ABSTRACT
Interleukin-6 (IL-6) is a pleiotropic cytokine that regulates immune and inflammatory responses, and its overproduction is a hallmark of inflammatory diseases. Inhibition of IL-6 signaling with the anti-IL-6 receptor antibody tocilizumab has provided some clinical benefit to patients; however, direct cytokine inhibition may be a more effective option. We used the systematic evolution of ligands by exponential enrichment (SELEX) process to discover slow off-rate modified aptamers (SOMAmers) with hydrophobic base modifications that inhibit IL-6 signaling in vitro. Two classes of IL-6 SOMAmers were isolated from modified DNA libraries containing 40 random positions and either 5-(N-benzylcarboxamide)-2'-deoxyuridine (Bn-dU) or 5-[N-(1-naphthylmethyl)carboxamide]-2'-deoxyuridine (Nap-dU) replacing dT. These modifications facilitate the high affinity binding interaction with IL-6 and provide resistance against degradation by serum endonucleases. Post-SELEX optimization of one Bn-dU and one Nap-dU SOMAmer led to improvements in IL-6 binding (10-fold) and inhibition activity (greater than 20-fold), resulting in lead SOMAmers with sub-nanomolar affinity (Kd = 0.2 nm) and potency (IC50 = 0.2 nm). Although similar in inhibition properties, the two SOMAmers have unique sequences and different ortholog specificities. Furthermore, these SOMAmers were stable in human serum in vitro for more than 48 h. Both SOMAmers prevented IL-6 signaling by blocking the interaction of IL-6 with its receptor and inhibited the proliferation of tumor cells in vitro as effectively as tocilizumab. This new class of IL-6 inhibitor may be an effective therapeutic alternative for patients suffering from inflammatory diseases.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Aptamers, Nucleotide/pharmacology , Interleukin-6/antagonists & inhibitors , Interleukin-6/immunology , Receptors, Interleukin-6/immunology , Amino Acid Sequence , Animals , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/metabolism , Aptamers, Nucleotide/chemistry , Aptamers, Nucleotide/metabolism , Base Sequence , CHO Cells , Cricetulus , Drug Discovery , Humans , Interleukin-6/chemistry , Interleukin-6/metabolism , Macaca fascicularis , Mice , Molecular Sequence Data , Rats , SELEX Aptamer Technique/methods , Serum/metabolismABSTRACT
3-[6-(2-Dimethylamino-1-imidazol-1-yl-butyl)-naphthalen-2-yloxy]-2,2-dimethyl-propionic acid and analogs were designed and synthesized as highly potent and selective CYP26 inhibitors, serving as retinoic acid metabolic blocking agents (RAMBAs), with demonstrated in vivo efficacy to increase the half-life of exogenous atRA.
Subject(s)
Enzyme Inhibitors/pharmacology , Naphthalenes/pharmacology , Propionates/pharmacology , Tretinoin/metabolism , Chromatography, High Pressure Liquid , Cytochrome P-450 Enzyme Inhibitors , Cytochrome P-450 Enzyme System , Enzyme Inhibitors/pharmacokinetics , Half-Life , Mass Spectrometry , Naphthalenes/pharmacokinetics , Propionates/pharmacokinetics , Retinoic Acid 4-Hydroxylase , Tretinoin/antagonists & inhibitorsABSTRACT
A series of [2-imidazol-1-yl-2-(6-alkoxy-naphthalen-2-yl)-1-methyl-ethyl]-dimethyl-amines were designed and synthesized as CYP26 inhibitors, serving as retinoic acid metabolic blocking agents (RAMBA's).
