Structure-activity relationship of dual inhibitors containing maleimide and imidazole motifs against glutaminyl cyclase and glycogen synthase kinase-3ß.

Alzheimer's disease (AD) is a major cause of dementia and one of the most common chronic diseases affecting the aging population. Because AD is considered a public health priority, there is a critical need to discover novel and effective agents for the treatment of this condition. In view of the known contribution of up-regulated glutaminyl cyclase (QC) and glycogen synthase kinase-3ß (GSK-3ß) to the initiation of AD, we previously evaluated a series of dual inhibitors containing maleimide and imidazole motifs as potential anti-AD agents. Here, we assessed another series of hybrids containing maleimide and imidazole motifs to gain an in-depth understanding of the structure-activity relationship (SAR). Based on the primary screening, the introduction of 5-methyl imidazole at one side of the molecule did not enhance the QC-specific inhibitory activity of these hybrids (2, IC50 = 1.22 µM), although the potency was increased by 2' substitution on the maleimide motif at the other side of the molecule. Interestingly, compounds containing 5-methyl imidazole exhibited stronger GSK-3ß-specific inhibitory activity (2, IC50 = 0.0021 µM), and the electron-withdrawing group and 2' and 3' substitution were favorable. Further investigation of substitutions on the maleimide motif in compounds 14-35 revealed that QC-specific inhibition in the presence of piperidine was improved by introduction of a methoxy group (R2). Increasing the linker length and introduction of a methoxy group (R2) also increased the GSK-3ß-specific inhibitory potency. These findings were further confirmed by molecular docking analysis of 33 and 24 with QC and GSK-3ß. Overall, these hybrids exhibited enhanced inhibitory potency against both QC and GSK-3ß, highlighting an important strategy for improving the potency of hybrids as dual-targeting anti-AD agents.

CAMIO: a transgenic CRISPR pipeline to create diverse targeted genome deletions in Drosophila.

The genome is the blueprint for an organism. Interrogating the genome, especially locating critical cis-regulatory elements, requires deletion analysis. This is conventionally performed using synthetic constructs, making it cumbersome and non-physiological. Thus, we created Cas9-mediated Arrayed Mutagenesis of Individual Offspring (CAMIO) to achieve comprehensive analysis of a targeted region of native DNA. CAMIO utilizes CRISPR that is spatially restricted to generate independent deletions in the intact Drosophila genome. Controlled by recombination, a single guide RNA is stochastically chosen from a set targeting a specific DNA region. Combining two sets increases variability, leading to either indels at 1-2 target sites or inter-target deletions. Cas9 restriction to male germ cells elicits autonomous double-strand-break repair, consequently creating offspring with diverse mutations. Thus, from a single population cross, we can obtain a deletion matrix covering a large expanse of DNA at both coarse and fine resolution. We demonstrate the ease and power of CAMIO by mapping 5'UTR sequences crucial for chinmo's post-transcriptional regulation.