Design, synthesis and insight into the structure-activity relationship of 1,3-disubstituted indazoles as novel HIF-1 inhibitors

Design, synthesis and insight into the structure-activity relationship (SAR) of 1,3-disubstituted indazoles as novel HIF-1 inhibitors are described. In particular, the substituted furan moiety on indazole skeleton as well as its substitution pattern turns out crucial for the high HIF-1 inhibition.     

The zinc chelator, N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine, increases the level of nonfunctional HIF-1alpha protein in normoxic cells

The hypoxia-inducible factor-1alpha (HIF-1alpha) subunit is activated in response to lack of oxygen. HIF-1alpha-specific prolyl hydroxylase and factor inhibiting HIF-1alpha (FIH-1) catalyze hydroxylation of the proline and asparagine residues of HIF-1alpha, respectively. The hydroxyproline then interacts with ubiquitin E3 ligase, the von Hippel-Lindau protein, leading to degradation of HIF-1alpha by ubiquitin-dependent proteasomes, while the hydroxylation of the asparagine residue prevents recruitment of the coactivator, cAMP-response element-binding protein (CBP), thereby decreasing the transactivation ability of HIF-1alpha. We found that the Zn-specific chelator, N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), enhances the activity of HIF-1alpha-proline hydroxylase 2 but the level of HIF-1alpha protein does not fall because TPEN also inhibits ubiquitination. Since the Zn chelator does not prevent FIH-1 from hydroxylating the asparagine residue of HIF-1alpha, its presence leads to the accumulation of HIF-1alpha that is both prolyl and asparaginyl hydroxylated and is therefore nonfunctional. In hypoxic cells, TPEN also prevents HIF-1alpha from interacting with CBP, so reducing expression of HIF-1alpha target genes. As a result, Zn chelation causes the accumulation of nonfunctional HIF-1alpha protein in both normoxia and hypoxia.     

Lower expression of CXCR4 in lymph node metastases than in primary breast cancers: potential regulation by ligand-dependent degradation and HIF-1alpha

Stromal-derived factor-1 (SDF-1) is a unique ligand of the CXC chemokine receptor 4 (CXCR4), which is critically involved in the metastasis of breast cancer. High levels of SDF-1 in the common destination organs of metastasis, such as the lymph nodes, lungs, liver, and bones, attract CXCR4-positive tumor cells. The interaction between SDF-1 and CXCR4 leads to the activation of specific signaling pathways, allowing for homing and metastatic progression. However, regulation of CXCR4 expression at the metastatic organ site is not well-documented. We detected the expression of CXCR4 and hypoxia inducible factor (HIF)-1alpha in breast tumor tissues by immunohistochemical staining and analyzed SDF-1 in primary tumors and lymph nodes using real-time RT-PCR. Compared to the corresponding metastasized tumors in the lymph nodes, primary invasive carcinomas showed more intense staining for CXCR4, particularly on the cellular membrane. Both primary tumors and lymph node metastases exhibited higher levels of CXCR4 expression compared to non-neoplastic breast tissues. Therefore, we hypothesized that the tumor environment in the lymph nodes may cause the reduction of CXCR4 levels in the metastatic tumor cells because of: (1) high SDF-1 levels and (2) lower levels of HIF-1alpha. Our in vitro data demonstrated that high levels of SDF-1 can induce the internalization and degradation of CXCR4 through the lysosome pathway. In addition, lower levels of HIF-1alpha in the lymph node metastases, probably induced by the less hypoxic environment, further lowered CXCR4 levels. These results indicate that ligand-dependent degradation and lower HIF-1alpha levels may be potential causes of lowered levels of CXCR4 in the lymph nodes compared to the primary tumors. Our study suggests that CXCR4 levels in tumor cells are regulated by its microenvironment. These findings may enhance our ability to understand the biological behavior of breast cancers.     

Hypoxia-induced HIF-1 alpha accumulation is augmented in a co-culture of keloid fibroblasts and human mast cells: involvement of ERK1/2 and PI-3K/Akt

Keloids represent a prolonged inflammatory fibrotic state with areas that display distinctive histological features characterized by an abundant extracellular matrix stroma, a local infiltration of inflammatory cells including mast cells, and a milieu of enriched cytokines. Previous studies from our laboratory demonstrated an intrinsic higher level of HIF-1alpha and VEGF protein expression in keloid tissues compared with their adjacent unremarkable skins. To further investigate the mechanisms underlying the elevated expression of HIF-1alpha and VEGF in keloids, we exposed a co-culture of keloid fibroblasts and mast cells (HMC-1) to hypoxic conditions and studied the expression of HIF-1alpha and its target gene, VEGF. Our results showed that hypoxia-dependent HIF-1alpha protein accumulation and VEGF expression is augmented in keloid fibroblasts when co-cultured with HMC-1 cells under the condition where direct cell-cell contact is allowed. But such augmentation is not observed in the transwell co-culture system whereas fibroblasts and HMC-1 cells were separated by a porous membrane. Our results also indicated that the enhancement of hypoxia-mediated activation of ERK1/2 and Akt requires direct cell-cell interaction between mast cells and keloid fibroblasts, and activation of both ERK1/2 and Akt is involved in the hypoxia-dependent HIF-1alpha protein accumulation and VEGF expression in the co-culture system. These findings suggest that under hypoxic conditions mast cells may contribute, at least in part, to an elevated expression of HIF-1alpha and VEGF protein in keloids via direct cell-cell interaction with fibroblasts.     

The effect of IL-1alpha on the expression of matrix metalloproteinases, plasminogen activators, and their inhibitors in osteoblastic ROS 17/2.8 cells

Interleukin-1 (IL-1) plays key roles in altering bone matrix turnover. This turnover is regulated by matrix metalloproteinases (MMPs), tissue inhibitor of matrix metalloproteinases (TIMPs), and the plasminogen activation system, including tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (uPA) , and plasminogen activator inhibitor type-1 (PAI-1). In this study, we examined the effect of IL-1alpha on the expression of the MMPs, TIMPs, tPA, uPA, and PAI-1 genes in osteoblasts derived from the rat osteosarcoma cell line ROS 17/2.8. The cells were cultured in alpha-minimum essential medium containing 10% fetal bovine serum with 0 or 100 U/ml of IL-1alpha for up to 14 days. The levels of MMPs, TIMPs, uPA, tPA, and PAI-1 expression were estimated by determining the mRNA levels using real-time RT-PCR and by determining protein levels using ELISA. In IL-1alpha cultures, the expression levels of MMP-1, -2, -3, -13, and -14 exceeded that of the control through day 14 of culture, and the expression of MMPs increased markedly from the proliferative to the later stages of culture. The TIMP-1, -2, and -3 expression levels increased from the initial to the proliferative stages of culture. The expression of tPA increased greatly during the proliferative stage of culture, and uPA expression increased throughout the culture period, increasing markedly from the proliferative to the later stages of culture. In contrast, PAI-1 expression decreased in the presence of IL-1alpha through day 14. These results suggest that IL-1alpha stimulate bone matrix turnover by increasing MMPs, tPA, and uPA production and decreasing PAI-1 production by osteoblasts, and incline the turnover to the resolution.     

Properties of the Trp290His variant of Fusarium NRRL 2903 galactose oxidase: interactions of the GOasesemi state with different buffers, its redox activity and ability to bind azide

The indole ring of Trp-290 in galactose oxidase has an important role in restricting entry to the substrate-binding (Cu) site of galactose oxidase via a short ～8?Å access pocket/channel. It also overlays and helps stabilise the radical-forming Cu-coordinated Tyr-272, reduction potential 400?mV. In this paper the effect of replacing Trp-290 by the less bulky His residue is explored at 25??°C, I=0.100?M (NaCl), and different effects are quantified. Interactions with buffers, not observed in the case of wild-type (WT) GOase, have been investigated by UV-Vis spectrophotometry on the non-radical GOase_semi (Cu^II) form of the Trp290His variant. Equilibrium constants K _eq/M^–1 from absorbance changes at 635?nm are for 1?:?1 interactions with the OH-containing buffers H₂PO₄ ^– (231), Hepes (43) and Tris (202), concentrations 0–60?mM. No similar interactions are observed with Mes, Lutidine and Ches, when significantly different UV-Vis spectra with no peak at ～635?nm are obtained. At pH 7.5 the reduction potential for the Trp290His GOase_ox/GOase_semi couple is 730?mV, which compares with 400?mV for the WT GOase couple. Consistent with the 730?mV value the GOase_semi form is not oxidised with [Fe(CN)₆]^3– (410?mV) or [W(CN)₈]^3– (530?mV), and much stronger oxidants such as [Mo(CN)₈]^3– (800?mV) and [IrCl₆]^2– (890?mV) are required. The GOase_ox product is unstable and decays within 20?min with re-formation of GOase_semi. From changes in UV-Vis spectra with pH, Trp290His GOase_semi gives a pK _a of 6.9, and rate constants for the oxidation of GOase_semi with [Mo(CN)₈]^3– are dependent on this same pK _a. The latter compares with 7.9 for WT GOase_semi, and is assigned here also as protonation of Tyr-495. The 1?:?1 binding of azide at the substrate-binding (H₂O) site of Trp290His GOase_semi was studied and gives a formation constant 330?M^–1 at pH 7.5, which is an order of magnitude less than the corresponding value for WT GOase_semi. The trends observed indicate less affinity of Trp290His GOase_semi for the ionic reactants H⁺ and N₃ ^–.     

Cloning,characterization, hypoxia and heat shock response of hypoxia inducible factor-1 (HIF-1) from the small abalone Haliotis diversicolor

In this study, hypoxia inducible factor-1α (HIF-1α) and hypoxia inducible factor-1β (HIF-1β) from small abalone Haliotis diversicolor were cloned. The cDNA of H. diversicolor HIF-1α (HdHIF-1α) is 2833 bp encoding a protein of 711aa and H. diversicolor HIF-1β (HdHIF-1β) is 1919 bp encoding a protein of 590aa. Similar to other species' HIF-1, HdHIF-1 has one basic helix–loop–helix (bHLH) domain and two Per-Arnt-Sim (PAS) domains, and HdHIF-1α has a oxygen-dependent degradation domain (ODDD) with two proline hydroxylation motifs and a C-terminal transactivation domain (C-TAD) with an asparagine hydroxylation motif. Under normoxic conditions, HdHIF-1α and HdHIF-1β mRNAs were constitutively present in all examined tissues. Under hypoxia (2.0 mg/L DO at 25 °C) stress, HdHIF-1α expression was up-regulated in gills at 4 h, 24 h and 96 h, and in hemocytes at 24 h and 96 h, while HdHIF-1β remained relatively constant. Under thermal stress (31 °C), HdHIF-1α expression was significantly increased in gills at 4 h, and hemocytes at 0 h and 4 h, while HdHIF-1β expression still remained relatively constant. These results suggested that HIF-1α may play an important role in adaption to poor environment in H. diversicolor.     

Structural insight into the novel iron‐coordination and domain interactions of transferrin‐1 from a model insect,Manduca sexta

Transferrins function in iron sequestration and iron transport by binding iron tightly and reversibly. Vertebrate transferrins coordinate iron through interactions with two tyrosines, an aspartate, a histidine, and a carbonate anion, and conformational changes that occur upon iron binding and release have been described. Much less is known about the structure and functions of insect transferrin‐1 (Tsf1), which is present in hemolymph and influences iron homeostasis mostly by unknown mechanisms. Amino acid sequence and biochemical analyses have suggested that iron coordination by Tsf1 differs from that of the vertebrate transferrins. Here we report the first crystal structure (2.05 Å resolution) of an insect transferrin. Manduca sexta (MsTsf1) in the holo form exhibits a bilobal fold similar to that of vertebrate transferrins, but its carboxyl‐lobe adopts a novel orientation and contacts with the amino‐lobe. The structure revealed coordination of a single Fe³⁺ ion in the amino‐lobe through Tyr90, Tyr204, and two carbonate anions. One carbonate anion is buried near the ferric ion and is coordinated by four residues, whereas the other carbonate anion is solvent exposed and coordinated by Asn121. Notably, these residues are highly conserved in Tsf1 orthologs. Docking analysis suggested that the solvent exposed carbonate position is capable of binding alternative anions. These findings provide a structural basis for understanding Tsf1 function in iron sequestration and transport in insects as well as insight into the similarities and differences in iron homeostasis between insects and humans.     

Molecular cloning,biochemical and structural analysis of elongation factor-1 alpha from Leishmania donovani: comparison with the mammalian homologue

The Src-homology 2 domain containing protein tyrosine phosphatase-1 (SHP-1) is involved in the pathogenesis of infection with Leishmania. Recently, we identified elongation factor-1 alpha (EF-1 alpha) from Leishmania donovani as a SHP-1 binding and activating protein [J. Biol. Chem. 277 (2002) 50190]. To characterize this apparent Leishmania virulence factor further, the cDNA encoding L. donovani EF-1 alpha was cloned and sequenced. Whereas nearly complete sequence conservation was observed amongst EF-1 alpha proteins from trypanosomatids, the deduced amino acid sequence of EF-1 alpha of L. donovani when compared to mammalian EF-1 alpha sequences showed a number of significant changes. Protein structure modeling-based upon the known crystal structure of EF-1 alpha for Saccharomyces cerevisiae-identified a hairpin loop present in mammalian EF-1 alpha and absent from the Leishmania protein which corresponded to a 12 amino acid deletion. Consistent with these structural differences, the sub-cellular distributions of L. donovani EF-1 alpha and host EF-1 alpha were strikingly different. Interestingly, infection of macrophages with L. donovani caused redistribution of host as well as pathogen EF-1 alpha. Since EF-1 alpha is essential for survival, the distinct biochemical and structural properties of Leishmania EF-1 alpha may provide a novel target for drug development.     

Membrane attachment and fusion of HIV-1, influenza A,and SARS-CoV-2: resolving the mechanisms with biophysical methods

Attachment to and fusion with cell membranes are two major steps in the replication cycle of many human viruses. We focus on these steps for three enveloped viruses, i.e., HIV-1, IAVs, and SARS-CoV-2. Viral spike proteins drive the membrane attachment and fusion of these viruses. Dynamic interactions between the spike proteins and membrane receptors trigger their specific attachment to the plasma membrane of host cells. A single virion on cell membranes can engage in binding with multiple receptors of the same or different types. Such dynamic and multivalent binding of these viruses result in an optimal attachment strength which in turn leads to their cellular entry and membrane fusion. The latter process is driven by conformational changes of the spike proteins which are also class I fusion proteins, providing the energetics of membrane tethering, bending, and fusion. These viruses exploit cellular and membrane factors in regulating the conformation changes and membrane processes. Herein, we describe the major structural and functional features of spike proteins of the enveloped viruses including highlights on their structural dynamics. The review delves into some of the case studies in the literature discussing the findings on multivalent binding, membrane hemifusion, and fusion of these viruses. The focus is on applications of biophysical tools with an emphasis on single-particle methods for evaluating mechanisms of these processes at the molecular and cellular levels.