Here, we describe the selection and optimization of a chemical series active in both a full-length and a fragmentbased Huntington's disease (HD) assay. Twenty-four thousand small molecules were screened in a phenotypic HD assay, identifying a series of compounds bearing a 3-hydroxy-3-trifluoromethylpyrazole moiety as able to revert the toxicity induced by full-length mutant Htt by up to 50%. A chemical exploration around the series led to the identification of compound 4f, which demonstrated to be active in a Htt171− 82Q rat primary striatal neuron assay and a PC12-Exon-1 based assay. This compound was selected for testing in R6/2 mice, in which it was well-tolerated and showed a positive effect on body weight and a positive trend in preventing ventricular volume enlargment. These studies provide strong rationale for further testing the potential benefits of 3-hydroxy-3-trifluoromethylpyrazoles in treating HD.

Huntington's Disease is a rare neurodegenerative disease caused by an abnormal expansion of CAG repeats encoding polyglutamine in the first exon of the huntingtin gene. N-terminal fragments containing polyglutamine (polyQ) sequences aggregate and can bind to cellular proteins, resulting in several pathophysiological consequences for affected neurons such as changes in gene transcription. One transcriptional pathway that has been implicated in HD pathogenesis is the CREB binding protein (CBP)/cAMP responsive element binding (CREB) pathway. We developed a phenotypic assay to screen for compounds that can reverse the transcriptional dysregulation of the pathway caused by induced mutated huntingtin protein (µHtt). 293/T-REx cells were stably co-transfected with an inducible full-length mutated huntingtin gene containing 138 glutamine repeats and with a reporter gene under control of the cAMP responsive element (CRE). One clone, which showed reversible inhibition of µHtt-induced reporter activity upon treatment with the neuroprotective Rho kinase inhibitor Y27632, was used for the development of a high-throughput phenotypic assay suitable for a primary screening campaign, which was performed on a library of 24,000 compounds. Several hit compounds were identified and validated further in a cell viability adenosine triphosphate assay. The assay has the potential for finding new drug candidates for the treatment of HD.

(19)F/(18)F isotope exchange is a useful method to label drug molecules containing (19)F-fluorine with (18)F without modifying the drug molecule itself. Sphingosine-1-phosphate (S1P) is an important cellular mediator that functions by signaling through cell surface receptors. S1P is involved in several cell responses and may be related to many central nervous system disorders, including neural malfunction in Alzheimer's disease. In this study, [(18)F]1-benzyl-N-(3,4-difluorobenzyl)-2-isopropyl-6-(2-methoxyethoxy)-1H-indole-3-carboxamide, a novel (18)F-labeled positron emission tomography tracer for the S1P3 receptor, was successfully synthesized using the (19)F/(18)F isotope exchange reaction. Parameters of the reaction kinetics were studied, and correlations between the initial (18)F-activity, the amount of precursor, radiochemical yield and specific activity (SA) were determined. Contrary to expectations, high initial (18)F-activity decreased the radiochemical yield, and only a minor increase of SA occurred. This is most probably due to the complexity of the molecule and the subsequent susceptibility to radiolytic bond disruption. On the basis of the present results, a convenient condition for the (19)F/(18)F exchange reaction is the use of 2 µmol precursor with 20 GBq of (18)F-activity. This afforded a radiochemical yield of ~10% with an SA of 0.3 GBq/µmol. Results from this study are of interest for new tracer development where high initial (18)F-activity and (19)F/(18)F isotope exchange is used.

We report the presence of Actinomycetes in degraded sandstone monuments, and on examination of 173 samples we identified Nocardia restricta as particularly prevalent. In our procedure, the extracted bacterial DNA was the template in polymerase chain reaction (PCR) experiments in order to amplify specific regions of the 16S rDNA. The fidelity of amplified fragment was confirmed by nested-PCR or restriction enzyme specific cutting. To confirm the specificity of the assay, the amplified fragments were cloned in a convenient plasmid vector, the sequence analysed and compared with the expected DNA genomic portion.