleotide reductase (RR) catalyses the rate-limiting step of dNTP synthesis, establishing it as an important cancer target. While RR is traditionally inhibited by nucleoside-based antimetabolites, we recently discovered a naphthyl salicyl acyl hydrazone-based inhibitor (NSAH) that binds reversibly to the catalytic site (C-site). Here we report the synthesis and in vitro evaluation of 13 distinct compounds (TP1-13) with improved binding to hRR over NSAH (TP8), with lower KD's and more predicted residue interactions. Moreover, TP6 displayed the greatest growth inhibiting effect in the Panc1 pancreatic cancer cell line with an IC50 of 0.393 µM. This represents more than a 2-fold improvement over NSAH, making TP6 the most potent compound against pancreatic cancer emerging from the hydrazone inhibitors. NSAH was optimised by the addition of cyclic and polar groups replacing the naphthyl moiety, which occupies the phosphate-binding pocket in the C-site, establishing a new direction in inhibitor design.

Nitro-Group-Containing Drugs.

The nitro group is considered to be a versatile and unique functional group in medicinal chemistry. Despite a long history of use in therapeutics, the nitro group has toxicity issues and is often categorized as a structural alert or a toxicophore, and evidence related to drugs containing nitro groups is rather contradictory. In general, drugs containing nitro groups have been extensively associated with mutagenicity and genotoxicity. In this context, efforts toward the structure-mutagenicity or structure-genotoxicity relationships have been undertaken. The current Perspective covers various aspects of agents that contain nitro groups, their bioreductive activation mechanisms, their toxicities, and approaches to combat their toxicity issues. In addition, recent advances in the field of anticancer, antitubercular and antiparasitic agents containing nitro groups, along with a patent survey on hypoxia-activated prodrugs containing nitro groups, are also covered.

1-Arylsulfonyl indoline-benzamides as a new antitubulin agents, with inhibition of histone deacetylase.

We report structure-activity relationships of 1-arylsulfonyl indoline based benzamides. The benzamide (9) exhibits striking tubulin inhibition with an IC50 value of 1.1 µM, better than that of combretastain A-4 (3), and substantial antiproliferative activity against a variety of cancer cells, including MDR-positive cell lines with an IC50 value of 49 nM (KB), 79 nM (A549), 63 nM (MKN45), 64 nM (KB-VIN10), 43 nM (KB-S15), and 46 nM (KB-7D). Dual inhibitory potential of compound 9 was found as it demonstrated significant inhibitory potential against HDAC1, 2 and 6 in comparison to MS-275 (6). Some key interactions of 9 with the amino acid residues of the active site of tubulin and with amino acid residues of HDAC 1 isoform have been figured out by molecular modeling. Compound 9 also demonstrated significant in vivo efficacy in the human non-small cell lung cancer A549 xenograft model as well as B-cell lymphoma BJAB xenograft tumor model.