The histone H3.1 variant regulates TONSOKU-mediated DNA repair during replication

The tail of replication-dependent histone H3.1 varies from that of replication-independent H3.3 at the amino acid located at position 31 in plants and animals, but no function has been assigned to this residue to demonstrate a unique and conserved role for H3.1 during replication. Here, we show that TONSOKU (TSK/TONSL), which rescues broken replication forks, specifically interacts with H3.1 via recognition of alanine 31 by its tetratricopeptide repeat domain. Our results indicate that genomic instability in the absence of ATXR5/ATXR6-catalyzed H3K27me1 in plants depends on H3.1, TSK and DNA polymerase theta (Pol θ). Overall, this work reveals an H3.1-specific function during replication and the common strategy used in multicellular eukaryotes for regulating post-replicative chromatin maturation and TSK, which relies on histone mono-methyltransferases and reading the H3.1 variant

PET Imaging of Fatty Acid Amide Hydrolase with [18F]DOPP in Nonhuman Primates

Fatty acid amide hydrolase (FAAH) regulates endocannabinoid signaling. [11C]CURB, an irreversibly binding FAAH inhibitor, has been developed for clinical research imaging with PET. However, no fluorine-18 labeled radiotracer for FAAH has yet advanced to human studies. [18F]DOPP ([18F]3-(4,5-dihydrooxazol-2-yl)phenyl (5-fluoropentyl)carbamate) has been identified as a promising 18F-labeled analogue based on rodent studies. The goal of this work is to evaluate [18F]DOPP in nonhuman primates to support its clinical translation. High specific activity [18F]DOPP (5–6 Ci·μmol–1) was administered intravenously (iv) to three baboons (2M/1F, 3–4 years old). The distribution and pharmacokinetics were quantified following a 2 h dynamic imaging session using a simultaneous PET/MR scanner. Pretreatment with the FAAH-selective inhibitor, URB597, was carried out at 200 or 300 μg/kg iv, 10 min prior to [18F]DOPP administration. Rapid arterial blood sampling for the first 3 min was followed by interval sampling with metabolite analysis to provide a parent radiotracer plasma input function that indicated ∼95% baseline metabolism at 60 min and a reduced rate of metabolism after pretreatment with URB597. Regional distribution data were analyzed with 1-, 2-, and 3-tissue compartment models (TCMs), with and without irreversible trapping since [18F]DOPP covalently links to the active site of FAAH. Consistent with previous findings for [11C]CURB, the 2TCM with irreversible binding was found to provide the best fit for modeling the data in all regions. The composite parameter λk3 was therefore used to evaluate whole brain (WB) and regional binding of [18F]DOPP. Pretreatment studies showed inhibition of λk3 across all brain regions (WB baseline: 0.112 mL/cm3/min; 300 μg/kg URB597: 0.058 mL/cm3/min), suggesting that [18F]DOPP binding is specific for FAAH, consistent with previous rodent data

Synthesis of [ 11 C]Bexarotene by Cu-Mediated [ 11 C]Carbon Dioxide Fixation and Preliminary PET Imaging

Chemistry and Chemical Biolog

¹¹CO bonds made easily for positron emission tomography radiopharmaceuticals

The positron-emitting radionuclide carbon-11 ((11)C, t1/2 = 20.3 min) possesses the unique potential for radiolabeling of any biological, naturally occurring, or synthetic organic molecule for in vivo positron emission tomography (PET) imaging. Carbon-11 is most often incorporated into small molecules by methylation of alcohol, thiol, amine or carboxylic acid precursors using [(11)C]methyl iodide or [(11)C]methyl triflate (generated from [(11)C]carbon dioxide or [(11)C]methane). Consequently, small molecules that lack an easily substituted (11)C-methyl group are often considered to have non-obvious strategies for radiolabeling and require a more customized approach. [(11)C]Carbon dioxide itself, [(11)C]carbon monoxide, [(11)C]cyanide, and [(11)C]phosgene represent alternative reactants to enable (11)C-carbonylation. Methodologies developed for preparation of (11)C-carbonyl groups have had a tremendous impact on the development of novel PET tracers and provided key tools for clinical research. (11)C-Carbonyl radiopharmaceuticals based on labeled carboxylic acids, amides, carbamates and ureas now account for a substantial number of important imaging agents that have seen translation to higher species and clinical research of previously inaccessible targets, which is a testament to the creativity, utility and practicality of the underlying radiochemistry

Practical Radiosynthesis and Preclinical Neuroimaging of [11C]isradipine, A Calcium Channel Antagonist

In the interest of developing in vivo positron emission tomography (PET) probes for neuroimaging of calcium channels, we have prepared a carbon-11 isotopologue of a dihydropyridine Ca2+-channel antagonist, isradipine. Desmethyl isradipine (4-(benzo[c][1,2,5]oxadiazol-4-yl)-5-(isopropoxycarbonyl)-2,6-dimethyl-1,4-dihydropyridine -3-carboxylic acid) was reacted with [11C]CH3I in the presence of tetrabutylammonium hydroxide in DMF in an HPLC injector loop to produce the radiotracer in a good yield (6 ± 3% uncorrected radiochemical yield) and high specific activity (143 ± 90 GBq·μmol−1 at end-of-synthesis). PET imaging of normal rats revealed rapid brain uptake at baseline (0.37 ± 0.08 %ID/cc (percent of injected dose per cubic centimeter) at peak, 15–60 s), which was followed by fast washout. After pretreatment with isradipine (2 mg·kg−1, i.p.), whole brain radioactivity uptake was diminished by 25–40%. This preliminary study confirms that [11C]isradipine can be synthesized routinely for research studies and is brain penetrating. Further work on Ca2+-channel radiotracer development is planned

Practical Radiosynthesis and Preclinical Neuroimaging of [11C]isradipine, a Calcium Channel Antagonist

In the interest of developing in vivo positron emission tomography (PET) probes for neuroimaging of calcium channels, we have prepared a carbon-11 isotopologue of a dihydropyridine Ca2+-channel antagonist, isradipine. Desmethyl isradipine (4-(benzo[c][1,2,5]oxadiazol-4-yl)-5-(isopropoxycarbonyl)-2,6-dimethyl-1,4-dihydropyridine -3-carboxylic acid) was reacted with [11C]CH3I in the presence of tetrabutylammonium hydroxide in DMF in an HPLC injector loop to produce the radiotracer in a good yield (6 ± 3% uncorrected radiochemical yield) and high specific activity (143 ± 90 GBq·µmol−1 at end-of-synthesis). PET imaging of normal rats revealed rapid brain uptake at baseline (0.37 ± 0.08% ID/cc (percent of injected dose per cubic centimeter) at peak, 15–60 s), which was followed by fast washout. After pretreatment with isradipine (2 mg·kg−1, i.p.), whole brain radioactivity uptake was diminished by 25%–40%. This preliminary study confirms that [11C]isradipine can be synthesized routinely for research studies and is brain penetrating. Further work on Ca2+-channel radiotracer development is planned

PET Imaging of Fatty Acid Amide Hydrolase with [¹⁸F]DOPP in Nonhuman Primates

Fatty acid amide hydrolase (FAAH) regulates endocannabinoid signaling. [¹¹C]­CURB, an irreversibly binding FAAH inhibitor, has been developed for clinical research imaging with PET. However, no fluorine-18 labeled radiotracer for FAAH has yet advanced to human studies. [¹⁸F]­DOPP ([¹⁸F]­3-(4,5-dihydrooxazol-2-yl)­phenyl (5-fluoropentyl)­carbamate) has been identified as a promising ¹⁸F-labeled analogue based on rodent studies. The goal of this work is to evaluate [¹⁸F]­DOPP in nonhuman primates to support its clinical translation. High specific activity [¹⁸F]­DOPP (5–6 Ci·μmol^–1) was administered intravenously (iv) to three baboons (2M/1F, 3–4 years old). The distribution and pharmacokinetics were quantified following a 2 h dynamic imaging session using a simultaneous PET/MR scanner. Pretreatment with the FAAH-selective inhibitor, URB597, was carried out at 200 or 300 μg/kg iv, 10 min prior to [¹⁸F]­DOPP administration. Rapid arterial blood sampling for the first 3 min was followed by interval sampling with metabolite analysis to provide a parent radiotracer plasma input function that indicated ∼95% baseline metabolism at 60 min and a reduced rate of metabolism after pretreatment with URB597. Regional distribution data were analyzed with 1-, 2-, and 3-tissue compartment models (TCMs), with and without irreversible trapping since [¹⁸F]­DOPP covalently links to the active site of FAAH. Consistent with previous findings for [¹¹C]­CURB, the 2TCM with irreversible binding was found to provide the best fit for modeling the data in all regions. The composite parameter <i>λk</i>₃ was therefore used to evaluate whole brain (WB) and regional binding of [¹⁸F]­DOPP. Pretreatment studies showed inhibition of <i>λk</i>₃ across all brain regions (WB baseline: 0.112 mL/cm³/min; 300 μg/kg URB597: 0.058 mL/cm³/min), suggesting that [¹⁸F]­DOPP binding is specific for FAAH, consistent with previous rodent data