Inhibition of Hsp110-STAT3 interaction in endothelial cells alleviates vascular remodeling in hypoxic pulmonary arterial Hypertension model.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a progressive and devastating disease characterized by pulmonary vascular remodeling which is associated with the malignant phenotypes of pulmonary vascular cells. Recently, the effects of heat shock protein 110 (Hsp110) in human arterial smooth muscle cells were reported. However, the underlying roles and mechanisms of Hsp110 in human pulmonary arterial endothelial cells (HPAECs) that was disordered firstly at the early stage of PAH remain unknown. METHODS: In this research, the expression of Hsp110 in PAH human patients and rat models was investigated, and the Hsp110 localization was determined both in vivo and in vitro. The roles and mechanism of elevated Hsp110 in excessive cell proliferation and migration of HPAECs were assessed respectively exposed to hypoxia. Small molecule inhibitors targeting Hsp110-STAT3 interaction were screened via fluorescence polarization, anti-aggregation and western blot assays. Moreover, the effects of compound 6 on HPAECs abnormal phenotypes in vitro and pulmonary vascular remodeling of hypoxia-indued PAH rats in vivo by interrupting Hsp110-STAT3 interaction were evaluated. RESULTS: Our studies demonstrated that Hsp110 expression was increased in the serum of patients with PAH, as well as in the lungs and pulmonary arteries of PAH rats, when compared to their respective healthy subjects. Moreover, Hsp110 levels were significantly elevated in HPAECs under hypoxia and mediated its aberrant phenotypes. Furthermore, boosted Hsp110-STAT3 interaction resulted in abnormal proliferation and migration via elevating p-STAT3 and c-Myc in HPAECs. Notably, we successfully identified compound 6 as potent Hsp110-STAT3 interaction inhibitor, which effectively inhibited HPAECs proliferation and migration, and significantly ameliorated right heart hypertrophy and vascular remodeling of rats with PAH. CONCLUSIONS: Our studies suggest that elevated Hsp110 plays a vital role in HPAECs and inhibition of the Hsp110-STAT3 interaction is a novel strategy for improving vascular remodeling. In addition, compound 6 could serve as a promising lead compound for developing first-in-class drugs against PAH.

Interdomain interactions dictate the function of the Candida albicans Hsp110 protein Msi3.

Heat shock proteins of 110 kDa (Hsp110s), a unique class of molecular chaperones, are essential for maintaining protein homeostasis. Hsp110s exhibit a strong chaperone activity preventing protein aggregation (the "holdase" activity) and also function as the major nucleotide-exchange factor (NEF) for Hsp70 chaperones. Hsp110s contain two functional domains: a nucleotide-binding domain (NBD) and substrate-binding domain (SBD). ATP binding is essential for Hsp110 function and results in close contacts between the NBD and SBD. However, the molecular mechanism of this ATP-induced allosteric coupling remains poorly defined. In this study, we carried out biochemical analysis on Msi3, the sole Hsp110 in Candida albicans, to dissect the unique allosteric coupling of Hsp110s using three mutations affecting the domain-domain interface. All the mutations abolished both the in vivo and in vitro functions of Msi3. While the ATP-bound state was disrupted in all mutants, only mutation of the NBD-SBDß interfaces showed significant ATPase activity, suggesting that the full-length Hsp110s have an ATPase that is mainly suppressed by NBD-SBDß contacts. Moreover, the high-affinity ATP-binding unexpectedly appears to require these NBD-SBD contacts. Remarkably, the "holdase" activity was largely intact for all mutants tested while NEF activity was mostly compromised, although both activities strictly depended on the ATP-bound state, indicating different requirements for these two activities. Stable peptide substrate binding to Msi3 led to dissociation of the NBD-SBD contacts and compromised interactions with Hsp70. Taken together, our data demonstrate that the exceptionally strong NBD-SBD contacts in Hsp110s dictate the unique allosteric coupling and biochemical activities.

Identification of selective homeodomain interacting protein kinase 2 inhibitors, a potential treatment for renal fibrosis.

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.

Novel pyrazolo[3,4-b]pyridine derivatives: Synthesis, structure-activity relationship studies, and regulation of the AMPK/70S6K pathway.

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.

Design, synthesis, and biological evaluation of 1,2,4-oxadiazole-containing pyrazolo[3,4-b]pyridinones as a new series of AMPKɑ1ß1γ1 activators.

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 AMPKɑ1ß1γ1 activators. The in vitro biological assay demonstrated that compounds 12k (EC50 [AMPKα1γ1ß1] = 180 nM) and 13q (EC50 [AMPKα1γ1ß1] = 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-ß 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.

Fluorofenidone protects against acute kidney injury.

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 vivo and in vitro. Compared with the model group, treatment with AKFPD effectively ameliorated kidney damages. In order to elucidate the mechanisms, we discovered that AKFPD treatment notably alleviated generation of reactive oxygen species, reduced the phosphorylation levels of MAPKs (ERK1 and 2, JNKs, and p38), suppressed inflammatory response, inhibited apoptosis, and abated the expression of CP transporters (organic cation transporter 2 and copper transport protein 1) compared with the model group. Moreover, because renal ischemia reperfusion injury (IRI)-induced AKI and LPS-induced AKI are the major models representative of renal transplantation-correlated AKI and sepsis-related AKI, which are also the main causes of AKI, we have also proved the effectiveness of AKFPD on these models. In conclusion, these findings suggest that AKFPD is a potent drug for CP-, IRI-, and LPS-caused AKI and elucidate the underlying mechanism.-Jiang, Y., Quan, J., Chen, Y., Liao, X., Dai, Q., Lu, R., Yu, Y., Hu, G., Li, Q., Meng, J., Xie, Y., Peng, Z., Tao, L. Fluorofenidone protects against acute kidney injury.

Drug repurposing and rediscovery: Design, synthesis and preliminary biological evaluation of 1-arylamino-3-aryloxypropan-2-ols as anti-melanoma agents.

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 ß 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.

Targeted degradation of CD147 proteins in melanoma.

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.

Binding pocket-based design, synthesis and biological evaluation of novel selective BRD4-BD1 inhibitors.

Bromodomain-containing protein 4 (BRD4), consisting of two tandem bromodomains (BD1 and BD2), is key epigenetic regulator in fibrosis and cancer, which has been reported that BD1 and BD2 have distinct roles in post-translational modification. But there are few selective inhibitors toward those two domains. Herein, this study designed and synthesized a series of novel selective BRD4-BD1 inhibitors, using computer-aided drug design (CADD) approach focused on exploring the difference of the binding pockets of BD1 and BD2, and finding the His437 a crucial way to achieve BRD4-BD1 selectivity. Our results revealed that the compound 3u is a potent selective BRD4-BD1 inhibitor with IC50 values of 0.56 µM for BD1 but >100 µM for BD2. The compound exhibited a broad spectrum of anti-proliferative activity against several human cancer and fibroblastic cell lines, which might be related to its capability of reducing the expression of c-Myc and collagen I. Furthermore, it could induce apoptosis in A375 cells. To the contrary, the selective BD2 inhibitor, RVX-208, did not indicate any of these activities. Our findings highlight that the function of BRD4-BD1 might be predominant in fibrosis and cancer. And it is rational to further develop novel selective BRD4-BD1 inhibitors.

Dual inhibitors of RAF-MEK-ERK and PI3K-PDK1-AKT pathways: Design, synthesis and preliminary anticancer activity studies of 3-substituted-5-(phenylamino) indolone derivatives.

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 ±â€¯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.

The Protective Effect of Fluorofenidone against Cyclosporine A-Induced Nephrotoxicity.

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)-ß1, 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-ß1, 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-ß1, 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-ß1, 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.

Synthesis and structure-activity relationships of pyrazolo-[3,4-b]pyridine derivatives as adenosine 5'-monophosphate-activated protein kinase activators.

A series of pyrazolo[3,4-b]pyridine derivatives were designed, synthesized, and evaluated for their activation activity toward adenosine 5'-monophosphate-activated protein kinase (AMPK). According to the enzyme activity, the pyrazole N-H exposure and para substitution on the diphenyl group were proved to be essential for the activation potency. Compound 17f showed equal activation compared with A-769662. In the molecular modeling study, compound 17f exhibited important hydrogen bond interaction with Lys29, Asp88, and Arg83. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays on the NRK-49F cell line showed that potent enzyme activators could effectively inhibit cell proliferation, especially for 17f (EC50 [AMPKα1γ1ß1] = 0.42 µM, efficacy = 79%; IC50 [NRK-49F cell line] = 0.78 µM). These results might provide new insights to explore novel AMPK activators.

Discovery of 1-(4-((3-(4-methylpiperazin-1-yl)propyl)amino)benzyl)-5-(trifluoromethyl)pyridin-2(1H)-one, an orally active multi-target agent for the treatment of diabetic nephropathy.

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.

[Research progress of NADPH oxidases and their inhibitors].

NADPH oxidases (NOXs) are the key enzymes of redox signaling in vivo and also the main source of reactive oxygen species (ROS) in the body. ROS plays a role of double-edged sword. On the one hand, ROS, at the level of physiological amount, has the effect of immune defense and also acts as a second messenger involved in the regulation of cellular signaling pathways. On the other hand, excessive ROS can cause oxidative stress, leading to the disorder of cellular functions. Recently, studies showed that ROS plays an important role in acceleration of some pathological reactions such as inflammation, fibrosis and tumor formation. As a major source of ROS, NOX has become a popular target in treating oxidative stress, inflammation, fibrosis and tumor. Herein, the role of NOX in these pathological processes and the research progress of NOX inhibitors are reviewed.

[Progress in the study of new cancer target Cdk5 and its inhibitors].

Cyclin-dependent kinase-5(Cdk5) is a kind of Ser/Thr kinases in the signaling pathway, which regulates the neural development. The recent studies have confirmed that hyperactivation of Cdk5 is closely associated with the evolution, progression and apoptosis of tumors. The Cdk5 inhibitors have been extensively studied in the drug discovery against cancer. The structure features of these inhibitors and molecular mechanisms of their activities have provided clues for the drug development. In the second generation Cdk5 inhibitors, the ATP-binding pocket, a highly conserved site, has been targeted in the drug design in most cases. In addition, a growing number of peptides has been generated by targeting the protein/protein interfaces of Cdk5.

[Research progress of p70 ribosomal protein S6 kinase inhibitors].

p70 ribosomal protein S6 kinase (p70S6K), an important member of AGC family, is a kind of multifunctional Ser/Thr kinases, which plays an important role in mTOR signaling cascade. The p70 ribosomal protein S6 kinase is closely associated with diverse cellular processes such as protein synthesis, mRNA processing, glucose homeostasis, cell growth and apoptosis. Recent studies have highlighted the important role of S6K in cancer, which arose interests of scientific researchers for the design and discovery of anti-cancer agents. Herein, the mechanisms of S6K and available inhibitors are reviewed.

Synthesis, pharmacophores, and mechanism study of pyridin-2(1H)-one derivatives as regulators of translation initiation factor 3A.

Twenty-seven 1,5-disubstituted-pyridin-2(1H)-one derivatives were synthesized and evaluated for their anti-cancer and anti-fibrosis activity by A549 and NIH3T3 cell viability assays, respectively. To study the selectivity between the cancer and fibrosis cell lines, pharmacophore models (F1-F4) were built in advance for compounds with pyridin-2(1H)-one scaffold, which revealed the relationship between the occupation of the aromatic sub-site F4 and potent anti-cancer activity. The relationship between structure and anti-cancer activity for all target compounds is also reported herein: 1-Phenyl-5-((m-tolylamino)methyl)pyridine-2(1H)-one (22) displayed both potency and selectivity (IC50=0.13 mM) toward the A549 cell line through the inhibition of translation initiation, especially by eIF3a suppression, and can be treated as a lead for the design of novel eIF3a regulators and anti-lung cancer agents.

Discovery of Ureido-Based Apcin Analogues as Cdc20-specific Inhibitors against Cancer.

Cdc20 is a promising drug target that plays an important role in the mid-anaphase process of cellular mitosis, and Apcin is the only reported core structure of the Cdc20-specific inhibitor. Some potent Apcin derivatives were obtained in our previous research, and a structure-activity relationship was determined. In this study, we designed and synthesized a series of ureido-based Apcin derivatives. The proliferation-inhibition experiments on four cancer-cell lines showed that ureido skeleton could promote the anti-proliferation activity of purine-substituted compounds, whereas the ureido analogues with pyrimidine substitutes showed no significant improvement in the inhibitory effect compared with the original ones. Further tests confirmed that ureido-based compounds can enhance the binding affinity to Cdc20 by increasing the levels of Cdc20 downstream proteins. Compound 27 revealed a remarkably antitumor activity pattern against Hela (IC50 = 0.06 ± 0.02 µM) and potent binding affinity to Cdc20. Moreover, compound 20 induced caspase-dependent apoptosis and cell-cycle arrest at the G2/M phase, and compound 27 induced caspase-dependent apoptosis and promoted microtubule polymerization. Finally, a molecular-docking simulation was performed for compounds 20 and 27 to predict the potential ligand-protein interactions with the active sites of the Cdc20 proteins.

Pulmonary artery smooth muscle cell phenotypic switching: A key event in the early stage of pulmonary artery hypertension.

Pulmonary arterial hypertension (PAH) is a currently incurable pulmonary vascular disease. Since current research on PAH is mainly aimed at the middle and late stages of disease progression, no satisfactory results have been achieved. This has led researchers to focus on the early stages of PAH. This review highlights for the first time a key event in the early stages of PAH progression, namely, the occurrence of pulmonary arterial smooth muscle cell (PASMC) phenotypic switching. Summarizing the related reports of phenotypic switching provides new perspectives and directions for the early pathological progression and treatment strategies for PAH.