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The chemokine receptor CXCR4 mediates the migration of hematopoietic cells to the stroma-derived factor 1A (SDF-1A) -producing bone marrow microenvironment. Using peptide-based CXCR4 inhibitors derived from the chemokine viral macrophage inflammatory protein II, we tested the hypothesis that the inhibition of CXCR4 increases sensitivity to chemotherapy by interfering with stromal/leukemia cell interactions. First, leukemic cells expressing varying amounts of surface CXCR4 were examined for their chemotactic response to SDF-1A or stromal cells, alone or in the presence of different CXCR4 inhibitors. Results showed that the polypeptide RCP168 had the strongest antagonistic effect on the SDF-1A -or stromal cell -induced chemotaxis of leukemic cells. Furthermore, RCP168 blocked the binding of anti-CXCR4 monoclonal antibody 12G5 to surface CXCR4 in a concentration-dependent manner and inhibited SDF-1A -induced AKT and extracellular signal-regulated kinase phosphorylation. Finally, RCP168 significantly enhanced chemotherapy-induced apoptosis in stroma-cocultured Jurkat, primary chronic lymphocytic leukemia, and in a subset of acute myelogenous leukemia cells harboring Flt3 mutation. Equivalent results were obtained with the small-molecule CXCR4 inhibitor AMD3465. Our data therefore suggest that the SDF-1A/CXCR4 interaction contributes to the resistance of leukemia cells to chemotherapy-induced apoptosis. Disruption of these interactions by the peptide CXCR4 inhibitor RCP168 represents a novel strategy for targeting leukemic cells within the bone marrow microenvironment. [Mol Cancer Ther 2006;5(12):3113 -21]

Human cytochrome P450 aromatase catalyzes with high specificity the synthesis of estrogens from androgens. Aromatase inhibitors (AIs) such as exemestane, 6-methylideneandrosta-1,4-diene-3,17-dione, are preeminent drugs for the treatment of estrogen-dependent breast cancer. The crystal structure of human placental aromatase has shown an androgen-specific active site. By utilization of the structural data, novel C6-substituted androsta-1,4-diene-3,17-dione inhibitors have been designed. Several of the C6-substituted 2-alkynyloxy compounds inhibit purified placental aromatase with IC50 values in the nanomolar range. Antiproliferation studies in a MCF-7 breast cancer cell line demonstrate that some of these compounds have EC50 values better than 1 nM, exceeding that for exemestane. X-ray structures of aromatase complexes of two potent compounds reveal that, per their design, the novel side groups protrude into the opening to the access channel unoccupied in the enzyme–substrate/exemestane complexes. The observed structure–activity relationship is borne out by the X-ray data. Structure-guided design permits utilization of the aromatase-specific interactions for the development of next generation AIs.

The chemokine receptor CXCR4 is critical for many biological functions, such as B-cell lymphopoiesis, regulation of neuronal cell migration, and vascular development (1-3). In addition, CXCR4 together with another chemokine receptor CCR5 are two principal co-receptors for the cellular entry of the human immunodeficiency virus type 1 (HIV-1) 1 (4 -7). The stromal cell-derived factor-1 (SDF-1␣) is the only known natural ligand of CXCR4 and plays important roles in migration, proliferation, and differentiation of leukocytes (8, 9). The viral macrophage inflammatory protein II (vMIP-II) encoded by human herpesvirus 8 (10) is an antagonistic chemokine ligand of CXCR4 (11, 12). vMIP-II also interacts with other chemokine receptors such as CCR5 and CCR3 and inhibits HIV-1 entry mediated by these co-receptors.CXCR4 and other chemokine receptors belong to the superfamily of seven transmembrane G-protein-coupled receptors (GPCRs) (13). These membrane proteins transmit signals from extracellular ligands to intracellular biological pathways via heterotrimeric G-proteins and have been a major class of therapeutic targets for a wide variety of human diseases (14). As such, characterizing the mechanism of biological recognition between these receptors and their ligands is essential for understanding the physiological or pathological processes that they mediate and devising novel strategies for clinical intervention. For CXCR4, studies have been carried out by a number of laboratories using chimeric chemokine receptors and site-specific mutants to study multiple domains of CXCR4 that are important for interacting with chemokine ligands and HIV-1 (15-23). However, because there is no high resolution crystal structure available for CXCR4 (or any other chemokine receptor) alone or complexed with ligands, the structural and biochemical basis of ligand binding and signaling through these important membrane receptors remains poorly understood.To further define the structure-function relationship of the chemokine receptor-ligand interaction, theoretical computer modeling and site-directed mutagenesis were combined to predict plausible structural models for chemokine receptors and their complexes with ligands, such as interleukin-8 receptor ␤ (24) and CCR5 (25,26). Structural models of CXCR4 and its complex with ligands were also proposed (27, 28). Complementary to modeling and mutational analyses of the receptors,

The entry of human immunodeficiency virus type 1 (HIV-1) into the cell is initiated by the interaction of the viral surface envelope protein with two cell surface components of the target cell, CD4 and a chemokine coreceptor, usually CXCR4 or CCR5. The natural ligand of CXCR4 is stromal cell-derived factor 1␣ (SDF-1␣). Whereas the overlap between HIV-1 and SDF-1␣ functional sites on the extracellular domains of CXCR4 has been well documented, it has yet to be determined whether there are sites in the transmembrane (TM) helices of CXCR4 important for HIV-1 and/or SDF-1␣ functions, and if such sites do exist, whether they are overlapping or distinctive for the separate functions of CXCR4.

BackgroundChemoresistance is one of the major obstacles for cancer therapy in the clinic. Nuclear paraspeckle assembly transcript 1 (NEAT1) has been reported as an oncogene in most malignancies such as lung cancer, esophageal cancer, and gastric cancer. This study is designed to investigate the function of NEAT1 in paclitaxel (PTX) resistance of ovarian cancer and its potential molecular mechanism.Patients and methodsThe expressions of NEAT1 and miR-194 in ovarian cancer tissues and cells were estimated by quantitative real-time polymerase chain reaction (qRT-PCR). MTT, flow cytometry, and Western blot assays were used to assess the effect of NEAT1 on PTX resistance in PTX-resistant ovarian cancer cells. Luciferase reporter assay was applied to examine the association between NEAT1, zinc finger E-box-binding homeobox 1 (ZEB1) and miR-194. Xenograft tumor model was established to confirm the biological role of NEAT1 in PTX resistance of ovarian cancer in vivo.ResultsNEAT1 was upregulated, and miR-194 was downregulated in PTX-resistant ovarian cancer tissues and cells. Functionally, NEAT1 knockdown enhanced cell sensitivity to PTX via promoting PTX-induced apoptosis in vitro. NEAT1 was identified as a molecular sponge of miR-194 to upregulate ZEB1 expression. Mechanistically, NEAT1-knockdown-induced PTX sensitivity was mediated by miR-194/ZEB1 axis. Moreover, NEAT1 knockdown improved PTX sensitivity of ovarian cancer in vivo.ConclusionNEAT1 contributed to PTX resistance of ovarian cancer cells at least partly through upregulating ZEB1 expression by sponging miR-194, elucidating a novel regulatory pathway of chemoresistance in PTX-resistant ovarian cancer cells and providing a possible long noncoding RNA (lncRNA)-targeted therapy for ovarian cancer.

The direct fusion of viral and target cell membranes required for human immunodeficiency virus type 1 (HIV-1) entry is initiated by the primary receptor, CD4, and a chemokine receptor, usually CXCR4 or CCR5. Chemokine receptors are members of the G-protein-coupled receptor (GPCR) superfamily that possess seven transmembrane (TM) domains. Because of its importance in the development of AIDS, CXCR4 has been explored as a new target for drug discovery to combat the AIDS epidemic (3,8,10). As the natural ligands of chemokine receptors, chemokines are small soluble proteins of about 70 amino acid residues that play prominent roles in leukocyte activation and inflammation (5, 11). Most of the known human chemokines are broadly categorized into the CXC and CC chemokines based on the position of two conserved cysteine residues in their amino (N)-terminal domains (3, 11). The natural chemokines of CXCR4 or CCR5 can inhibit HIV-1 infection (4, 13) by blocking HIV-1 gp120 binding sites (2, 14) and/or inducing receptor internalization (1, 9).Despite their important roles in the pathogenesis of AIDS and other human diseases, the lack of receptor selectivity of natural chemokines has made their direct clinical applications problematic. It is common knowledge that a chemokine receptor can often be recognized by multiple ligands, while a chemokine ligand binds to several different receptors (15), illustrating the apparent redundancy and the lack of selectivity in the chemokine ligand-receptor interaction network. As such, we have been working toward the development of a systematic chemical biology approach based on chemokine protein structures and chemistry to generate synthetically and modularly modified (SMM) chemokines that have higher receptor binding selectivity and improved pharmacological profiles compared with natural chemokines. This SMM chemokine approach was recently applied to generate novel ligands selective for CXCR4 or CCR5 by modifying the N-terminal (1-10) sequence module of viral macrophage inflammatory protein II (vMIP-II) or stromal cell-derived factor 1␣ (SDF-1␣) (unpublished data). Importantly, some of these SMM chemokines,