ABSTRACT
Promutagenic O6-alkylguanine adducts in DNA are repaired in humans by O6-methylguanine-DNA-methyltransferase (MGMT) in an irreversible reaction. Here we describe the synthesis of a phosphoramidite that allows the preparation of oligodeoxyribonucleotides (ODNs) containing a novel tricyclic thio analogue of O6-methylguanine in which the third ring bridges the 6-thio group and C7 of a 7-deazapurine. These ODNs are very poor substrates for MGMT and poorly recognised by the alkyltransferase-like protein, Atl1. Examination of the active sites of both MGMT and Atl1 suggest large steric clashes hindering binding of the analogue. Such analogues, if mutagenic, are likely to be highly toxic.
Subject(s)
Alkyl and Aryl Transferases/chemistry , Guanine/analogs & derivatives , O(6)-Methylguanine-DNA Methyltransferase/chemistry , Oligodeoxyribonucleotides/chemistry , Sulfhydryl Compounds/chemistry , Alkyl and Aryl Transferases/metabolism , Guanine/chemistry , Guanine/metabolism , Humans , Models, Molecular , Molecular Structure , O(6)-Methylguanine-DNA Methyltransferase/metabolism , Oligodeoxyribonucleotides/chemical synthesis , Oligodeoxyribonucleotides/metabolism , Sulfhydryl Compounds/metabolismABSTRACT
The development of tools for the early diagnosis of pancreatic adenocarcinoma is an urgent need in order to increase treatment success rate and reduce patient mortality. Here, we present a modular nanosystem platform integrating soft nanoparticles with a targeting peptide and an active imaging agent for diagnostics. Biocompatible single-chain polymer nanoparticles (SCPNs) based on poly(methacrylic acid) were prepared and functionalized with the somatostatin analogue PTR86 as the targeting moiety, since somatostatin receptors are overexpressed in pancreatic cancer. The gamma emitter 67Ga was incorporated by chelation and allowed in vivo investigation of the pharmacokinetic properties of the nanoparticles using single photon emission computerized tomography (SPECT). The resulting engineered nanosystem was tested in a xenograph mouse model of human pancreatic adenocarcinoma. Imaging results demonstrate that accumulation of targeted SCPNs in the tumor is higher than that observed for nontargeted nanoparticles due to improved retention in this tissue.
Subject(s)
Adenocarcinoma/genetics , Nanoparticles/administration & dosage , Pancreatic Neoplasms/genetics , Somatostatin/biosynthesis , Adenocarcinoma/diagnosis , Adenocarcinoma/pathology , Animals , Cell Line, Tumor , Early Detection of Cancer , Gene Expression Regulation, Neoplastic/drug effects , Humans , Mice , Nanoparticles/chemistry , Pancreatic Neoplasms/diagnosis , Pancreatic Neoplasms/pathology , Polymers/chemistry , Polymethacrylic Acids/administration & dosage , Polymethacrylic Acids/chemistry , Somatostatin/chemistry , Xenograft Model Antitumor Assays , Pancreatic NeoplasmsABSTRACT
The need for test systems for nanoparticle biocompatibility, toxicity, and inflammatory or adaptive immunological responses is paramount. Nanoparticles should be free of microbiological and chemical contaminants, and devoid of toxicity. Nevertheless, in the absence of contamination, these particles may still induce undesired immunological effects in vivo, such as enhanced autoimmunity, hypersensitivity reactions, and fibrosis. Here we show that artificial particles of specific sizes affect immune cell recruitment as tested in a dermal air pouch model in mice. In addition, we demonstrate that the composition of nanoparticles may influence immune cell recruitment in vivo. Aside from biophysical characterizations in terms of hydrodynamic diameter, zeta potential, concentration, and atomic concentration of metals, we show that - after first-line in vitro assays - characterization of cellular and molecular effects by dermal air pouch analysis is straightforward and should be included in the quality control of nanoparticles. We demonstrate this for innate immunological effects such as neutrophil recruitment and the production of immune-modulating matrix metalloproteases such as MMP-9; we propose the use of air pouch leukocytosis analysis as a future standard assay.
Subject(s)
Air , Biological Assay/methods , Leukocytosis/chemically induced , Materials Testing/methods , Nanoparticles/toxicity , Toxicity Tests/methods , Animals , Biological Assay/instrumentation , Materials Testing/instrumentation , Mice , Particle Size , Polystyrenes , Toxicity Tests/instrumentationABSTRACT
We show that DNA containing a conformationally-locked anti analogue of O(6)-alkylguanine is a poor substrate for human O(6)-methylguanine-DNA methyltransferase (MGMT) and the alkyltransferase-like protein, Atl1. This highlights the requirement for the syn conformation and rationalises why certain O(6)-alkylguanines are poor MGMT substrates.
Subject(s)
Alkyl and Aryl Transferases/metabolism , Guanine/metabolism , O(6)-Methylguanine-DNA Methyltransferase/metabolism , Oligodeoxyribonucleotides/biosynthesis , Crystallography, X-Ray , Guanine/analogs & derivatives , Humans , Protein Binding , Protein Structure, TertiaryABSTRACT
The syntheses of novel tricyclic pyrrolo[2,3-d]pyrimidine analogues of O(6)-methylguanine and S(6)-methylthioguanine are described. The crystal structures and pK(a) values of these analogues are reported. In a standard substrate assay with the human repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) only the oxygen-containing analogue displayed activity.
Subject(s)
Guanine/analogs & derivatives , Crystallography, X-Ray , Drug Design , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Guanine/chemical synthesis , Guanine/chemistry , Guanine/pharmacology , Humans , In Vitro Techniques , Kinetics , Molecular Structure , O(6)-Methylguanine-DNA Methyltransferase/antagonists & inhibitorsABSTRACT
The syntheses of novel tricyclic pyrrolo[2,3-d]pyrimidine analogues of S6-methylthioguanine are described. The crystal structures and pKa values of these and related O6-methylguanine analogues are reported. All compounds display higher pKa values than O6-methylguanine with the sulfur-containing analogues being the more basic and exhibiting higher stability in aqueous solution. In a standard substrate assay with the human repair protein O6-methylguanine-DNA methyltransferase (MGMT) only the oxygen-containing analogue displayed activity.
