Discovery of novel pyrrolo[2,1-c][1,4]benzodiazepine-3,11-dione (PBD) derivatives as selective HDAC6 inhibitors for the efficient treatment of idiopathic pulmonary fibrosis (IPF) in vitro and in vivo.

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease characterized by a progressive fibrotic phenotype. Immunohistochemical studies on HDAC6 overexpression in IPF lung tissues confirmed that IPF is associated with aberrant HDAC6 activity. We herein developed a series of novel HDAC6 inhibitors that can be used as potential pharmacological tools for IPF treatment. The best-performing derivative H10 showed good selectivity for multiple isoforms of the HDAC family. The structural analysis and structure-activity relationship studies of H10 will contribute to optimizing the binding mode of the new molecules. The pharmacological mechanism of H10 to inhibit pulmonary fibrosis was validated, and its ability to inhibit the IPF phenotype was also demonstrated. Moreover, H10 showed satisfactory metabolic stability. The efficacy of H10 was also determined in a mouse model of bleomycin-induced pulmonary fibrosis. The results highlighted in this paper may provide a reference for the identification of new drug molecules for the treatment of IPF.

Discovery of novel tryptamine derivatives as GluN2B subunit-containing NMDA receptor antagonists via pharmacophore-merging strategy with orally available therapeutic effect of cerebral ischemia.

A series of tryptamine derivatives has been designed and synthesized as novel GluN2B subunit-containing NMDA receptor (GluN2B-NMDAR) antagonists, which could simultaneously manifest the receptor-ligand interactions of representative GluN2B-NMDAR antagonists ifenprodil (1) and EVT-101 (3). In the present study, the neuroprotective potential of these compounds was explored through chemical synthesis and pharmacological characterization. Compound Z25 with significantly better neuroprotective activity than the positive control drug (percentage of protection: 55.8 ± 0.6% vs. 41.0 ± 2.7%) was considered to be an effective antagonist of the human GluN2B-NMDA receptor. Judging from in vitro pharmacological profiling, Z25 could downregulate NMDA-induced increased intracellular Ca2+ concentration, and Z25 could also upregulate NMDA-induced decreased intracellular p-ERK 1/2 expression, which suggested that Z25 is an antagonist of the GluN2B-NMDA receptor. Furthermore, the in vitro preliminary evaluation of the drug-like properties of compound Z25 showed remarkable plasma stability. Based on in vivo pharmacokinetic and pharmacodynamic studies in C57 mice, compound Z25 exhibited a relatively short half-life and a low F value (3.12 ± 0.01%), while administration of Z25 substantially improved the cognitive performance of mice in a series of tests of cerebral ischemic injury. Overall, these results support the further development of compound Z25 as a potential lead compound to treat the cerebral ischemic injury by antagonizing GluN2B-NMDA receptor.

Novel pyrrolo[2,1-c][1,4]benzodiazepine-3,11-dione (PBD) derivatives as selective HDAC6 inhibitors to suppress tumor metastasis and invasion in vitro and in vivo.

Selective inhibition of histone deacetylase 6 (HDAC6) has been emerged as a promising approach to cancer treatment. As a pivotal strategy for drug discovery,molecular hybridization was introduced in this study and a series of pyrrolo[2,1-c][1,4] benzodiazepine-3,11-diones (PBDs) based hydroxamic acids was rationally designed and synthesizedas novel selective HDAC6 inhibitors. Preliminary in vitro enzyme inhibition assay and structure-activity relationship (SAR) discussion confirmed our design strategy and met the expectation. Several of the compounds showed high potent against HDAC6 enzyme in vitro, and compound A7 with a long aliphatic linker was revealed to have the similar activity as the positive control tubastatin A. Further in vitro characterization of A7 demonstrates the metastasis inhibitory potency in MDA-MB-231 cell line and western blotting showed that A7 could induce the upregulation of Ac-α-tubulin, but not induce the excessive acetylation of histone H3, which indicated that the compound had HDAC6 targeting effect in MDA-MB-231 cells. In vivo study revealed that compound A7 has satisfactory inhibitory effects onliver and lung metastasis of breast cancer in mice. Molecular docking released that A7 could fit well with the receptor and interact with some key residues, which lays a foundation for further structural modifications to elucidate the interaction mode between compounds and target protein. This pharmacological investigation workflow provided a reasonable and reference methodto examine the pharmacological effects of inhibiting HDAC6 with a single molecule, either in vitro or in vivo. All of these results suggested that A7 is a promising lead compound that could lead to the further development of novel selective HDAC6 inhibitors for the treatment of tumor metastasis.

Repurposing of cefpodoxime proxetil as potent neuroprotective agent through computational prediction and in vitro validation.

In recent reports, NR2B-NMDA receptor antagonists showed more research value because of its strong targeting ability and less side effects potential. In 2016, EVT-101 was reported to bind in an almost entirely new binding region of this target. Whether strikingly different binding modes can improve targeting and reduce side effects is worth studying. In our preliminary work, we explored the binding patterns of ifenprodil and EVT-101, found the key amino acids and summarized the pharmacophores, hoping to find such antagonists that target the two binding modes simultaneously. In this study, we developed a scalable virtual screening workflow in the FDA-approved drugs library to identify novel NR2B-NMDAR antagonists based on the combination of two pharmacophores. Cefpodoxime proxetil (5) was identified as the hit compound, and it was found for the first time that 5 might have neuroprotective activity as a NR2B-NMDAR antagonist. This result interested us to make further study, the ligand-receptor interactions modeled by molecular docking studies showed that the compound could perfectly merge both the pharmacophore characteristics of ifenprodil and EVT-101 at the binding cavity between the ATDs of GluN1 and GluN2B. The accuracy of molecular docking results and binding stability of ligand-receptor complexes were validated through 100 ns molecular dynamics simulation and binding free energy calculation. Afterwards, MTT assay (49.8%±0.1%, 5 µM) on NMDA injured SH-SY5Y cells and evidence of the effect on attenuating Ca2+ influx induced by NMDA were applied to validate the computational results, further investigation showed that 5 could suppress the NR2B upregulation induced by NMDA. [Formula: see text] Communicated by Ramaswamy H. Sarma.

Discovery of specific HDAC6 inhibitor with anti-metastatic effects in pancreatic cancer cells through virtual screening and biological evaluation.

Histone deacetylase 6 (HDAC6) has been demonstrated to play a major role in cell motility and aggresome formation, and HDAC6 inhibition is therefore considered as a promising epigenetic strategy for cancer treatment. At present, only a minority of compounds have been reported as HDAC6 inhibitors, so specific HDAC6 inhibitors with safety profile need to be discovered urgently. In this paper, HDAC6 inhibitors with diverse structures were used to generate the pharmacophore model by ligand-based method, which contained two hydrogen bond acceptors and two hydrophobic groups. A combined virtual screening based on pharmacophore model and molecular docking was adopted to screen potential HDAC6 inhibitors. Subsequently, the HDAC6 inhibitory activity of the hit compounds were evaluated using an in vitro enzyme binding inhibition assay. The experimental results illustrated that cefoperazone sodium had the strongest inhibitory effect on HDAC6 among the six screened compounds, and its IC50 value was 8.59 ± 1.06 µM. Cefoperazone sodium significantly catalyzed the hyperacetylation of α-tubulin but not histone H3, proving that cefoperazone sodium was a selective inhibitor of HDAC6. Since the expression of HDAC6 plays an important role in cancer metastasis, the effects of cefoperazone sodium on migration and invasion of human pancreatic cancer PANC-1 cells were further investigated by wound healing and transwell chamber assays. It was found that cefoperazone sodium could evidently inhibit the migration and invasion of PANC-1 cells. Furthermore, the binding pattern of inhibitor at the active site of the crystal structure was revealed by molecular docking, providing a reference value for the structural design and optimization of HDAC6 inhibitors. This study provides a systematic virtual screening approach for discovering HDAC6 active inhibitors, and by which the specific effect of cefoperazone sodium against HDAC6 was found, suggesting its potential application on cancer therapy.

Design, synthesis and biological evaluation of 1-benzyl-5-oxopyrrolidine-2-carboximidamide derivatives as novel neuroprotective agents.

A series of 1-benzyl-5-oxopyrrolidine-2-carboximidamide derivatives were designed and synthesized. Their protective activities against N-methyl-d-aspartic acid (NMDA)-induced cytotoxicity were investigated in vitro. All of the compounds exhibited neuroprotective activities, especially 12k, which showed higher potency than reference compound 1 (ifenprodil). Further investigation showed that 12k could attenuate Ca2+ influx and suppress the NR2B upregulation induced by NMDA. The docking results indicated that 12k could fit well into binding site of 1 in the NR2B-NMDA receptor. Additionally, 12k exhibited excellent metabolic stability. Furthermore, the results of behavioral tests showed that compound 12k could significantly improve learning and memory in vivo. These results suggested that 12k is a promising neuroprotective drug candidate and that the NR2B-NMDA receptor is a potential target of 12k.