SignificanceAptamers are now used ubiquitously as binding agents for a broad range of applications. Natural (unmodified) DNA and RNA aptamers have considerably less chemical diversity than protein-based ligands such as antibodies, limiting their utility. Aptamers possessing a single chemical modification have helped bridge this diversity gap. We report the selection and identification of aptamers with two diversity-enhancing chemical modifications that bind and inhibit proprotein convertase subtilisin/kexin type 9 (PCSK9), a representative human therapeutic protein target. The addition of a second modification, especially in certain pairwise combinations, resulted in significant improvements in affinity, ligand efficiency, epitope coverage, metabolic stability, and inhibitory activity. Extensively chemically functionalized aptamers have the potential to become the next generation of nucleic-acid–based ligands.
It has recently been shown that the transcription factor Erg, an Ets family member, drives constitutive expression of the intercellular adhesion molecule 2 (ICAM-2) in human umbilical vein endothelial cells (HUVECs) and that its expression is downregulated by the pleiotropic cytokine tumor necrosis factor ␣ (TNF-␣). To identify other Erg target genes and to define its function in the endothelium, a combined approach of antisense oligonucleotides (GeneBloc) and differential gene expression was used. Treatment of HUVECs with Erg-specific GeneBloc for 24, 48, and 72 hours suppressed Erg mRNA and protein levels at all time points. Total RNA extracted from HUVECs treated with Ergspecific or control GeneBloc was analyzed for differences in gene expression using high-density, sequence-verified cDNA arrays containing 482 relevant genes. Inhibition of Erg expression resulted in decreased expression of ICAM-2, as predicted. Four more genes decreased in Erg-deficient HUVECs were the extracellular matrix proteins SPARC and thrombospondin, the adhesive glycoprotein von Willebrand factor, and the small GTPase RhoA. Each of these molecules has been directly or indirectly linked to angiogenesis because of its role in vascular remodeling, adhesion, or shape change. Therefore, the role of Erg in vascular remodeling was tested in an in vitro model, and the results showed that HUVECs treated with Erg GeneBloc had a decreased ability to form tubulelike structures when grown on Matrigel. These results suggest that Erg may be a mediator of the TNF-␣ effects on angiogenesis in vivo. IntroductionThe endothelium plays a key role in a large number of diseases, among them cancer and metastasis, rheumatoid arthritis, atherosclerosis, and thrombosis, by regulating several key processes such as leukocyte recruitment, hemostasis, vascular permeability, and angiogenesis. During inflammation, several mediators can activate the endothelium to promote a proinflammatory, prothrombotic state and to regulate the angiogenic response. Angiogenesis (or new vessel formation) is a tightly regulated process that requires the integration of signals triggered by growth factors and adhesion events, namely modulation of cell-cell contact and interaction with the extracellular matrix. 1 Angiogenesis is a critical aspect of the development, growth, and repair of new tissues; it occurs physiologically in circumstances such as wound healing and the menstrual cycle. It is also a key component of many diseases driven by tissue proliferation, such as cancer and rheumatoid arthritis. 2,3 In such diseases, new vessel formation supports tissue growth and cellular infiltration, and it contributes to the destructive proliferation of the inflammatory tissue. The link between angiogenesis and inflammation has been suggested by various studies that focus on common features such as the cellular infiltrate, proliferation, and overlapping roles of inflammatory mediators and growth factors. [4][5][6] One such mediator is the pleiotropic cytokine tumor necrosis factor ␣ (TNF-␣),...
Ribozymes are catalytic RNA molecules that can be designed to cleave specific RNA sequences. To investigate the potential use of synthetic stabilized ribozymes for the treatment of chronic hepatitis C virus (HCV) infection, we designed and synthesized hammerhead ribozymes targeting 15 conserved sites in the 5Ј untranslated region (UTR) of HCV RNA. This region forms an internal ribosome entry site that allows for efficient translation of the HCV polyprotein. The 15 synthetic ribozymes contained modified nucleotides and linkages that stabilize the molecules against nuclease degradation. All 15 ribozymes were tested for their ability to reduce expression in an HCV 5Ј UTR/ luciferase reporter system and for their ability to inhibit replication of an HCV-poliovirus (HCV-PV) chimera. Treatment with several ribozymes resulted in significant downregulation of HCV 5Ј UTR/luciferase reporter expression (range 40% to 80% inhibition, P F .05). Moreover, several ribozymes showed significant inhibition (G90%, Chronic infection with hepatitis C virus (HCV) can lead to cirrhosis, liver failure and/or hepatocellular carcinoma over a period of 10 to 20 years. 1,2 The Centers for Disease Control recently reported the number of chronically infected Americans to be approximately 4.5 million; thus, HCV infection is over four times as prevalent as human immunodeficiency virus infection. 3 Worldwide the prevalence of chronic HCV is similar to that found in the United States. 3 Thus, chronic HCV infection represents an important public health problem throughout the world.PTreatment of chronic HCV infection with interferon alfa leads to sustained viral clearance in only approximately 12% of patients. 4 Newer therapeutic regimes, such as the combination of interferon alfa and ribavirin, can lead to 38% to 43% of patients having a sustained virological response. 5,6 However, even with current combination regimes, approximately 60% of patients have no sustained virological benefit. Additionally, treatment with interferon alfa and ribavirin leads to significant toxicities. 5,6 Therefore, there still remains a great need for improved therapeutic modalities.HCV is a 9.5-kb, plus-strand, RNA virus that is a member of the human flavivirus family. 7,8 Although the sequence of the HCV-RNA genome is highly variable among clinical isolates, the 5Ј untranslated region (UTR) of the genome is highly conserved with respect to RNA sequence identity. 9,10 The conserved sequence/structure of the 5Ј UTR of HCV RNA contains an internal ribosome entry site (IRES) to mediate translation independent of a 5Ј-cap structure. 10,11 The HCV IRES does not require any viral protein for initiation of translation 12 and IRES elements also occur in cellular messenger RNAs. 13,14 Because the components of IRES-mediated translation are shared between cellular and HCV messenger RNAs, targeting the mechanism of HCV-IRES-mediated translation can be problematic. However, because the HCV-IRES sequence is highly conserved among viral genotypes, it is an excellent target for ribo...
Inhibition of the checkpoint kinase Chk1, both as a monotherapy and in combination with DNA damaging cytotoxics, is a promising therapeutic approach for the treatment of a wide array of human cancers. However, much remains to be elucidated in regard to the patient populations that will respond best to a Chk1 inhibitor and the optimal therapeutics to combine with a Chk1 inhibitor. In an effort to discover sensitizing mutations and novel combination strategies for Chk1 inhibition, an siRNA screen was performed in combination with the selective Chk1 inhibitor AR458323. This screen employed a custom made library of siRNAs targeting 195 genes, most of which are involved in cell-cycle control or DNA damage repair. One of the most prominent and consistent hits across runs of the screen performed in three different cancer cell lines was Wee1 kinase. MK-1775 is a small molecule inhibitor of Wee1 that is currently in early stage clinical trials. In confirmation of the results obtained from the siRNA screen, AR458323 and MK-1775 synergistically inhibited proliferation in multiple cancer cell types. This antiproliferative effect correlated with a synergistic induction of apoptosis. In cellular mechanistic studies, the combination of the two molecules resulted in dramatic decreases in inhibitory phosphorylation of cyclin-dependent kinases, an increase in DNA damage, alterations in cell-cycle profile, and collapse of DNA synthesis. In conclusion, the clinical combination of a Chk1 inhibitor and a Wee1 inhibitor holds promise as an effective treatment strategy for cancer.
IL-1α is an essential cytokine that contributes to inflammatory responses and is implicated in various forms of pathogenesis and cancer. Here we report a naphthyl modified DNA aptamer that specifically binds IL-1α and inhibits its signaling pathway. By solving the crystal structure of the IL-1α/aptamer, we provide a high-resolution structure of this critical cytokine and we reveal its functional interaction interface with high-affinity ligands. The non-helical aptamer, which represents a highly compact nucleic acid structure, contains a wealth of new conformational features, including an unknown form of G-quadruplex. The IL-1α/aptamer interface is composed of unusual polar and hydrophobic elements, along with an elaborate hydrogen bonding network that is mediated by sodium ion. IL-1α uses the same interface to interact with both the aptamer and its cognate receptor IL-1RI, thereby suggesting a novel route to immunomodulatory therapeutics.
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