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HIPK2 is a serine/threonine kinase, located in the nucleus, that was initially found to be able to phosphorylate p53 at Ser46 to promote apoptosis; it has been widely studied. It has been reported that HIPK2 can simultaneously regulate TGF-β/Smad3, Wnt/β-catenin, Notch and NF-κB pathways in the kidney to initiate inflammation and fibrosis, resulting in the development of chronic kidney disease (CKD). Therefore, inhibition of HIPK2 is strongly considered an effective method for the treatment of CKD. In brief, this review summarizes the progress of HIPK2 in CKD as well as the reported HIPK2 inhibitors and their role in different CKD models.

NADPH oxidase (NOX) is the only enzyme in the human body that can directly produce reactive oxygen species. Recent studies have shown that NADPH oxidase is a very potential target for the treatment of Diabetic Nephropathy (DN). Here, a series of quinoline (quinolinone) derivatives were designed and synthesized based on pharmacophore modelling. And the antiproliferative effect against NRK-49F cell-line was evaluated as their potential anti-DN activity. 19d, the most potent compounds with an effective cell proliferation inhibitory activity (IC50 = 5.25 uM), showed a promising inhibitory activity on NOXs (IC50 = 1.12 µM). The 3D-QSAR model based on the anti-proliferation activity of NRK-49F exhibited the good predictive ability. In conclusion, 19d may have therapeutic effects on diabetic nephropathy like other NOX inhibitors in clinical trials.

Utilizing fragment-based hybrid designing strategies, 24 N-benzyl pyridine-2-one containing derivatives were synthesized by successfully incorporating 6-(4H-1,2,4-triazol-3-yl) pyridin-2-amine of scaffold of ASK1 inhibitor (GS-444217). These newly synthesized compounds were screened in cell-free ASK1 and PDK1 kinase and cellular vitality assays. Among all compounds tested, both 21c and 21d displayed single digit potency of 9.13, 1.73 nM in inhibiting ASK1, and exhibited excellent enzyme inhibitory activity against PDK1 (the inhibition rates at 10 μM were 13.63% and 23.80%, respectively). Specifically, both compounds inhibited the TGF-β1 induced fibrotic response and blocked the up-regulated protein expression levels of ASK1-p38/JNK signaling pathways and possessed the potency in reducing PDK1/Akt phosphorylation. The results herein showed the potential lead characteristics of 21c or 21d as dual inhibitors ASK1/PDK1 kinases.

Fluorofenidone (AKF-PD) is a novel pyridone agent that reduces the deposition of extracellular matrix (ECM) in various models of renal fibrosis. However, there are no reports on the effect of AKF-PD in preventing fibrosis in the folic acid nephropathy model. Besides, the mechanisms of action of AKF-PD in preventing renal fibrosis are not fully understood. In the study, we observed that AKF-PD reduced folate-induced kidney injury, ameliorated the deterioration of renal function, and suppressed the deposition of ECM by decreasing the expression of collagen I, collagen III, transforming growth factor-β (TGF-β), fibronectin (FN), and alpha smooth muscle actin (α-SMA) in the folic acid nephropathy model. Additionally, AKF-PD suppressed the activation of the NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome to reduce the production of caspase-1 and IL-1β, and alleviated mitochondrial oxidative damage by promoting mitochondrial energy metabolism and reducing the expression of NADPH oxidase 4 (NOX4). The results of in vitro experiments demonstrated that AKF-PD suppressed NLRP3 inflammasome activation in activated peritoneal-derived macrophages (PDMs) and renal tubular epithelial cells (RTECs). AKF-PD increased the intracellular ATP content and decreased the expression of NOX4, while preventing the excessive production of mitochondrial reactive oxygen species (mtROS) in activated PDMs. In conclusion, this study demonstrated that AKF-PD inhibited renal fibrosis by suppressing the mtROS-NLRP3 pathway in the folic acid nephropathy model. These findings provide new evidence in support of the clinical use of AKF-PD in the treatment of diseases related to renal fibrosis.

A series of novel pyrazolo[3,4-b]pyridine derivatives were designed, synthesized, and biologically evaluated for anti-lung cancer activity. Structure-activity relationship and AutoGPA models were constructed based on the in vitro antiproliferative potency of the compounds against a human lung adenocarcinoma cell line (A549). Compound 9d exhibits improved potency for A549 cell growth inhibition (3.06 ± 0.05 μM) compared with A-769662 (45.29 ± 2.14 μM). Compound 9d can elevate the phosphorylation levels of adenosine monophosphate-activated protein kinase (AMPK) and its substrate acetyl-CoA carboxylase and reduce the level of phosphorylated ribosomal S6 kinase (p-70S6K) at 1 μM, which is comparable to the activity of A-769662 at 20 μM. 9d induced G2/M cell cycle arrest, which was rescued when co-incubated with "Compound C," a potent AMPK inhibitor. Taken together, compound 9d showed potential anti-lung cancer activity via inducing cell cycle arrest by regulation of the AMPK/70S6K pathway in A549 cells, which could provide a new lead for the discovery of anti-lung cancer agents.

Current pulmonary arterial hypertension (PAH) therapeutic strategies mainly focus on vascular relaxation with less emphasis on vascular remodeling, which results in poor prognosis. Hence, dual pathway regulators with vasodilation effect via soluble guanylate cyclase (sGC) stimulation and vascular remodeling regulation effect by AMP-activated protein kinase (AMPK) inhibition provide more advantages and potentialities. Herein, we designed and synthesized a series of novel pyrazolo[3,4-b] pyridine derivatives based on sGC stimulator and AMPK inhibitor scaffolds. In vitro, 2 exhibited moderate vasodilation activity and higher proliferation and migration suppressive effects compared to riociguat. In vivo, 2 significantly decreased right ventricular systolic pressure (RVSP), attenuated pulmonary artery medial thickness (PAMT), and right ventricular hypertrophy (RVH) in hypoxia-induced PAH rat models (i.g.). Given the unique advantages of significant vascular remodeling inhibition and moderate vascular relaxation based on the dual pathway regulation, we proposed 2 as a promising lead for anti-PAH drug discovery.

Objectives The in vitro evaluation of new drugs is an important step in the drug development pipeline. Orbitazine is a derivative of PAC-1 that has substituted the functional group homopiperazine ring with a piperazine ring. The purpose of this study was to assess the metabolic profile of orbitazine. Methods Metabolism was characterized in vitro by incubating liver microsomes with metabolize orbitazine or the classical metabolic enzyme substrates. High performance liquid chromatography (HPLC) and LC-MS/MS were used to identify the parent drugs and metabolites of orbitazine or metabolic enzyme substrates. Key findings There was no difference in metabolic stability or metabolites across different species. The metabolites included a debenzyl compound and several hydroxyl compounds, defined as M1(316), M2(440), M3(422), M4(422) and M5(422). We found that orbitazine was metabolized by CYP3A4, CYP2C9 and CYP2D6 in a human liver microsomes incubation system. Orbitazine had no significant inhibitory effect on CYP1A2, CYP2B6, CYP2C9, or CYP2C19 in human liver microsomes, but showed a dose-dependent inhibition of CYP2C8, CYP2D6 and CYP3A4; and there was no orbitazine-mediated induction of CYP1A2, CYP2B6, CYP3A4 or mRNA expression in hepatocytes. Conclusions This in vitro data on the metabolism of orbitazine may provide valuable information to support further clinical progression as a potential therapeutic molecule.

Homeodomain interacting protein kinase 2 (HIPK2) has emerged as a promising target for the discovery of anti-renal fibrosis drugs. Herein, to develop specific pharmacologic inhibitors of HIPK2, we designed and synthesized a series of compounds containing benzimidazole and pyrimidine scaffolds via fragment-based drug design strategy. Kinase assay was applied to evaluate the inhibitory activity of target compounds against HIPKs enzyme. The molecular docking study suggest the contribution of tyrosine residues beside the active sites of HIPK1-3 to the selectivity of active compounds. Compound 15q displayed good selectivity and potent inhibitory activity against HIPK2 compared to other two subtype enzymes. 15q could downregulate phosphorylated p53, the direct substrate of HIPK2, and decrease the fibrosis-related downstream of HIPK2, such as p-Smad3 and α-SMA in NRK-49F cells. 15q showed no effect on the cell apoptosis in fibrotic or cancer cell lines, suggesting little cancer risk of 15q. Notably, 15q displayed encouraging in vivo anti-fibrotic effects in the unilateral ureteral obstruction mouse model, which could be used as a potential lead for structural optimization and candidate for the development of selective HIPK2 inhibitors.

Pulmonary arterial hypertension (PAH) is a rapidly progressing disease with limited therapeutic options. Studies have elucidated the multifactorial pathological characteristics of PAH, indicating the complexity and difficulty of PAH treatment. Currently available treatments focus primarily on vasodilation rather than on vascular remodeling, although several drugs have been developed for the latter. This paradigm for management leads to PAH remaining an incurable disease; thus, there is an urgent need to explore new strategies for coping with this devastating disease. In this review, we discuss current strategies and options for PAH therapy and emerging novel therapeutic approaches in PAH treatment. This viewpoint suggests a shift in PAH treatment strategy from mono-activity to dual effects on vasoconstriction and vascular remodeling.

Cisplatin (CP) is one of the most effective chemotherapeutics in the treatment of human cancers. However, the beneficial effects of CP are limited by the toxic effects, especially nephrotoxicity. Fluorofenidone (AKFPD) is a promising multifunctional antifibrosis pyridinone drug discovered by our group. But there is no evidence of its protective effects against acute kidney injury (AKI). Therefore, we investigated the protective effects of AKFPD on CP-induced AKI

In first-line treatment of advanced renal cell carcinoma (aRCC), the KEYNOTE-426 study demonstrated a significant progression-free survival and overall survival for pembrolizumab plus axitinib in comparison with sunitinib. The objective of the current study was to evaluate the cost effectiveness of pembrolizumab plus axitinib versus sunitinib for previously untreated patients with aRCC in China. A Markov model was used to estimate the costs and health outcomes of treatment of aRCC with sunitinib or pembrolizumab plus axitinib. Univariable and probabilistic sensitivity analyses were performed to determine the robustness of the model outcomes. Additional subgroup analyses were also performed. The primary outputs of the model included the total cost, life-years (LYs), quality-adjusted LYs (QALYs), and incremental cost-effectiveness ratio (ICER). Pembrolizumab plus axitinib provided an additional 2.461 LYs (1.650 QALYs). The total cost per patient was US$178,725 for pembrolizumab plus axitinib and US$87,693 for sunitinib. The ICER for pembrolizumab plus axitinib was US$55,185/QALY versus sunitinib. Sensitivity analyses found the results to be most sensitive to pembrolizumab cost and overall hazard ratio. The results of subgroup analyses showed that the ICER remained greater than US$32,000/QALY across the all patient subgroups. Pembrolizumab plus axitinib is not likely to be cost effective versus sunitinib for patients with previously untreated aRCC at a threshold value of US$29,306/QALY.

Non-small-cell lung cancer (NSCLC) is an aggressive subtype of pulmonary carcinomas with high mortality. However, chemotherapy drug resistance and high recurrence rates hinder the curative effect of platinum-based first-line chemotherapy, which makes it urgent to develop new antitumor drugs for NSCLC. 9za, a new candidate drug synthesized by our research group, has been verified with potent antilung cancer activity in preliminary experiments. However, the underlying molecular mechanism of 9za remains largely vague. This work revealed that 9za could play important cytotoxic and proapoptotic roles in NSCLC cells. Moreover, 9za could induce autophagy and promote autophagy flux. Interestingly, the cytotoxic and proapoptotic roles were significantly dependent on 9za-induced cytoprotective autophagy. That is, the coadministration of 9za with an autophagy inhibitor such as chloroquine or 3-methyladenine exhibited increased cytotoxic and proapoptotic effects compared with 9za treatment alone. In addition, 9za exposure suppressed the phosphorylation of phosphoinositide-dependent protein kinase 1 (PDK1), protein kinase B (Akt), mammalian targets of rapamycin (mTOR), p70 S6 kinase, and 4E binding protein 1 by a dose-dependent way, manifesting that the Akt/mTOR axis was implicated in 9za-induced autophagy. In addition, the overexpression of PDK1 resulted in increased phosphorylation of PDK1 and Akt and blocking of 9za-mediated autophagy. These data showed that the PDK1/Akt/mTOR pathway was involved in 9za-induced autophagy. Hence, this work provides a theoretical basis for exploiting 9za as a new antilung cancer candidate drug and hints that the combination of 9za with an autophagy inhibitor is a feasible alternative approach for the therapy of NSCLC.

The dysfunction and mutual compensatory activation of RAF-MEK-ERK and PI3K-PDK1-AKT pathways have been demonstrated as the hallmarks in several primary and recurrent cancers. The strategy of concurrent blocking of these two pathways shows clinical merits on effective cancer therapy, such as combinatory treatments and dual-pathway inhibitors. Herein, we report a novel prototype of dual-pathway inhibitors by means of merging the core structural scaffolds of a MEK1 inhibitor and a PDK1 inhibitor. A library of 43 compounds that categorized into three series (Series I–III) was synthesized and tested for antitumor activity in lung cancer cells. The results from structure-activity relationship (SAR) analysis showed the following order of antitumor activity that 3-hydroxy-5-(phenylamino) indolone (Series III) > 3-alkenyl-5-(phenylamino) indolone (Series I) > 3-alkyl-5-(phenylamino) indolone (Series II). A lead compound 9za in Series III showed most potent antitumor activity with IC50 value of 1.8 ± 0.8 µM in A549 cells. Moreover, antitumor mechanism study demonstrated that 9za exerted significant apoptotic effect, and cellular signal pathway analysis revealed the potent blockage of phosphorylation levels of ERK and AKT in RAF-MEK-ERK and PI3K-PDK1-AKT pathways, respectively. The results reported here provide robust experimental basis for the discovery and optimization of dual pathway agents for anti-lung cancer therapy.

Background/Aims: Cyclosporine A (CsA) is an immunosuppressant drug that is used during organ transplants. However, its utility is limited by its nephrotoxic potential. This study aimed to investigate whether fluorofenidone (AKF-PD) could provide protection against CsA-induced nephrotoxicity. Methods: Eighty-five male Sprague-Dawley rats were divided into 5 groups: drug solvent, CsA, CsA with AKF-PD (250, 500 mg/kg/day), and CsA with pirfenidone (PFD, 250 mg/kg/day). Tubulointerstitial injury index, extracellular matrix (ECM) deposition, expression of type I and IV collagen, transforming growth factor (TGF)-β1, platelet-derived growth factor (PDGF), Fas ligand (FASL), cleaved-caspase-3, cleaved-poly(ADP-ribose) polymerase (PARP)-1, and the number of transferase-mediated nick end-labeling (TUNEL)-positive renal tubule cells were determined. In addition, levels of TGF-β1, FASL, cleaved-caspase-3, cleaved-PARP-1, and number of annexin V-positive cells were determined in rat proximal tubular epithelial cells (NRK-52E) treated with CsA (20 μmol/L), AKF-PD (400 μg/mL), PFD (400 μg/mL), and GW788388 (5 μmol/L). Results: AKF-PD (250, 500 mg/kg/day) significantly reduced tubulointerstitial injury, ECM deposition, expression of type I and IV collagen, TGF-β1, PDGF, FASL, cleaved-caspase-3, cleaved-PARP-1, and number of TUNEL-positive renal tubule cells in the CsA-treated kidneys. In addition, AKF-PD (400 μg/mL) significantly decreased TGF-β1, FASL, cleaved-caspase-3, and PARP-1 expression in NRK-52E cells and further reduced the number of annexin V-positive cells. Conclusion: AKF-PD protect kidney from fibrosis and apoptosis in CsA-induced kidney injury.

Background A novel dual MEK/PDK1 inhibitor named 9za has been synthesized by our research team. Preliminary study showed that 9za possessed potent cytotoxicity and proapoptosis in non-small cell lung cancer (NSCLC) cells. Nevertheless, the precise underlying mechanism is vague. Methods In this work, we adopted the MTT assay, the Cell Cycle Detection Kit, and the JC-1 staining assay to detect the cell viability, the cell cycle distribution and the mitochondrial membrane potential (MMP), respectively. Cell apoptosis was measured by the morphology observation under a light microscope, Annexin V-FITC/propidium iodide (PI) apoptosis detection and the colorimetric TUNEL assay. Western blot was used to monitor the cell cycle-, apoptosis-related proteins and relevant proteins involved in the signaling pathways. Results The MTT assay demonstrated that 9za sharply decreased the viability of NSCLC cells. Cell cycle analysis revealed that low concentrations of 9za arrested the cell cycle at the G0/G1 phase , which was further confirmed by the decreased levels of Cyclin D1, cyclin-dependent kinase 4 (CDK4) and cyclin-dependent kinase 6 (CDK6). Additionally, morphological observations, Annexin V-FITC/propidium iodide (PI) apoptosis analysis and TUNEL assays indicated that high concentrations of 9za induced cell apoptosis. Furthermore, the JC-1 staining assay revealed that the mitochondrial membrane potential was downregulated following 9za exposure. Western blot also showed that 9za markedly decreased the expression levels of total Bcl-2, Cytochrome C in the mitochondria and BCL2 associated X (BAX) in the cytoplasm. However, the levels of BAX in the mitochondria, Cytochrome C in the cytoplasm, active caspase-9, active caspase-3 and cleaved–PARP showed the opposite changes. Moreover, the dose-dependent decreased phosphorylation levels of PDK1, protein kinase B (Akt), MEK and extracellular signal regulated kinase 1/2 (ERK1/2) after 9za treatment verified that 9za was indeed a dual MEK/PDK1 inhibitor, as we expected. Compared with a single MEK inhibitor PD0325901 or a single PDK1 inhibitor BX517, the dual MEK/PDK1 inhibitor 9za could strengthen the cytotoxic and proapoptotic effect, indicating that the double blocking of the MEK and PDK1 signaling pathways plays stronger cell growth inhibition and apoptosis induction roles than the single blocking of the MEK or PDK1 signaling pathway in NSCLC cells. Our work elucidated the molecular mechanisms for 9za as a novel drug candidate against NSCLC.

Current pulmonary arterial hypertension (PAH) therapeutic strategies mainly focus on vascular relaxation with less emphasis on vascular remodeling, which results in poor prognosis. Hence, dual pathway regulators with vasodilation effect via soluble guanylate cyclase (sGC) stimulation and vascular remodeling regulation effect by AMP-activated protein kinase (AMPK) inhibition provide more advantages and potentialities. Herein, we designed and synthesized a series of novel pyrazolo[3,4

CD147 is a transmembrane glycoprotein and a member of immunoglobulin superfamily, is strongly expressed in melanoma cells. CD147 has a pivotal role in tumor development. Therefore, it is a potential drug target for melanoma. In this article, we report the discovery of the first CD147 protein proteolysis targeting chimeras (PROTACs) derived from the natural product pseudolaric acid B (PAB). The representative compound 6a effectively induced degradation of CD147 and inhibited melanoma cells in vitro and in vivo. 6a could be used as the novel type of anticancer agent or as a part of the molecular biology research toolkit used in the gain-of-function study of the dynamic roles of CD147 in cancer networks.

Aim We explored whether Fluorofenidone reduced interleukin-1β (IL-1β) production by interacting with NLRP3 inflammasome in unilateral ureteral obstruction (UUO). Methods Ureteral obstruction rats were treated with Fluorofenidone (500 mg/kg per day) for 3, 7 days. Morphologic analysis and leukocytes infiltration were assessed in ligated kidneys. Furthermore, plasmids of NLRP3, ASC, pro-Caspase-1, pro-IL-1β were co-transfected into 293 T cells, and then treated with Fluorofenidone (2 mM). The expression of NLRP3, ASC, pro-caspase-1, cleavage caspase-1, pro-IL-1β and cleavage IL-1β were measured by Western blot or real-time PCR in vivo and in vitro. Moreover the interaction of NLRP3 inflammasome-assembly was detected by co-immunoprecipitation and confocal immunofluorescence. Results Fluorofenidone treatment significantly attenuated renal fibrosis and leukocytes infiltration in UUO model. Fluorofenidone had no effect on the expression of pro-IL-1β. Interestingly, Fluorofenidone inhibited the activation of NLRP3 inflammasome, downregulated Caspase-1 levels and thereby decreased the cleavage of pro-IL-1β into IL-1β in vivo and in vitro. Fluorofenidone treatment distinctively weakened the interaction between NLRP3 and ASC, as well as ASC and pro-Caspase-1 in vivo. However, Fluorofenidone treatment only significantly weakened the interaction between ASC and pro-Caspase-1 in co-transfected 293 T cells. Conclusion Fluorofenidone serves as a novel anti-inflammatory agent that attenuates IL-1β production in UUO model by interacting with NLRP3 inflammasome.

Oxidative stress, inflammation and fibrosis can cause irreversible damage on cell structure and function of kidney and are key pathological factors in Diabetic Nephropathy (DN). Therefore, multi-target agents are urgently need for the clinical treatment of DN. Using Pirfenidone as a lead compound and based on the previous research, two novel series (5-trifluoromethyl)-2(1H)-pyridone analogs were designed and synthesized. SAR of (5-trifluoromethyl)-2(1H)-pyridone derivatives containing nitrogen heterocyclic ring have been established for in vitro potency. In addition, compound 8, a novel agent that act on multiple targets of anti-DN with IC50 of 90μM in NIH3T3 cell lines, t1/2 of 4.89±1.33h in male rats and LD50>2000mg/kg in mice, has been advanced to preclinical studies as an oral treatment for DN.

Homeodomain-Interacting Protein Kinase 2 (HIPK2) is a critical regulator of multiple pathways upstream of Chronic Kidney Disease (CKD). With the purpose of seeking HIPK2 inhibitors with novel scaffolds, a multistep virtual screening from the SPECS library was conducted. The screening process generally included similarity screening, random forest modeling, docking and ADMET prediction. Ten compounds were finally purchased and evaluated in biological activity test. The activity results demonstrated that the compound 1 (SPECS ID AG-401/37408043) can inhibit enzymatic activity and cell viability of high-glucose-induced rat normal renal interstitial fibroblast cells (NRK49F) with moderate activity. Its binding modes with the HIPK2 ATP binding site were analyzed. Overall, the compound 1 with a rather new scaffold might be used as the lead for the future structural optimization to seek potent HIPK2 inhibitors.

A series of 3-methylene-2-oxoindoline-5-carboxamide derivatives were synthesized in appreciable yield by using 4-aminobenzoic acid as a starting material. The preliminary biological test results showed that most compounds displayed potent inhibitory activity against proliferation of human lung adenocarcinoma epithelial cell line (A549) in MTT assay. Compound 6l displayed the highest potency (IC50 = 3.0 μM). The western blot analysis demonstrated a correlation between anti-proliferative activity of active compounds and blockade of the phosphorylation of extracellular signal-regulated kinases (ERK1/2). The docking study also provides new insights into further optimization of 3-methylene-2-oxoindoline-5-carboxamide derivatives for the discovery of more potent RAF/MEK/ERK pathway regulators as anti-lung cancer agents.

Background: Lung carcinoma is the leading cause of malignant tumor related mortality in China in recent decades, and the development of new and effective therapies for patients with advanced lung carcinoma is needed. We recently found that fluorofenidone (FD), a newly developed pyridine compound, reduced the activation of Stat3 (Signal transducer and activator of transcription 3) in fibroblasts. Stat3 plays a crucial role in the development of lung cancer and may represent a new therapeutic target. In this study, we examined the effect of FD on human lung adenocarcinoma cells in vivo and in vitro. Methods: The effect of FD on the growth of lung cancer cells was measured with a CCK-8 assay, colony formation assay and xenograft tumor model. A flow cytometry analysis was performed to study cell cycle arrest and apoptosis. Western blotting and immunohistochemistry were used to observe the expression of Stat3. Changes in the expression of RNA induced by FD were assessed using gene chip and real-time RT-PCR assays. Results: In vitro, FD inhibited the growth of lung adenocarcinoma A549 and SPC-A1 cells in a dose-dependent manner. After treatment with FD, the A549 and SPC-A1 cells were arrested in the G1 phase, and apoptosis was induced. In vivo, this compound significantly inhibited the growth of tumors that were subcutaneously implanted in mice. Moreover, FD decreased Stat3 activity in lung cancer cells and xenograft tumor tissue, and microarray chip results showed that FD altered the gene expression profile of lung cancer cells. Specifically, NUPR1, which plays a significant role in cancer development, was down-regulated by FD in lung cancer cells. Conclusion: Our study supports the clinical evaluation of FD as a potential lung adenocarcinoma therapy.

Mefunidone is a new pyridone agent that attenuates renal tubulointerstitial fibrosis. However, the signaling pathways involved in the effect of mefunidone on renal tubulointerstitial fibrosis have not been well explained. Inflammatory response initiates and promotes renal tubulointerstitial fibrosis, and the inhibitor of nuclear factor kappa-B kinase beta (IKKβ) is a master regulator of inflammation. This study is determined to clarify the influence of mefunidone on renal inflammation and the phosphorylation of IKKβ. Experimental renal tubulointerstitial fibrosis was induced by unilateral ureteral obstruction (UUO) for 3, 7 and 14days in sprague dawley rat. Treatment with mefunidone was conducted simultaneously. Obstructed kidneys were harvested for the assessment. Our results showed that treatment with mefunidone ameliorated renal inflammatory injury, renal tubular lesions and interstitial fibrosis. Further studies indicated that treatment with mefunidone mitigated the expressions of tumor necrosis factorα (TNFα) and interleukin-1β (IL-1β) in the kidney. The phosphorylation of IKKβ and inhibitor of kappa-B (IκB) and the expression of NOD-like receptor family, pyrin domain containing 3 (NALP3) were also reduced in vivo after treatment with mefunidone. In vitro, peritoneal macrophages were incubated with necrotic cells or lipopolysaccharide in the presence or absence of mefunidone. Mefunidone markedly decreased necrotic cell or LPS induced IL-1β production and LPS induced TNFα production in primary peritoneal macrophages. Furthermore, mefunidone significantly inhibited the phosphorylation of IKKβ/IκB and nuclear transition of NF-κB p65 in peritoneal macrophages stimulated by necrotic cell or lipopolysaccharide. In conclusion, mefunidone serves as a novel anti-inflammatory agent that attenuates UUO-induced renal interstitial inflammation and fibrosis, possibly through suppressing IKKβ phosphorylation.

Soluble Guanylate Cyclase (sGC) is the intracellular receptor of Nitric Oxide (NO). The activation of sGC results in the conversion of Guanosine Triphosphate (GTP) to the secondary messenger cyclic Guanosine Monophosphate (cGMP). cGMP modulates a series of downstream cascades through activating a variety of effectors, such as Phosphodiesterase (PDE), Protein Kinase G (PKG) and Cyclic Nucleotide-Gated Ion Channels (CNG). NO-sGC-cGMP pathway plays significant roles in various physiological processes, including platelet aggregation, smooth muscle relaxation and neurotransmitter delivery. With the approval of an sGC stimulator Riociguat for the treatment of Pulmonary Arterial Hypertension (PAH), the enthusiasm in the discovery of sGC modulators continues for broad clinical applications. Notably, through activating the NO-sGC-cGMP pathway, sGC stimulator and activator potentiate for the treatment of various diseases, such as PAH, Heart Failure (HF), Diabetic Nephropathy (DN), Systemic Sclerosis (SS), fibrosis as well as other diseases including Sickle Cell Disease (SCD) and Central Nervous System (CNS) disease. Here, we review the preclinical and clinical studies of sGC stimulator and activator in recent years and prospect for the development of sGC modulators in the near future.

Adenosine monophosphate-activated protein kinase (AMPK) plays a key role in maintaining whole-body homeostasis and has been regarded as a therapeutic target for the treatment of diabetic nephropathy (DN). Herein, a series of 1,2,4-oxadiazole-containing pyrazolo[3,4-b]pyridinone derivatives is reported as AMPKɑ1β1γ1 activators. The in vitro biological assay demonstrated that compounds 12k (EC50 [AMPKα1γ1β1] = 180 nM) and 13q (EC50 [AMPKα1γ1β1] = 2 nM) displayed significant enzyme activation. Mechanism studies indicated that both compounds reduced the levels of reactive oxygen species in a rat kidney fibroblast cell line (NRK-49F) stimulated by transforming growth factor-β and induced early apoptosis of NRK-49F cells at 10 μM. Molecular docking studies suggested that 13q exhibited critical hydrogen-bond interactions with the critical amino acid residues Lys29, Lys31, Asn111, and Asp88 at the binding site of the AMPK protein. These results enrich the structure pool of AMPK activators and provide novel lead compounds for the subsequent development of compounds with a promising therapeutic potential against DN.

Twenty-eight novel 1,5-disubstituted-2(1H)-pyridone derivatives were designed and synthesized for discovering more potent anti-lung cancer agents combined with anti-fibrotic profiles. The in vitro antiproliferative activities of the derivatives against A549 and NIH3T3 cell lines were tested by MTT assays. The majority of the tested analogues exhibited equivalent or an improved anti-lung cancer activity. Prominently, compound 4l displayed the best potency and selectivity toward A549 with an IC50 value of 20 μM, nearly comparable to the positive control cisplatin (IC50 = 10 μM) and even superior to the lead compound 22 (IC50 = 130 μM). Simultaneously, compound 4l showed significant inhibitory activity against NIH3T3 (IC50 = 55 μM), which may contribute to hindering the proliferation of lung cancer cells fundamentally. What is more, the 3D-QSAR models established on the activity data may provide new insights into the design of novel 2(1H)-pyridone derivatives and lay a theoretical foundation for further studies of promising anti-lung cancer activity with the maintenance of anti-fibrotic effect.

Interleukin (IL)-1β plays an important role in the pathogenesis of idiopathic pulmonary fibrosis. The production of IL-1β is dependent upon caspase-1-containing multiprotein complexes called inflammasomes and IL-1R1/MyD88/NF-κB pathway. In this study, we explored whether a potential anti-fibrotic agent fluorofenidone (FD) exerts its anti-inflammatory and anti-fibrotic effects through suppressing activation of NACHT, LRR and PYD domains-containing protein 3 (NALP3) inflammasome and the IL-1β/IL-1R1/MyD88/NF-κB pathway in vivo and in vitro. Male C57BL/6J mice were intratracheally injected with Bleomycin (BLM) or saline. Fluorofenidone was administered throughout the course of the experiment. Lung tissue sections were stained with haemotoxylin and eosin and Masson's trichrome. Cytokines were measured by ELISA, and α-smooth muscle actin (α-SMA), fibronectin, collagen I, caspase-1, IL-1R1, MyD88 were measured by Western blot and/or RT-PCR. The human actue monocytic leukaemia cell line (THP-1) were incubated with monosodium urate (MSU), with or without FD pre-treatment. The expression of caspase-1, IL-1β, NALP3, apoptosis-associated speck-like protein containing (ASC) and pro-caspase-1 were measured by Western blot, the reactive oxygen species (ROS) generation was detected using the Flow Cytometry, and the interaction of NALP3 inflammasome-associated molecules were measured by Co-immunoprecipitation. RLE-6TN (rat lung epithelial-T-antigen negative) cells were incubated with IL-1β, with or without FD pre-treatment. The expression of nuclear protein p65 was measured by Western blot. Results showed that FD markedly reduced the expressions of IL-1β, IL-6, monocyte chemotactic protein-1 (MCP-1), myeloperoxidase (MPO), α-SMA, fibronectin, collagen I, caspase-1, IL-1R1 and MyD88 in mice lung tissues. And FD inhibited MSU-induced the accumulation of ROS, blocked the interaction of NALP3 inflammasome-associated molecules, decreased the level of caspase-1 and IL-1β in THP-1 cells. Besides, FD inhibited IL-1β-induced the expression of nuclear protein p65. This study demonstrated that FD, attenuates BLM-induced pulmonary inflammation and fibrosis in mice via inhibiting the activation of NALP3 inflammasome and the IL-1β/IL-1R1/MyD88/ NF-κB pathway.

The overproduction of cortisol is associated with many severe and life-threatening diseases, such as Cushing’s syndrome (CS) and chronic wound healing. 11β-Hydroxylase (CYP11B1) is considered as an attractive target for treating these diseases, since it is a key enzyme responsible for the last step in cortisol biosynthesis. Nowadays, medical therapy has become increasingly important for CS patients, especially for those who are in need of surgery or suffer from surgery failure and those in early phases of radiation therapy. In clinic, steroidogenesis blockers including CYP11B1 inhibitors are utilized most frequently. Nevertheless, drugs that inhibit CYP11B1 are inevitable with side effects due to lack of selectivity over other steroidogenesis enzymes. Recent advances in the development of novel CYP11B1 inhibitors might overcome these limitations. In addition, the beneficial effects of down-regulation of cortisol levels to wound closure have been recently disclosed and have stimulated topical application of CYP11B1 inhibitors as a novel therapeutic strategy for curing chronic wounds. Herein, we provide a review of the current CYP11B1 inhibitors in clinic combating CS and the latest development of novel CYP11B1 inhibitors for treating CS and chronic wounds.

Malignant melanoma (MM) presents as the highest morbidity and mortality type in skin cancer. Herein, inspired by the previously reported anti-melanoma effect of propranolol, a widely applied β adrenergic receptor antagonist as cardiovascular drug, we set out to exploit its potential as anti-melanoma therapy based on the drug repurposing strategy. Structural optimization of propranolol yielded 5m, which exhibits dramatically improved potency on human melanoma cell growth (1.98–3.70 μM), compared to propranolol (59.5–75.8 μM). Further investigation demonstrated that 5m could inhibit colony formation of melanoma cell line (completely abolished at 2 μM for 5m, partially inhibited at 50 μM for propranolol), induce cell apoptosis and cell cycle arrest in the G2/M phase (both observed at 1 μM). Preliminary mechanism study indicated that 5m could disrupt the cellular microtubule network, which suggested tubulin as a potential target. Docking study provided a structural insight into the interaction between 5m and tubulin. In summary, our study presents a drug repurposing case that redirects a cardiovascular agent to an anti-melanoma agent.

The aim of the present research was to study the therapeutic impacts of fluorofenidone (AKF-PD) on pig serum (PS)-induced liver fibrosis in rats and the complex molecular mechanisms of its effects on hepatic stellate cells (HSCs). Wistar rats were randomly divided into normal control, PS and PS/AKF-PD treatment groups. The activated human HSC LX-2 cell line was also treated with AKF-PD. The expression of collagen I and III, and α-smooth muscle actin (α-SMA) was determined by immunohistochemical staining and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Western blotting and/or RT-qPCR analyses were used to determine the expression of transforming growth factor (TGF)-β1, α-SMA, collagen I, mothers against decapentaplegic homolog (Smad)-3, extracellular signal-regulated kinase (ERK)1/2, p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun N-terminal kinase (JNK). AKF-PD attenuated the degree of hepatic fibrosis and liver injury in vivo, which was associated with the downregulation of collagen I and III, and α-SMA at the mRNA and protein levels. In vitro, AKF-PD treatment significantly reduced the TGF-β1-induced activation of HSCs, as determined by the reduction in collagen I and α-SMA protein expression. The TGF-β1-induced upregulation of the phosphorylation of Smad 3, ERK1/2, p38 and JNK was attenuated by AKF-PD treatment. These findings suggested that AKF-PD attenuated the progression of hepatic fibrosis by suppressing HSCs activation via the TGF-β1/Smad and MAPK signaling pathways, and therefore that AKF-PD may be suitable for use as a novel therapeutic agent against liver fibrosis.

Interleukin-1 beta (IL-1β) is a potent pro-inflammatory and pro-fibrotic cytokine that plays an important role in renal fibrosis. Fluorofenidone (AKF-PD) is a novel pyridone agent that exerts a strong renal anti-fibrotic effect. We previously found that administration of AKF-PD could significantly attenuate IL-1β production in vitro and in vivo. However, the underlying mechanism is not fully understood. Here we show that AKF-PD has no effect on the expression of pro-IL-1β in activated mouse macrophages in vitro. Instead, AKF-PD inhibits the inflammasome, lowering caspase-1 levels and thereby decreasing cleavage of pro-IL-1β into IL-1β. AKF-PD was found to block inflammasome activity induced by various signals, including ATP, alum crystals, and Salmonella typhimurium. These results provide a novel mechanistic insight into how AKF-PD exerts its anti-inflammatory and anti-fibrotic activities, and suggest that AKF-PD might block IL-1β production via suppression of inflammasomes in renal fibrosis. In addition, the results suggest that AKF-PD may be of therapeutic potential in other inflammasome-related diseases.