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Radiolabeled Aminopyrazoles as Novel Isoform Selective Probes for pJNK3 Quantification.

ABSTRACT: The c-Jun N-terminal kinase 3 (JNK3) is a stress-activated kinase primarily expressed in the brain and implicated as an early mediator of neuronal apoptosis. We sought to develop a PET tracer to visualize pathological JNK3 activation. Because regional JNK3 activation precedes apoptosis, such an imaging agent might enable the detection of "at risk" brain regions prior to neuronal death. We prepared a set of ¹⁹F-containing compounds on the basis of the reported aminopyrazoles. The candidate, F3, was tritiated and used in autoradiography experiments to demonstrate regional and temporal changes in JNK3 activation in a mouse model of Parkinson's disease. A significant increase in ^pJNK3 B _max versus control animals in multiple brain regions was observed at 8 months, including the ventral midbrain. Pathological activation of JNK3 in these regions preceded statistically significant neuron loss. Analyses of brain concentrations of [¹⁸F]-F3 in naïve rats following intravenous injection revealed a small but detectable signal over the background, but was likely not sufficient to support PET imaging.

SUBMITTER: Bales BC

PROVIDER: S-EPMC9575163 | biostudies-literature | 2022 Oct

REPOSITORIES: biostudies-literature

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Radiolabeled Aminopyrazoles as Novel Isoform Selective Probes for pJNK3 Quantification.

Bales Brian C BC Cotero Victoria V Meyer Dan E DE Roberts Jeannette C JC Rodriguez-Silva Monica M Siclovan Tiberiu M TM Chambers Jeremy W JW Rishel Michael J MJ

ACS medicinal chemistry letters 20220928 10

The c-Jun N-terminal kinase 3 (JNK3) is a stress-activated kinase primarily expressed in the brain and implicated as an early mediator of neuronal apoptosis. We sought to develop a PET tracer to visualize pathological JNK3 activation. Because regional JNK3 activation precedes apoptosis, such an imaging agent might enable the detection of "at risk" brain regions prior to neuronal death. We prepared a set of <sup>19</sup>F-containing compounds on the basis of the reported aminopyrazoles. The candi ...[more]

PMID: 36262398

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Project description:OBJECTIVES:Drugs for Neglected Diseases initiative (DNDi) has identified three chemical lead series, the nitroimidazoles, benzoxaboroles and aminopyrazoles, as innovative treatments for visceral leishmaniasis. The leads discovered using phenotypic screening, were optimised following disease- and compound-specific criteria. Several leads of each series were progressed and preclinical drug candidates have been nominated. Here we evaluate the efficacy of the lead compounds of each of these three chemical classes in in vitro and in vivo models of cutaneous leishmaniasis. METHODS:The in vitro activity of fifty-five compounds was evaluated against the intracellular amastigotes of L. major, L. aethiopica, L. amazonensis, L. panamensis, L. mexicana and L. tropica. The drugs demonstrating potent activity (EC50 < 5 μM) against at least 4 of 6 species were subsequently evaluated in vivo in different L. major - BALB/c mouse models using a 5 or 10-day treatment with either the oral or topical formulations. Efficacy was expressed as lesion size (measured daily using callipers), parasite load (by quantitative PCR - DNA) and bioluminescence signal reduction relative to the untreated controls. RESULTS:The selected drug compounds (3 nitroimidazoles, 1 benzoxaborole and 3 aminopyrazoles) showed consistent and potent activity across a range of Leishmania species that are known to cause CL with EC50 values ranging from 0.29 to 18.3 μM. In all cases, this potent in vitro antileishmanial activity translated into high levels of efficacy with a linear dose-response against murine CL. When administered at 50 mg/kg/day, DNDI-0690 (nitroimidazole), DNDI-1047 (aminopyrazole) and DNDI-6148 (benzoxaborole) all resulted in a significant lesion size reduction (no visible nodule) and an approximate 2-log-fold reduction of the parasite load as measured by qPCR compared to the untreated control. CONCLUSIONS:The lead compounds DNDI-0690, DNDI-1047 and DNDI-6148 showed excellent activity across a range of Leishmania species in vitro and against L. major in mice. These compounds offer novel potential drugs for the treatment of CL.

Project description:BackgroundNorthern blotting is still used as a gold standard for validation of the data obtained from high-throughput whole transcriptome-based methods. However, its disadvantages of lower sensitivity, labor-intensive operation, and higher quality of RNA required limit its utilization in a routine molecular biology laboratory to monitor gene expression at RNA level. Therefore, it is necessary to optimize the traditional Northern protocol to make the technique more applicable for standard use.ResultsIn this paper, we report modifications and tips used to improve the traditional Northern protocol for the detection of mRNAs in total RNA. To maximize the retention of specifically bound radiolabeled probes on the blot, posthybridization washes were performed under only with moderate-stringency until the level of radioactivity retained on the filter decreased to 20~50 counts per second, rather than normally under high and low stringency sequentially for scheduled time or under only high stringent condition. Successful detection of the low-expression gene using heterologous DNA probes in 20 µg of total RNA after a two-day exposure suggested an improvement in detection sensitivity. Quantitatively controlled posthybridization washes combined with an ethidium bromide-prestaining RNA procedure to directly visualize prestained RNA bands at any time during electrophoresis or immediately after electrophoresis, which made the progress of the Northern procedure to be monitored and evaluated step by step, thereby making the experiment reliable and controllable. We also report tips used in the modified Northern protocol, including the moderate concentration of formaldehyde in the gel, the accessory capillary setup, and the staining jar placed into an enamel square tray with a lid used for hybridization. Using our modified Northern protocol, eight rounds of rehybridization could be performed on a single blot. The modification made and tips used ensured the efficient proceeding of the experiment and the resulting good performance, but without using special reagents or equipment.ConclusionsThe modified Northern protocol improved detection sensitivity and made the experiment easy, less expensive, reliable, and controllable, and can be employed in a routine molecular biology laboratory to detect low-expressed mRNAs with heterologous DNA probes in total RNA.

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Radiolabeled Aminopyrazoles as Novel Isoform Selective Probes for pJNK3 Quantification.

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Radiolabeled Aminopyrazoles as Novel Isoform Selective Probes for pJNK3 Quantification.

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