Labeling of Synthetic Oligonucleotides Using the Klenow Fragment of E. coli DNA Polymerase I

In this method, a short primer is hybridized to an oligonucleotide template whose sequence is the complement of the desired radiolabeled probe. The primer is then extended using the Klenow fragment to incorporate [α-32P]dNTPs in a template-directed manner. After the reaction, the template and product are separated by denaturation followed by electrophoresis through a polyacrylamide gel under denaturing conditions. With this method, it is possible to generate oligonucleotide probes that contain several radioactive atoms per molecule of oligonucleotide and to achieve specific activities as high as 2 × 1010 cpm/µg of probe. Because the end product of the reaction is dsDNA, whose strands must be separated and the labeled product isolated, this method is generally not used to prepare nonradiolabeled oligonucleotides.

Chemical tools for epichaperome-mediated interactome dysfunctions of the central nervous system

Diseases are a manifestation of how thousands of proteins interact. In several diseases, such as cancer and Alzheimer’s disease, proteome-wide disturbances in protein-protein interactions are caused by alterations to chaperome scaffolds termed epichaperomes. Epichaperome-directed chemical probes may be useful for detecting and reversing defective chaperomes. Here we provide structural, biochemical, and functional insights into the discovery of epichaperome probes, with a focus on their use in central nervous system diseases. We demonstrate on-target activity and kinetic selectivity of a radiolabeled epichaperome probe in both cells and mice, together with a proof-of-principle in human patients in an exploratory single group assignment diagnostic study (ClinicalTrials.gov Identifier: NCT03371420). The clinical study is designed to determine the pharmacokinetic parameters and the incidence of adverse events in patients receiving a single microdose of the radiolabeled probe administered by intravenous injection. In sum, we introduce a discovery platform for brain-directed chemical probes that specifically modulate epichaperomes and provide proof-of-principle applications in their use in the detection, quantification, and modulation of the target in complex biological systems.

Synthesis and Fundamental Evaluation of Radioiodinated Rociletinib (CO-1686) as a Probe to Lung Cancer with L858R/T790M Mutations of Epidermal Growth Factor Receptor (EGFR)

Rociletinib (CO-1686), a 2,4-diaminopyrimidine derivative, is a highly potent tyrosine kinase inhibitor (TKI) that acts on epidermal growth factor receptor (EGFR) with L858R/T790M mutations. We supposed radioiodinated CO-1686 would function as a useful tool for monitoring EGFR L858R/T790M mutations. To aid in patient selection before therapy with EGFR-TKIs, this study aimed to develop a 125I-labeled derivative of CO-1686, N-{3-[(2-{[4-(4-acetylpiperazin-1-yl)-2-methoxyphenyl]amino}-5-(trifluoromethyl)pyrimidine-4-yl] amino}-5-([125I]iodophenyl)acrylamide ([125I]ICO1686) and evaluate its selectivity toward EGFR L858R/T790M. Radiosynthesis was performed by iododestannylation of the corresponding tributylstannyl precursor with [125I]NaI and N-chlorosuccinimide. The selectivity of the tracer for detecting EGFR L858R/T790M was evaluated using three relevant non-small cell lung cancer (NSCLC) cell lines—H1975, H3255 and H441 overexpressing the dual mutation EGFR L858R/T790M, active mutant EGFR L858R and wild-type EGFR, respectively. The nonradioactive ICO1686 and the precursor compound were successfully synthesized. A novel radiolabeled probe, [125I]ICO1686, was prepared with high radiochemical yield (77%) and purity (>99%). ICO1686 exhibited high cytotoxicity toward H1975 (IC50 0.20 ± 0.05 μM) and H3255 (IC50 0.50 ± 0.21 μM), which is comparable to that of CO-1686. In contrast, the cytotoxicity of ICO1686 toward H441 was 10-fold lower than that toward H1975. In the cell uptake study, the radioactivity uptake of [125I]ICO1686 in H1975 was 101.52% dose/mg, whereas the uptakes in H3255 and H441 were 33.52 and 8.95% dose/mg, respectively. The uptake of [125I]ICO1686 in H1975 was greatly reduced to 45.61% dose/mg protein by treatment with excess CO-1686. In vivo biodistribution study of the radiotracer found that its accumulation in H1975 tumor (1.77 ± 0.43% ID/g) was comparable to that in H3255 tumor (1.63 ± 0.23% ID/g) and the accumulation in H1975 tumor was not reduced by pretreatment with an excess dose of CO-1686. Although this radiotracer exhibited highly specific in vitro uptake in target cancer cells, structural modification is required to improve in vivo biodistribution.

Non-invasive molecular imaging and reporter genes

Molecular-genetic imaging in living organisms has become a new field with the exceptional growth over the past 5 years. Modern imaging is based on three technologies: nuclear, magnetic resonance and optical imaging. Most current molecular-genetic imaging strategies are “indirect,” coupling a “reporter gene” with a complimentary “reporter probe.” The reporter transgene usually encodes for an enzyme, receptor or transporter that selectively interacts with a radiolabeled probe and results in accumulation of radioactivity in the transduced cell. In addition, reporter systems based on the expression of fluorescence or bioluminescence proteins are becoming more widely applied in small animal imaging. This review begins with a description of Positron Emission Tomography (PET)-based imaging genes and their complimentary radiolabeled probes that we think will be the first to enter clinical trials. Then we describe other imaging genes, mostly for optical imaging, which have been developed by investigators working with a variety of disease models in mice. Such optical reporters are unlikely to enter the clinic, at least not in the near-term. Reporter gene constructs can be driven by constitutive promoter elements and used to monitor gene therapy vectors and the efficacy of gene targeting and transduction, as well as to monitor adoptive cell-based therapies. Inducible promoters can be used as “sensors” to monitor endogenous cell processes, including specific intracellular molecular-genetic events and the activity of signaling pathways, by regulating the magnitude of reporter gene expression.