Bisabosqual A: A novel asparagine synthetase inhibitor suppressing the proliferation and migration of human non-small cell lung cancer A549 cells.

Asparagine synthetase (ASNS) is a crucial enzyme for the de novo biosynthesis of endogenous asparagine (Asn), and ASNS shows the positive relationship with the growth of several solid tumors. Most of ASNS inhibitors are analogs of transition-state in ASNS reaction, but their low cell permeability hinders their anticancer activity. Therefore, novel ASNS inhibitors with a new pharmacophore urgently need to be developed. In this study, we established and applied a system for in vitro screening of ASNS inhibitors, and found a promising unique bisabolane-type meroterpenoid molecule, bisabosqual A (Bis A), able to covalently modify K556 site of ASNS protein. Bis A targeted ASNS to suppress cell proliferation of human non-small cell lung cancer A549 cells and exhibited a synergistic effect with L-asparaginase (L-ASNase). Mechanistically, Bis A promoted oxidative stress and apoptosis, while inhibiting autophagy, cell migration and epithelial-mesenchymal transition (EMT), impeding cancer cell development. Moreover, Bis A induced negative feedback pathways containing the GCN2-eIF2α-ATF4, PI3K-AKT-mTORC1 and RAF-MEK-ERK axes, but combination treatment of Bis A and rapamycin/torin-1 overcame the potential drug resistance triggered by mTOR pathways. Our study demonstrates that ASNS inhibition is promising for cancer chemotherapy, and Bis A is a potential lead ASNS inhibitor for anticancer development.

Laeverin/aminopeptidase Q induces indoleamine 2,3-dioxygenase-1 in human monocytes.

Human extravillous trophoblast (EVT) invades the maternal endometrium and reconstructs uterine spiral arteries cooperatively with maternal immune cells. Although EVT has allogeneic paternal antigens, the maternal immune system does not reject it. Here, we found that laeverin (LVRN), an EVT-specific cell surface peptidase, interacts with monocytes to produce indoleamine 2,3-dioxygenase-1 (IDO1). LVRN-transfected Swan71 cells, a cytotrophoblast-derived cell line, and increased IDO1 expression in PBMC under cell-to-cell interacting conditions. Soluble recombinant LVRN (r-LVRN) interacted with CD14-positive monocytes and induced their IDO1 expression without the intervention of other immune cell populations. LVRN-induced IDO1 production was promoted in PMA-activated monocyte-like THP-1 cells. Furthermore, r-LVRN decreased the tryptophan level and increased the kynurenine/tryptophan ratio in the culture media of the PMA-treated THP-1 cells. These findings suggest that LVRN is one of the key molecules that mediate the interaction between EVT and monocytes/macrophages and creates an immunosuppressive environment at the maternal-fetal interface in the uterus.

Establishment of an MR1 Presentation Reporter Screening System and Identification of Phenylpropanoid Derivatives as MR1 Ligands.

Mucosal-associated invariant T (MAIT) cells are innate-like T cells that are modulated by ligands presented on MHC class I-related proteins (MR1). These cells have attracted attention as potential drug targets because of their involvement in the initial response to infection and various disorders. Herein, we have established the MR1 presentation reporter assay system employing split-luciferase, which enables the efficient exploration of MR1 ligands. Using our screening system, we identified phenylpropanoid derivatives as MR1 ligands, including coniferyl aldehyde, which have an ability to inhibit the MR1-MAIT cell axis. Further, the structure-activity relationship study of coniferyl aldehyde analogs revealed the key structural features of ligands required for MR1 recognition. These results will contribute to identifying a broad range of endogenous and exogenous MR1 ligands and to developing novel MAIT cell modulators.

Spheroid formation induces chemokine production in trophoblast-derived Swan71 cells.

PROBLEM: In the cell column of anchoring villi, the cytotrophoblast differentiates into extravillous trophoblast (EVT) and invades the endometrium in contact with maternal immune cells. Recently, chemokines were proposed to regulate the decidual immune response. To investigate the roles of chemokines around the anchoring villi, we examined the expression profiles of chemokines in the first-trimester trophoblast-derived Swan71 cells using a three-dimensional culture model. METHOD OF STUDY: The gene expressions in the spheroid-formed Swan71 cells were examined by microarray and qPCR analyses. The protein expressions were examined by immunochemical staining. The chemoattractant effects of spheroid-formed Swan71 cells were examined by migration assay using monocyte-derived THP-1 cells. RESULTS: The expressions of an EVT marker, laeverin, and matrix metalloproteases, MMP2 and MMP9, were increased in the spheroid-cultured Swan71 cells. Microarray and qPCR analysis revealed that mRNA expressions of various chemokines, CCL2, CCL7, CCL20, CXCL1, CXCL2, CXCL5, CXCL6, CXCL8, and CXCL10, in the spheroid-cultured Swan71 cells were up-regulated as compared with those in the monolayer-cultured Swan71 cells. These expressions were significantly suppressed by hypoxia. Migration assay showed that culture media derived from the spheroid-formed Swan71 cells promoted THP-1 cell migration. CONCLUSION: This study indicated that chemokine expressions in Swan71 cells increase under a spheroid-forming culture and the culture media have chemoattractant effects. Since three-dimensional cell assembling in the spheroid resembles the structure of the cell column, this study also suggests that chemokines play important roles in the interaction between EVT and immune cells in their early differentiation stage.

Absolute Lymphocyte Count Is an Independent Prognostic Factor for ER-positive HER2-negative Advanced Breast Cancer Patients Treated With CDK4/6 Inhibitors.

BACKGROUND/AIM: The aim of this study was to elucidate the clinical significance of peripheral blood biomarkers, including absolute lymphocyte count (ALC), neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR) and C-reactive protein (CRP) in patients with estrogen receptor-positive human epidermal growth factor receptor 2-negative advanced breast cancer treated with the CDK4/6 inhibitors, abemaciclib and palbociclib. PATIENTS AND METHODS: A total of 83 patients treated with fulvestrant plus abemaciclib or palbociclib were included in this study. Progression-free survival (PFS) and overall survival (OS) were compared in relation to baseline levels of ALC, NLR, PLR and CRP. RESULTS: The cut-off values of ALC, NLR, PLR, and CRP for PFS were determined from the receiver operating characteristic curve using the Youden index for area under the curve and set at 1,212/µl, 1.964, 170 and 0.220 mg/dl, respectively. In the abemaciclib-treated group, ALC-high patients showed significantly better PFS than ALC-low patients (p=0.0151) and multivariate analysis revealed that ALC was an independent prognostic factor for PFS (p=0.0085). In the palbociclib-treated group, there was no significant relationship between any peripheral blood biomarkers and PFS. In both treatment groups, ALC-high patients showed significantly better OS than ALC-low patients (p=0.0169 and 0.0290, respectively). Multivariate analysis revealed ALC was an independent prognostic factor for OS in both abemaciclib- and palbociclib-treated groups (p=0.0112 and 0.0202, respectively). CONCLUSION: ALC is an independent prognostic factor for estrogen receptor-positive human epidermal growth factor receptor 2-negative advanced breast cancer patients treated with the CDK4/6 inhibitors abemaciclib and palbociclib.

Design, synthesis, and target identification of new hypoxia-inducible factor 1 (HIF-1) inhibitors containing 1-alkyl-1H-pyrazole-3-carboxamide moiety.

Hypoxia-inducible factor 1 (HIF-1) is a promising drug target for cancer chemotherapy. In our screening program aimed at identifying new HIF-1 inhibitors by using a hypoxia-responsive luciferase reporter gene assay, KUSC-5001 containing the 1-alkyl-1H-pyrazole-3-carboxamide moiety was found as a potential hit molecule. During an extensive structure-activity relationship (SAR) study, we developed a more effective HIF-1 inhibitor KUSC-5037 (IC50 = 1.2 µM). Under hypoxic conditions, KUSC-5037 suppressed the HIF-1α (a regulatory subunit of HIF-1) mRNA, causing decreases in the gene expression of HIF-1 target genes such as carbonic anhydrase 9 (CA9) and vascular endothelial growth factor (VEGF) genes. Furthermore, by applying our fluorescent and bifunctional probes, ATP5B, a catalytic ß subunit of mitochondrial FoF1-ATP synthase, was identified as a target protein of KUSC-5037. These results indicate that the derivatives of KUSC-5037 containing the 1-alkyl-1H-pyrazole-3-carboxamide moiety are promising lead molecules for the inhibition of HIF-1 signaling via FoF1-ATP synthase suppression.

The effects of 5-OP-RU stereochemistry on its stability and MAIT-MR1 axis.

Mucosal-associated invariant T (MAIT) cells are an abundant subset of innate-like T lymphocytes. MAIT cells are activated by microbial riboflavin-derived antigens, such as 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU), when presented by the major histocompatibility complex (MHC) class I-related protein (MR1). We have synthesized all stereoisomers of 5-OP-RU to investigate the effects of its stereochemistry on the MR1-dependent MAIT cell activation and MR1 upregulation. The analysis of MAIT cell activation by these 5-OP-RU isomers revealed that the stereocenters at the 2'- and 3'-OH groups in the ribityl tail are crucial for the recognition of MAIT-TCR, whereas that of 4'-OH group does not significantly affect the regulation of MAIT cell activity. Furthermore, kinetic analysis of complex formation between the ligands and MR1 suggested that 5-OP-RU forms a covalent bond to MR1 in cells within 1 hour. These findings provide guidelines for designing ligands that regulate MAIT cell functions.

Reciprocal Expression Patterns of Placental Leucine Aminopeptidase/Insulin-Regulated Aminopeptidase and Vasopressin in the Murine Brain.

Placental leucine aminopeptidase/insulin-regulated aminopeptidase (P-LAP/IRAP) regulates vasopressin and oxytocin levels in the brain and peripheral tissues by controlled degradation of these peptides. In this study, we determined the relationship between P-LAP/IRAP and vasopressin levels in subregions of the murine brain. P-LAP/IRAP expression was observed in almost all brain regions. The expression patterns of P-LAP/IRAP and vasopressin indicated that cells expressing one of these protein/peptide were distinct from those expressing the other, although there was significant overlap between the expression regions. In addition, we found reciprocal diurnal rhythm patterns in P-LAP/IRAP and arginine vasopressin (AVP) expression in the hippocampus and pituitary gland. Further, synchronously cultured PC12 cells on treatment with nerve growth factor (NGF) showed circadian expression patterns of P-LAP/IRAP and enzymatic activity during 24 h of incubation. Considering that vasopressin is one of the most efficient peptide substrates of P-LAP/IRAP, these results suggest a possible feedback loop between P-LAP/IRAP and vasopressin expression, that regulates the function of these substrate peptides of the enzyme via translocation of P-LAP/IRAP from intracellular vesicles to the plasma membrane in brain cells. These findings provide novel insights into the functions of P-LAP/IRAP in the brain and suggest the involvement of these peptides in modulation of brain AVP functions in hyperosmolality, memory, learning, and circadian rhythm.

Final results of a phase II study of nivolumab in Japanese patients with relapsed or refractory classical Hodgkin lymphoma.

BACKGROUND: Many patients with classical Hodgkin lymphoma show increased programmed death-1 ligand expression in Reed-Sternberg cells. We report the final results of a phase II study of nivolumab, an anti-programmed death-1 monoclonal antibody, in Japanese patients with relapsed or refractory classical Hodgkin lymphoma. METHODS: Japanese patients with previously treated classical Hodgkin lymphoma (aged ≥ 20 years) were administered nivolumab (3 mg/kg on Day 1 of 14-day cycles) until progressive disease, an unacceptable adverse event, or another clinically relevant reason. Treatment could continue beyond progressive disease at the investigator's discretion in selected patients. RESULTS: Seventeen patients (median age: 63.0 years) were enrolled. The median follow-up was 38.8 months. One patient with non-Hodgkin lymphoma was excluded from efficacy analyses. The centrally assessed overall response rate in 16 classical Hodgkin lymphoma patients was 87.5% (95% confidence interval = 61.7-98.4%) and the disease control rate was 93.8% (95% confidence interval = 69.8-99.8%). The median (95% confidence interval) duration of response and progression-free survival were 8.5 (2.4-12.6) and 11.7 (1.8-42.3) months, respectively. The 3-year overall survival rate was 80.4% (95% confidence interval = 50.6-93.2%). Nivolumab was continued beyond progressive disease in seven patients; six were alive at the data cut-off. Adverse drug reactions occurred in all 17 patients with grades 3-4 adverse drug reactions in eight patients and no grade 5 adverse drug reactions. Pulmonary toxicities occurred in five patients; four of these occurred ≥17 months after starting nivolumab. CONCLUSION: Nivolumab is effective and tolerable in Japanese relapsed or refractory classical Hodgkin lymphoma patients. Continued monitoring may be necessary to detect late-onset pulmonary toxicities. CLINICAL TRIAL REGISTRATION: JapicCTI-142755 (Japan Pharmaceutical Information Center).

Promoting Roles of Embryonic Signals in Embryo Implantation and Placentation in Cooperation with Endocrine and Immune Systems.

Embryo implantation in the uterus is an essential process for successful pregnancy in mammals. In general, the endocrine system induces sufficient embryo receptivity in the endometrium, where adhesion-promoting molecules increase and adhesion-inhibitory molecules decrease. Although the precise mechanisms remain unknown, it is widely accepted that maternal-embryo communications, including embryonic signals, improve the receptive ability of the sex steroid hormone-primed endometrium. The embryo may utilize repulsive forces produced by an Eph-ephrin system for its timely attachment to and subsequent invasion through the endometrial epithelial layer. Importantly, the embryonic signals are considered to act on maternal immune cells to induce immune tolerance. They also elicit local inflammation that promotes endometrial differentiation and maternal tissue remodeling during embryo implantation and placentation. Additional clarification of the immune control mechanisms by embryonic signals, such as human chorionic gonadotropin, pre-implantation factor, zona pellucida degradation products, and laeverin, will aid in the further development of immunotherapy to minimize implantation failure in the future.

Ubiquitin carboxyl-terminal hydrolase L1 promotes hypoxia-inducible factor 1-dependent tumor cell malignancy in spheroid models.

Hypoxia-inducible factor 1 (HIF-1) is a critical heterodimeric transcription factor for tumor malignancy. Recently, ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) has been reported to function as a deubiquitinating enzyme for the stabilization of its α subunit (HIF-1α). In the present study, we showed that UCHL1 inhibition can be an effective therapeutic strategy against HIF-1-dependent tumor malignancy. In 2D monolayer culture, a UCHL1 inhibitor suppressed HIF activity and decreased the transcription of HIF downstream genes by inhibiting the UCHL1-mediated accumulation of HIF-1α. Phenotypically, UCHL1 inhibition remarkably blocked cell migration. In 3D spheroid culture models, ectopic expression of UCHL1 significantly upregulated malignancy-related factors such as solidity, volume, as well as viable cell number in an HIF-1α-dependent manner. Conversely, inhibition of the UCHL1-HIF-1 pathway downregulated these malignancy-related factors and also abolished UCHL1-mediated cell proliferation and invasiveness. Finally, inhibition of UCHL1 promoted HIF-1α degradation and lowered the expression of HIF-1 target genes in the 3D model, as also observed in 2D monolayer culture. Our research indicates that the UCHL1-HIF-1 pathway plays a crucial role in tumor malignancy, making it a promising therapeutic target for cancer chemotherapy.

Isolation, Structure Elucidation, and Conformational Regulation of Myropeptins, Lipopeptides from the Fungus Myrothecium roridum.

Myropeptins, novel lipopeptides, were isolated from the culture broth of a fungus Myrothecium roridum F27113. Myropeptin A1 comprises a linear 20 amino acid-peptide chain and a lauric acid capping the N-terminus. Myropeptin A1 formed a helix structure and showed biological activities including antifungal and hemolysis. Myropeptin B, a shorter analogue by two amino acid residues, showed neither helicity nor biological activity. These two amino acids at the C-terminus regulate the molecular function of myropeptin A1.

Acute-phase protein-like properties of endoplasmic reticulum aminopeptidase 1.

Endoplasmic reticulum aminopeptidase 1 (ERAP1) is a multi-functional enzyme. In this study, we analysed its role in lipopolysaccharide-induced inflammatory response in wild-type and ERAP1-knockout mice. Following lipopolysaccharide injection, ERAP1 was secreted into the blood, increasing leucine aminopeptidase activity and NO synthesis therein. Among the amino acids tested, arginine concentration was significantly increased in wild-type mice compared to ERAP1-knockout mice. These results suggest that ERAP1 behaves similar to acute-phase proteins, which are secreted into the blood in response to infectious/inflammatory stimuli and are involved in enhancing NO synthesis as a host defense mechanism.

Factors Regulating Human Extravillous Trophoblast Invasion: Chemokine-peptidase and CD9-integrin Systems.

The invasion of an extravillous trophoblast (EVT) into maternal decidual tissues, especially towards maternal spiral arteries, is an essential process in the human placental formation and subsequent normal fetal development. However, the precise regulatory mechanisms to induce EVT invasion towards arteries and/or to protect EVT from further invasion are not well understood. We found that a chemokine receptor, CCR1, was specifically expressed on EVT migrating towards maternal arteries. Using EVT isolated from a primary villous explant culture, RANTES, which is one of the ligands for CCR1, was shown to enhance EVT invasion. Furthermore, we observed that the platelets were deposited among intravascular EVT and platelet-derived soluble factors, which contained RANTES, enhanced EVT invasion. On the one hand, dipeptidyl peptidase IV (DPPIV), which can metabolize RANTES on the cell surface, was expressed on non-invading EVT and was demonstrated to suppress EVT invasion. In contrast, laeverin/aminopeptidase Q, which is specifically expressed on EVT, was shown to induce EVT invasion. Also, CD9 which is a cell surface marker of platelets and a regulator of integrin function was expressed on EVT and gene knockdown of the CD9 molecule enhanced EVT invasion. These findings suggest that the chemokine-chemokine receptor, chemokine-peptidase, and CD9-integrin systems play important roles in the regulation of EVT invasion during early human placental formation.

CD9 suppresses human extravillous trophoblast invasion.

During human placentation, the extravillous trophoblast (EVT) invades the maternal decidua and reconstructs maternal spiral arteries. However, the precise mechanisms that control EVT behavior have not yet been elucidated in detail. CD9 has been reported to be a cell-motility-related molecule. Since we previously observed that CD9 was expressed on human EVT, we examined the possible involvement of CD9 in the invasion process of EVT. Placental and umbilical samples were obtained from patients who underwent legal abortions, normal delivery, or hysterectomy. The expression of CD9 at the implantation site and on isolated EVT from a villous explant culture, an EVT-derived immortalized cell line, Swan71, and HUVEC was examined by immunocytochemical staining, flow cytometry, and RT-PCR. The effects of anti-CD9 functional antibody (ALB6) on EVT and Swan71 cell invasion were further examined by matrigel invasion assay along with shRNAmir gene knockdown treatment. CD9 was highly expressed on EVT at the boundary region of EVT invasion and intravascular EVT. EVT and Swan71 cell invasions were promoted by ALB6 or shRNAmir treatment. CD9 expression on Swan71 cells was reduced under hypo-oxygenic conditions, while its expression was increased by the co-culture with HUVEC. These findings suggest that CD9 could attenuate EVT invasion under the influence of an oxygen environment and maternal endothelial cells, proposing that CD9 is a potential regulator of human placental formation.

Cardiovascular events occur independently of high on-aspirin platelet reactivity and residual COX-1 activity in stable cardiovascular patients.

Several studies have indicated that approximately 25 % of patients treated with aspirin exhibit high on-treatment platelet reactivity (HTPR), which is potentially associated with cardiovascular events (CVEs). However, this association is still controversial, since the mechanisms by which HTPR contributes to CVEs remain unclear and a no standardised definition of HTPR has been established. To determine whether HTPR is associated with CVE recurrence and what type of assay would best predict CVE recurrence, we conducted a multicentre prospective cohort study of 592 stable cardiovascular outpatients treated with aspirin monotherapy for secondary prevention. Their HTPR was determined by arachidonic acid- or collagen-induced aggregation assays using two different agonist concentrations. Residual cyclooxygenase (COX)-1 activity was assessed by measuring serum thromboxane (TX)B2 or urinary 11-dehydro TXB2. Shear-induced platelet thrombus formation was also examined. We followed all patients for two years to evaluate how these seven indexes were related to the recurrence of CVEs (cerebral infarction, transient ischaemic attack, myocardial infarction, unstable angina, revascularisation, other arterial thrombosis, or cardiovascular death). Of 583 patients eligible for the analysis, CVEs occurred in 69 (11.8 %). A Cox regression model identified several classical risk factors associated with CVEs. However, neither HTPR nor high residual COX-1 activity was significantly associated with CVEs, even by applying cut-off values suggested in previous reports or a receiver-operating characteristic analysis. In conclusion, recurrence of CVEs occurred independently of HTPR and residual COX-1 activity. Thus, our findings do not support the use of platelet or COX-1 functional testing for predicting clinical outcomes in stable cardiovascular patients.

Structure Elucidation of Verucopeptin, a HIF-1 Inhibitory Polyketide-Hexapeptide Hybrid Metabolite from an Actinomycete.

The transcriptional factor, hypoxia inducible factor-1 (HIF-1), is a promising target for cancer chemotherapy. From an actinomycete, verucopeptin (1) was identified as a HIF-1 signaling inhibitor. By a combination of chemical degradation and spectroscopic analyses, the absolute stereochemistry of metabolite 1 was determined to be 10R, 15S, 16S, 23S, 27S, 28R, 31S, 33S, 35R. Moreover, metabolite 1 was revealed to attenuate the HIF-1α and mTORC1 pathway, indicating that verucopeptin (1) would be a potent lead compound for anticancer chemotherapy.

Involvement of Phenylalanine 297 in the Construction of the Substrate Pocket of Human Aminopeptidase B.

Aminopeptidase B (APB, EC 3.4.11.6) preferentially hydrolyzes the N-terminal basic amino acids of synthetic and peptide substrates and requires a physiological concentration of NaCl for optimal activity. In this study, we used site-directed mutagenesis and molecular modeling to search for an amino acid residue that is critical for the enzymatic properties of human APB. Substitution of Phe297 with Tyr caused a significant decrease in hydrolytic activity toward synthetic and peptide substrates as well as chloride anion sensitivity. Molecular modeling suggests that Phe297 contributes to the construction of the substrate pocket of APB, which is wide enough to hold a chloride anion and allow the interaction of Gln169 with the N-terminal Arg residue of the substrate through bridging with the chloride anion. These results indicate that Phe297 is crucial for the optimal enzymatic activity and chloride anion sensitivity of APB via formation of the optimal structure of the catalytic pocket.

Expression, purification and enzymatic characterization of a recombinant human ubiquitin-specific protease 47.

In this study, the physicochemical and enzymatic properties of recombinant human ubiquitin (Ub)-specific protease (USP) 47, a novel member of the C19 family of de-ubiquitinating enzymes (DUB), were characterized for the first time. Recombinant human USP47 was expressed in a baculovirus expression system and purified to homogeneity. The purified protein was shown to be a monomeric protein with a molecular mass of ∼146 kDa on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. USP47 released Ub from Ub-aminoacyl-4-metheylcoumaryl-7-amide and Ub-tagged granzyme B. The substitution of the potential nucleophile Cys109 with Ser severely abrogated the Ub-releasing activity of USP47, indicating that USP47 is indeed a cysteine DUB. An assay using Ub dimer substrates showed that the enzyme cleaved a variety of isopeptide bonds between 2 Ub molecules, including the Lys48- and Lys63-linked isopeptide bonds. USP47 also released a Ub moiety from Lys48- and Lys63-linked polyUb chains. Of the inhibitors tested, N-ethylmaleimide, Zn ion and Ub aldehyde revealed a dose-dependent inhibition of USP47. In this study, clear differences in the enzymatic properties between USP47 and USP7 (the most closely related proteins among DUBs) were also found. Therefore, our results suggest that USP47 may play distinct roles in Ub-mediated cellular processes via DUB activity.

Design, synthesis, and structure-activity relationships of 1-ethylpyrazole-3-carboxamide compounds as novel hypoxia-inducible factor (HIF)-1 inhibitors.

Hypoxia-inducible factor (HIF)-1 is well known as a promising target for cancer chemotherapy. By screening an in-house chemical library using a hypoxia-responsive luciferase reporter gene assay, we identified CLB-016 (1) containing 1-ethylpyrazole-3-carboxamide as a HIF-1 inhibitor (IC50=19.1µM). In a subsequent extensive structure-activity relationship (SAR) study, we developed compound 11Ae with an IC50 value of 8.1µM against HIF-1-driven luciferase activity. Compounds 1 and 11Ae were shown to significantly suppress the HIF-1-mediated hypoxia response, including carbonic anhydrase IX (CAIX) gene expression and migration of human sarcoma HT1080 cells. These results revealed 1-ethylpyrazole-3-carboxamide as a novel scaffold to develop promising anti-cancer drugs targeting the HIF-1 signaling pathway.