SiRNA-HIF-1α delivered by attenuated Salmonella enhances the efficacy of Lenvatinib against hepatocellular carcinoma.

The treatment of hepatocellular carcinoma (HCC) remains a major challenge in the medical field. Lenvatinib, a multi-target tyrosine kinase inhibitor, has demonstrated anti-HCC effects by targeting and inhibiting pathways such as vascular endothelial growth factor receptor 1-3 (VEGFR1-3). However, the therapeutic efficacy of Lenvatinib is subject to various influences, with the hypoxic microenvironment of the tumor being a pivotal factor. Consequently, altering the hypoxic milieu of the tumor emerges as a viable strategy to augment the efficacy of Lenvatinib. Hypoxia-inducible factor-1α (HIF-1α), synthesized by tumor cells in response to oxygen-deprived conditions, regulates the expression of resistance genes, promotes tumor angiogenesis and cell proliferation, enhances tumor cell invasion, and confers resistance to radiotherapy and chemotherapy. Thus, we constructed a self-designed siRNA targeting HIF-1α to suppress its expression and improve the efficacy of Lenvatinib in treating HCC. The therapeutic efficacy of siRNA-HIF-1α in combination with Lenvatinib on HCC were evaluated through in vivo and in vitro experiments. The results showed that the recombinant Salmonella delivering siRNA-HIF-1α in combination with Lenvatinib effectively inhibited tumor growth and prolonged the survival of tumor-bearing mice. This treatment approach reduced cell proliferation and angiogenesis in HCC tissues while promoting tumor cell apoptosis. Additionally, this combined therapy significantly increased the infiltration of T lymphocytes and M1 macrophages within the tumor microenvironment, as well as elevated the proportion of immune cells in the spleen, thereby potentiating the host's immune response against the tumor.

Morusin suppresses the stemness characteristics of gastric cancer cells induced by hypoxic microenvironment through inhibition of HIF-1α accumulation.

Gastric cancer (GC) is a common, life-threatening malignancy that contributes to the global burden of cancer-related mortality, as conventional therapeutic modalities show limited effects on GC. Hence, it is critical to develop novel agents for GC therapy. Morusin, a typical prenylated flavonoid, possesses antitumor effects against various cancers. The present study aimed to demonstrate the inhibitory effect and mechanism of morusin on the stemness characteristics of human GC in vitro under hypoxia and to explore the potential molecular mechanisms. The effects of morusin on cell proliferation and cancer stem cell-like properties of the human GC cell lines SNU-1 and AGS were assessed by MTT assay, colony formation test, qRT-PCR, flow cytometry analysis, and sphere formation test under hypoxia or normoxia condition through in vitro assays. The potential molecular mechanisms underlying the effects of morusin on the stem-cell-like properties of human GC cells in vitro were investigated by qRT-PCR, western blotting assay, and immunofluorescence assay by evaluating the nuclear translocation and expression level of hypoxia-inducible factor-1α (HIF-1α). The results showed that morusin exerted growth inhibitory effects on SNU-1 and AGS cells under hypoxia in vitro. Moreover, the proportions of CD44+/CD24- cells and the sphere formation ability of SNU-1 and AGS reduced in a dose-dependent manner following morusin treatment. The expression levels of stem cell-related genes, namely Nanog, OCT4, SOX2, and HIF-1α, gradually decreased, and the nuclear translocation of the HIF-1α protein was apparently attenuated. HIF-1α overexpression partially reversed the abovementioned effects of morusin. Taken together, morusin could restrain stemness characteristics of GC cells by inhibiting HIF-1α accumulation and nuclear translocation and could serve as a promising compound for GC treatment.

An AMPK agonist suppresses the progress of colorectal cancer by regulating the polarization of TAM to M1 through inhibition of HIF-1α and mTOR signal pathway.

OBJECTIVE: This study aimed to evaluate the impact of an adenosine monophosphate-activated protein kinase (AMPK) agonist, metformin (MET), on the antitumor effects of macrophages and to determine the underlying mechanism involved in the process. MATERIALS AND METHODS: M0 macrophages were derived from phorbol-12-myristate-13-acetate-stimulated THP-1 cells. RESULTS: The levels of tumor necrosis factor-alpha (TNF-α) and human leukocyte antigen-DR (HLA-DR) were decreased in macrophages incubated with HCT116 cells, whereas those of arginase-1 (Arg-1), CD163, and CD206 were elevated; these effects were reversed by MET. The transfection of small interfering (si) RNA abrogated the influence of MET on the expression of the M1/M2 macrophage biomarkers. MET significantly suppressed the proliferation and migration abilities of HCT116 cells incubated with M0 macrophages; these actions were reversed by siRNA transfection against AMPK. The hypoxia-inducible factor 1-alpha (HIF-1α), phosphorylated protein kinase B (p-AKT), and phosphorylated mammalian target of rapamycin (p-mTOR) levels were reduced by the introduction of MET and promoted by siRNA transfection against AMPK. In addition, the levels of HIF-1α, p-AKT, and p-mTOR suppressed by MET were markedly increased following the transfection of siRNA against AMPK. CONCLUSION: These findings indicate that MET can repress the progression of colorectal cancer by transforming tumor-associated macrophages to the M1phenotype via inhibition of the HIF-1α and mTOR signaling pathways.

Advances in antitumor research of HIF-1α inhibitor YC-1 and its derivatives.

Generally, hypoxia-inducible factor-1α (HIF-1α) is highly expressed in solid tumors, it plays a key role in the occurrence and development of tumors, hindering cancer treatment in various ways. The antitumor activity and pharmacological mechanism of YC-1 [3-(5'-hydroxymethyl-2'-furyl)-1­benzyl indazole], an HIF-1α inhibitor, and the design and synthesis of its derivatives have attracted tremendous attention in the field of antitumor research. YC-1 is a potential drug candidate and a lead compound for tumor therapy. Hence, the multifaceted mechanism of action of YC-1 and the structure activity relationship (SAR) of its derivatives are important factors to be considered for the development of HIF-1α inhibitors. Therefore, this review aimed to provide a comprehensive overview of the various antitumor mechanisms of YC-1 in antitumor research and an in-depth summary of the SAR for the development of its derivatives. A full understanding and discussion of these aspects are expected to provide potential ideas for developing novel HIF-1α inhibitors and antitumor drugs belonging to the YC-1 class. The review also highlighted the application prospects of the YC-1 class of potential antitumor candidates, and provided some unique insights about these antitumor agents.

Molecular dynamics and 3D-QSAR studies on indazole derivatives as HIF-1α inhibitors.

Hypoxia-inducible factor (HIF) is a transcriptional factor which plays a crucial role in tumour metastasis thereby responsible for development of various forms of cancers. Indazole derivatives have been reported in the literature as potent HIF-1α inhibitor via interaction with key residues of the HIF-1α active site. Taking into consideration the role HIF-1α in cancer and potency of indazole derivative against HIF-1α; it was considered of interest to correlate structural features of known indazole derivatives with specified HIF-1α inhibitory activity to map pharmacophoric features through Three-dimensional quantitative structural activity relationship (3D-QSAR) and pharmacophore mapping. Field and Gaussian based 3D-QSAR studies were performed to realize the variables influencing the inhibitory potency of HIF-1α inhibitors. Field and Gaussian- based 3D-QSAR models were validated through various statistical measures generated by partial least square (PLS). The steric and electrostatic maps generated for both 3D-QSAR provide a structural framework for designing new inhibitors. Further; 3D-maps were also helpful in understanding variability in the activity of the compounds. Pharmacophore mapping also generates a common five-point pharmacophore hypothesis (A1D2R3R4R5_4) which can be employed in combination with 3D-contour maps to design potent HIF-1α inhibitors. Molecular docking and molecular dynamics (MD) simulation of the most potent compound 39 showed good binding efficiency and was found to be quite stable in the active site of the HIF-1α protein. The developed 3D-QSAR models; pharmacophore modelling; molecular docking studies along with the MD simulation analysis may be employed to design lead molecule as selective HIF-1α inhibitors for the treatment of Cancer.

Targeted inhibition of the GRK2/HIF-1α pathway is an effective strategy to alleviate synovial hypoxia and inflammation.

G-protein coupled receptor (GPCR) kinases (GRKs) and hypoxia-inducible factor-1α (HIF-1α) play key roles in rheumatoid arthritis (RA). Several studies have demonstrated that HIF-1α expression is positively regulated by GRK2, suggesting its posttranscriptional effects on HIF-1α. In this study, we review the role of HIF-1α and GRK2 in RA pathophysiology, focusing on their proinflammatory roles in immune cells and fibroblast-like synoviocytes (FLS).We then introduce several drugs that inhibit GRK2 and HIF-1α, and briefly outline their molecular mechanisms. We conclude by presenting gaps in knowledge and our prospects for the pharmacological potential of targeting these proteins and the relevant downstream signaling pathways.Future research is warranted and paramount for untangling these novel and promising roles for GRK2 and HIF-1α in RA.

Melatonin through blockade of Hif-1α signaling mediates the anti-fibrosis under hypoxia in canine Sertoli cells.

The hypoxic microenvironment of cryptorchidism is an important factor in the impairment and fibrosis of Sertoli cells which result in blood-testis barrier (BTB) destruction and spermatogenesis loss. Recent studies have shown that melatonin, a well-known pineal hormone exerts beneficial effects against pathological fibrosis in a various of organs. However, it is still unknown whether melatonin can regulate hypoxia-induced fibrosis of Sertoli cells. In this study we evaluate melatonin levels, and its synthesizing enzymes, AANAT and HIOMT expression patterns in canine cryptorchidism and contralateral normal testis. Results show abdominal testes presented low melatonin levels and AANAT and HIOMT expression compared with testes located in the scrotum. Moreover, we established a hypoxia-induced fibrosis model in canine Sertoli cells induced by cobalt chloride (CoCl2) and found that melatonin inhibited the EMT markers expression and ECM production as well as Hif-1α expression of Sertoli cells in a dose-dependent manner. Furthermore, use of Lificiguat (synonyms YC-1, Hif-1α inhibitor) to interfere with the Hif-1α pathway showed a similar effect with melatonin suppression of the fibrosis in Sertoli cells. The results indicate that melatonin supplementation can alleviate the fibrosis process of Sertoli cells caused by hypoxia, which is associated with regulating the inhibition of Hif-1α signaling.

A Novel Hypoxia-inducible Factor 1α Inhibitor KC7F2 Attenuates Oxygen-induced Retinal Neovascularization.

Purpose: KC7F2 is a novel molecule compound that can inhibit the translation of hypoxia-inducible factor 1α (HIF1α). It has been reported to exhibit potential antiangiogenic effect. We hypothesized that KC7F2 could inhibit oxygen-induced retinal neovascularization (RNV). The purpose of this study was to investigate this assumption. Methods: Oxygen-induced retinopathy (OIR) models in C57BL/6J mice and Sprague-Dawley rats were used for in vivo study. After intraperitoneal injections of KC7F2, RNV was detected by immunofluorescence and hematoxylin and eosin staining. Retinal inflammation was explored by immunofluorescence. EdU incorporation assay, cell counting kit-8 assay, scratch test, transwell assay, and Matrigel assay were used to evaluate the effect of KC7F2 on the proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVEC) induced by vascular endothelial growth factor (VEGF) in vitro. Protein expression was examined by Western blot. Results: KC7F2 treatment (10 mg/kg/d) in OIR mice significantly attenuated pathological neovascularization and decreased the number of preretinal neovascular cell nuclei, without changing the avascular area, which showed the same trends in OIR rats. Consistently, after the KC7F2 intervention (10 µM), cell proliferation was inhibited in VEGF-induced HUVEC, which was in agreement with the trend observed in the retinas of OIR mice. Meanwhile, KC7F2 suppressed VEGF-induced HUVEC migration and tube formation, and decreased the density of leukocytes and microglia colocalizing neovascular areas in the retinas. Moreover, the HIF1α-VEGF pathway activated in retinas of OIR mice and hypoxia-induced HUVEC, was suppressed by KC7F2 treatment. Conclusions: The current study revealed that KC7F2 was able to inhibit RNV effectively via HIF1α-VEGF pathway, suggesting that it might be an effective drug for RNV treatment.

Mathematical description of the effect of HIF inhibition on the radiobiological response of LNCaP cells.

According to the National Institute of Public Health, prostate cancer (PCa) is the leading cause of cancer death in Mexican men, highly associated with aggressiveness, resistance to treatment, and metastatic spread (Bharti et al., 2019) mediated by activation of the hypoxia-inducible factor 1 (HIF-1α). The objective of the present study was to evaluate the participation of HIF-1α activation in the radiobiological response of the human prostate adenocarcinoma cell line LNCaP, describing the phenomena with a mathematical model. Four groups were formed under different exposure conditions, including hypoxic cells treated with CoCl2 (300 µM for 22 h) with or without hypoxia-inducible factor inhibitor (150 nM chetomin for 4 h added after an incubation period of 18 h with CoCl2, just before completing the incubation period of 22 h). They were exposed to a source of 60Co in a dose range between 2 and 10 Gy to obtain survival curves that are fitted to a mathematical model. CoCl2 or chetomin treatments do not affect the viability of LNCaP cells that remained unchanged after irradiation. CoCl2 induced hypoxia reduces the survivability of LNCaP, and obstruction of HIF-1α signaling with chetomine produces a slight radioprotective effect. As others report, the genetic reprogramming induced by HIF-1α activation acts as an intrinsic agent that selects cells with more aggressive behavior (Pressley et al., 2017), while chetomin protects cells from death due to its scavenger properties. Interestingly, treatment with chetomin of cells induced to hypoxia (HIF-1 activation with CoCl2) produces a significant reduction in the radioresistance of LNCaP cells, demonstrating that the simultaneous use of chetomin and gamma radiation is an effective option for the treatment of hypoxic prostate cancer. At the molecular level, we suggest that the selective force exerted by HIF-1α depends on the production of free radicals by radiation. The proposed mathematical model showed that the rate of change in cell survival as a function of radiation dose is proportional to the product of two functions, one that describes cell death and the other that describes natural or artificial resistance to radiation.

DYRK1A suppression attenuates HIF­1α accumulation and enhances the anti­liver cancer effects of regorafenib and sorafenib under hypoxic conditions.

Hypoxia promotes drug resistance and induces the expression of hypoxia inducible factor (HIF)­1α in liver cancer cells. However, to date, no selective HIF­1α inhibitor has been clinically approved. The aim of this study is to investigate a drug­targetable molecule that can regulate HIF­1α under hypoxia. The present study demonstrated that hyperactivation of dual­specificity tyrosine­phosphorylation­regulated kinase 1A (DYRK1A)/HIF­1α signaling was associated with an increased risk of liver cancer. In addition, DYRK1A knockdown using small interfering RNA transfection or treatment with harmine, a natural alkaloid, significantly reduced the protein expression levels of HIF­1α in liver cancer cells under hypoxic conditions in vitro. Conversely, DYRK1A overexpression­vector transfection in liver cancer cell lines notably induced HIF­1α expression under the same conditions. Furthermore, DYRK1A was shown to interact and activate STAT3 under hypoxia to regulate HIF­1α expression. These findings indicated that DYRK1A may be a potential upstream activator of HIF­1α and positively regulate HIF­1α via the STAT3 signaling pathway in liver cancer cells. Additionally, treatment with harmine attenuated the proliferative ability of liver cancer cells under hypoxic conditions using sulforhodamine B and colony formation assay. Furthermore, DYRK1A knockdown could significantly enhance the anti­liver cancer effects of regorafenib and sorafenib under hypoxia. Co­treatment with harmine and either regorafenib or sorafenib also promoted cell death via the STAT3/HIF­1α/AKT signaling pathway under hypoxia using PI staining and western blotting. Overall, the results from the present study suggested that DYRK1A/HIF­1α signaling may be considered a novel pathway involved in chemoresistance, thus providing a potentially effective therapeutic regimen for treating liver cancer.

Hypoxia-induced macropinocytosis represents a metabolic route for liver cancer.

Hepatocellular carcinoma (HCC) invariably exhibits inadequate O2 (hypoxia) and nutrient supply. Hypoxia-inducible factor (HIF) mediates cascades of molecular events that enable cancer cells to adapt and propagate. Macropinocytosis is an endocytic process initiated by membrane ruffling, causing the engulfment of extracellular fluids (proteins), protein digestion and subsequent incorporation into the biomass. We show that macropinocytosis occurs universally in HCC under hypoxia. HIF-1 activates the transcription of a membrane ruffling protein, EH domain-containing protein 2 (EHD2), to initiate macropinocytosis. Knockout of HIF-1 or EHD2 represses hypoxia-induced macropinocytosis and prevents hypoxic HCC cells from scavenging protein that support cell growth. Germline or somatic deletion of Ehd2 suppresses macropinocytosis and HCC development in mice. Intriguingly, EHD2 is overexpressed in HCC. Consistently, HIF-1 or macropinocytosis inhibitor suppresses macropinocytosis and HCC development. Thus, we show that hypoxia induces macropinocytosis through the HIF/EHD2 pathway in HCC cells, harnessing extracellular protein as a nutrient to survive.

Inhibition of HIF-1α-AQP4 axis ameliorates brain edema and neurological functional deficits in a rat controlled cortical injury (CCI) model.

Traumatic brain injury (TBI) is an important cause of death in young adults and children. Till now, the treatment of TBI in the short- and long-term complications is still a challenge. Our previous evidence implied aquaporin 4 (AQP4) and hypoxia inducible factor-1α (HIF-1α) might be potential targets for TBI. In this study, we explored the roles of AQP4 and HIF-1α on brain edema formation, neuronal damage and neurological functional deficits after TBI using the controlled cortical injury (CCI) model. The adult male Sprague Dawley rats were randomly divided into sham and TBI group, the latter group was further divided into neutralized-AQP4 antibody group, 2-methoxyestradiol (2-ME2) group, and their corresponding control, IgG and isotonic saline groups, respectively. Brain edema was examined by water content. Hippocampal neuronal injury was assessed by neuron loss and neuronal skeleton related protein expressions. Spatial learning and memory deficits were evaluated by Morris water maze test and memory-related proteins were detected by western blot. Our data showed that increased AQP4 protein level was closely correlated with severity of brain edema after TBI. Compared with that in the control group, both blockage of AQP4 with neutralized-AQP4 antibody and inhibition of HIF-1α with 2-ME2 for one-time treatment within 30-60 min post TBI significantly ameliorated brain edema on the 1st day post-TBI, and markedly alleviated hippocampal neuron loss and spatial learning and memory deficits on the 21st day post-TBI. In summary, our preliminary study revealed the short-term and long-term benefits of targeting HIF-1α-AQP4 axis after TBI, which may provide new clues for the selection of potential therapeutic targets for TBI in clinical practice.

HIF-1α inhibition attenuates severity of Achilles tendinopathy by blocking NF-κB and MAPK pathways.

Tendinopathy is a common disease influencing life quality and tendon function of patients, especially in the elderly and athletes. Inflammation is an important pathologic process of tendinopathy. Hypoxia inducible factor-1 alpha (HIF-1α) participates actively in inflammatory process. However, little is known about the role of HIF-1α in tendinopathy. To address this issue, we verified the expression level of HIF-1α in tendinopathy in vivo and in vitro. Furthermore, the severity of tendinopathy in vivo and in vitro was assessed after HIF-1α inhibition. At the same time, inflammatory signaling cascades were evaluated. The expression level of HIF-1α increased in tendinopathy in vivo and in vitro. The migration and proliferation of tendon cells (TDCs) were reduced after HIF-1α inhibition. In the meantime, HIF-1α inhibition alleviated the severity of tendinopathy and promoted tendon repairing. We also found that HIF-1α inhibition reduced the phosphorylation levels of p65 in NF-κB signaling pathway and the phosphorylation levels of extracellular signal-regulated kinase 1/2 (ERK1/2), p38, and Jun N-terminal kinase (JNK) in MAPK signaling pathway. These findings suggest that HIF-1α increases in tendinopathy in vivo and vitro, and HIF-1α inhibition can suppress the severity of tendinopathy by blocking NF-κB and MAPK signaling pathways.

microRNA-671-5p reduces tumorigenicity of ovarian cancer via suppressing HDAC5 and HIF-1α expression.

OBJECTIVE: microRNA (miR)-based therapeutic reference has been established and expanded in the treatment of cancers. For this reason, we explored how miR-671-5p regulated tumorigenicity of ovarian cancer (OC) through regulating histone deacetylase 5 (HDAC5) and hypoxia-inducible factor-1α (HIF-1α). METHODS: miR-671-5p, HDAC5 and HIF-1α expression levels were determined in OC clinical tissues. The OC cell line H8910 was screened and transfected with the vectors that altered miR-671-5p, HDAC5 and HIF-1α levels. Finally, the proliferation, migration, invasion and apoptosis of the transfected H8910 cells were determined and the role of miR-671-5p and HDAC5 in vivo tumor growth was further discussed. RESULTS: Low expression miR-671-5p and high expression HDAC5 and HIF-1α levels were tested in OC tissues. Up-regulating miR-671-5p or down-regulating HDAC5 or HIF-1α suppressed proliferation, migration, invasion and augmented apoptosis of H8910 cells while silenced miR-671-5p or enhanced HDAC5 caused the opposite consequences. Overexpression of HDAC5 reduced while depletion of HDAC5 enhanced the influence of up-regulated miR-671-5p on OC cell growth. In animal models, suppressing miR-671-5p or promoting HDAC5 encouraged OC tumor growth. CONCLUSION: A summary delineates that miR-671-5p reduces tumorigenicity of OC via suppressing HDAC5 and HIF-1α expression levels.

Synthesis of sp3-rich chiral bicyclo[3.3.1]nonanes for chemical space expansion and study of biological activities.

Chiral sp3-rich bicyclo[3.3.1]nonane scaffolds 10-12 were synthesized as single diastereomers from aldehyde 9, which was prepared from 4,4-dimethoxycyclohexa-2,5-dienone through a copper-catalyzed enantioselective reduction. Three different types of intramolecular addition reactions were studied: SmI2-mediated reductive cyclization, base-promoted aldol reaction, and one-pot Mannich reaction. We succeeded in introducing three side-chains to scaffold 11 and construct an sp3-rich compound library in both enantiomeric variants by simply changing the chirality of the ligands. The biological evaluation revealed that all synthesized compounds exhibited a concentration-dependent inhibition of hypoxia-inducible factor-1 (HIF-1) transcriptional activity, with IC50 values in the range of 17.2-31.7 µM, whereas their effects on cell viability were varied (IC50 = 3.5 to > 100 µM). The most active compound 16f inhibits the accumulation of HIF-1α protein and mRNA in hypoxia, indicating that it has a mechanism of action distinctly different from other known compounds bearing the common bicyclo[3.3.1]nonane skeleton.

Capsaicin inhibits HIF-1α accumulation through suppression of mitochondrial respiration in lung cancer cells.

Hypoxia inducible factor (HIF)-1α is an important transcription factor regulating cancer metabolism in hypoxic environment. Capsaicin is known to inhibit hypoxia-induced HIF activity in lung cancer. Hence, in this study we tried to elucidate its inhibitory mechanism of action. In lung cancer cells, including H1299, H23, A549, and H2009 cells, capsaicin inhibited cell growth and HIF activation. Under hypoxic conditions, capsaicin reduced the accumulation of HIF-1α protein and the expression of its target genes, including pyruvate dehydrogenase kinase 1 (PDK1) and glucose transporter 1 (GLUT1), with no effect on overall HIF-1α mRNA levels in the H1299 cells. In addition, capsaicin increased intracellular oxygen levels by suppressing mitochondrial respiration, resulting in a reduction of HIF-1α accumulation. Furthermore, mitochondrial ATP production was reduced by capsaicin through the inhibition of mitochondrial respiration in the H1299, H23, A549, and H2009 cells. These results indicate that capsaicin potentially exhibits anticancer therapeutic effects in lung cancer under hypoxic conditions.

Hypoxia- and hyperoxia-related gene expression dynamics during developmental critical windows of the tropical gar Atractosteus tropicus.

Aquatic hypoxia is both a naturally-occurring and anthropogenically-generated event. Fish species have evolved different adaptations to cope with hypoxic environments, including gill modifications and air breathing. However, little is known about the molecular mechanisms involved in the respiration of embryonic and larval fishes during critical windows of development. We assessed expression of the genes hif-1α, fih-1, nhe1, epo, gr and il8 using the developing tropical gar as a piscine model during three developmental periods (fertilization to hatch, 1 to 6 days post hatch (dph) and 7 to 12 dph) when exposed to normoxia (~7.43 mg/L DO), hypoxia (~2.5 mg/L DO) or hyperoxia (~9.15 mg/L DO). All genes had higher expression when fish were exposed to either hypoxia or hyperoxia during the first two developmental periods. However, fish continuously exposed to hypoxia had increased expression of the six genes by hatching and 6 dph, and by 12 dph only hif-1α still had increased expression. The middle developmental period was the most hypoxia-sensitive, coinciding with several changes in physiology and morphology. The oldest larvae were the most resilient to gene expression change, with little variation in expression of the six genes compared. This study is the first to relate the molecular response of an air-breathing fish to oxygen availability to developmental critical windows and contributes to our understanding of some molecular responses of developing fish to changes in oxygen availability.

An efficient strategy for digging protein-protein interactions for rational drug design - A case study with HIF-1α/VHL.

The ubiquitination of the hypoxia-inducible factor-1α (HIF-1α) is mediated by interacting with the von Hippel-Lindau protein (VHL), and is associated with cancer, chronic anemia, and ischemia. VHL, an E3 ligase, has been reported to degrade HIF-1 for decades, however, there are few successful inhibitors currently. Poor understanding of the binding pocket and a lack of in-depth exploration of the interactions between two proteins are the main reasons. Hence, we developed an effective strategy to identify and design new inhibitors for protein-protein interaction targets. The hydroxyproline (Hyp564) of HIF-1α contributed the key interaction between HIF-1α and VHL. In this study, detailed information of the binding pocket were explored by alanine scanning, site-directed mutagenesis and molecular dynamics simulations. Interestingly, we found the interaction(s) between Y565 and H110 played a key role in the binding of VHL/HIF-1α. Based on the interactions, 8 derivates of VH032, 16a-h, were synthesized by introducing various groups bounded to H110. Further assay on protein and cellular level exhibited that 16a-h accessed higher binding affinity to VHL and markable or modest improvement in stabilization of HIF-1α or HIF-1α-OH in HeLa cells. Our work provides a new orientation for the modification or design of VHL/HIF-1α protein-protein interaction inhibitors.

AMSP-30 m as a novel HIF-1α inhibitor attenuates the development and severity of adjuvant-induced arthritis in rats: Impacts on synovial apoptosis, synovial angiogenesis and sonic hedgehog signaling pathway.

Growing evidence indicates that synovial hypoxia-inducible factor 1α (HIF-1α) can be as a promising target for RA therapy. We previously reported that AMSP-30 m as a novel HIF-1α inhibitor had potent activities of anticancer metastasis. This study clarified the therapeutic effects of HIF-1α inhibitor AMSP-30 m on adjuvant-induced arthritis (AIA) in rats and explored the possible mechanisms. AMSP-30 m was given intraperitoneally to AIA rats, and its therapeutic effects and anti-inflammatory activity were evaluated. The influences of AMSP-30 m on synovial apoptosis, angiogenesis and sonic hedgehog (Shh) pathway were examined. We found that, accompanied with the inhibition of synovial HIF-1α expression, AMSP-30 m had potent anti-arthritic and anti-inflammatory effects on AIA rats, evidenced by the reduction in paw swelling, arthritis index, histopathological scores, and the production of IL-1ß, IL-6, TNF-α in serum and synovial tissues. AMSP-30 m reduced synovial Ki67 expression and increased TUNEL-positive index, indicating its anti-proliferative and pro-apoptotic effects on AIA synovial cells, which was related to reducing Bcl-2 protein level and increasing Bax, cleaved caspase 3 protein levels. Additionally, AMSP-30 m showed anti-angiogenic effects within AIA synovium, indicated by the reduction of synovial VEGF expression and blood vessels number (especially CD31+/αSMA- immature vessels, but not CD31+/αSMA+ mature vessels). Moreover, AMSP-30 m inhibited the activation of synovial Shh pathway, suggested by the reduction of pathway-related proteins, like Shh, Smo, Gli-1, cyclin D1 and c-Myc. Collectively, HIF-1α inhibitor AMSP-30 m exerted potent anti-arthritic effects on AIA rats possibly by promoting synovial apoptosis, reducing synovial angiogenesis and inhibiting Shh pathway.

Synthesis, Binding Affinity Analysis, and 18 F Radiosynthesis of Small-Molecular-Weight HIF-1α-Binding Compounds.

Eleven small-molecular-weight compounds and three cyclic peptides were synthesized and evaluated for binding to hypoxia-inducible factor-1α (HIF-1α). Microscale thermophoresis analysis identified peptide [19 F]SFB-link-c-(Ppg)LLFVY 3 and small-molecule inhibitor 5 as potent HIF-1α binding compounds with KD values of 0.46±0.2 µM and 7.8±3.4 µM, respectively. Both compounds represent novel HIF-1α-targeting compounds that are predicted to interact with the PAS-B region of HIF-1α, as confirmed with molecular docking studies. Lead structures 3 and 5 were further radiolabelled with fluorine-18 for positron emission tomography (PET) imaging agents targeting HIF-1α in vivo.