Tuning Reactivity of Diphenylpropynone Derivatives with Metal-Associated Amyloid‑β Species via Structural Modifications

A diphenylpropynone derivative, <b>DPP2</b>, has been recently demonstrated to target metal-associated amyloid-β (metal–Aβ) species implicated in Alzheimer’s disease (AD). <b>DPP2</b> was shown to interact with metal–Aβ species and subsequently control Aβ aggregation (reactivity) in vitro; however, its cytotoxicity has limited further biological applications. In order to improve reactivity toward Aβ species and lower cytotoxicity, along with gaining an understanding of a structure-reactivity-cytotoxicity relationship, we designed, prepared, and characterized a series of small molecules (<b>C1</b>/<b>C2</b>, <b>P1</b>/<b>P2</b>, and <b>PA1</b>/<b>PA2</b>) as structurally modified <b>DPP2</b> analogues. A similar metal binding site to that of <b>DPP2</b> was contained in these compounds while their structures were varied to afford different interactions and reactivities with metal ions, Aβ species, and metal–Aβ species. Distinct reactivities of our chemical family toward in vitro Aβ aggregation in the absence and presence of metal ions were observed. Among our chemical series, the compound (<b>C2</b>) with a relatively rigid backbone and a dimethylamino group was observed to noticeably regulate both metal-free and metal-mediated Aβ aggregation to different extents. Using our compounds, cell viability was significantly improved, compared to that with <b>DPP2</b>. Lastly, modifications on the <b>DPP</b> framework maintained the structural properties for potential blood-brain barrier (BBB) permeability. Overall, our studies demonstrated that structural variations adjacent to the metal binding site of <b>DPP2</b> could govern different metal binding properties, interactions with Aβ and metal–Aβ species, reactivity toward metal-free and metal-induced Aβ aggregation, and cytotoxicity of the compounds, establishing a structure-reactivity-cytotoxicity relationship. This information could help gain insight into structural optimization for developing nontoxic chemical reagents toward targeting metal–Aβ species and modulating their reactivity in biological systems

Engineered <i>Saccharomyces cerevisiae</i> as a Biosynthetic Platform of Nucleotide Sugars

Glycosylation of biomolecules can greatly alter their physicochemical properties, cellular recognition, subcellular localization, and immunogenicity. Glycosylation reactions rely on the stepwise addition of sugars using nucleotide diphosphate (NDP)-sugars. Making these substrates readily available will greatly accelerate the characterization of new glycosylation reactions, elucidation of their underlying regulation mechanisms, and production of glycosylated molecules. In this work, we engineered Saccharomyces cerevisiae to heterologously express nucleotide sugar synthases to access a wide variety of uridine diphosphate (UDP)-sugars from simple starting materials (i.e., glucose and galactose). Specifically, activated glucose, uridine diphosphate d-glucose (UDP-d-Glc), can be converted to UDP-d-glucuronic acid (UDP-d-GlcA), UDP-d-xylose (UDP-d-Xyl), UDP-d-apiose (UDP-d-Api), UDP-d-fucose (UDP-d-Fuc), UDP-l-rhamnose (UDP-l-Rha), UDP-l-arabinopyranose (UDP-l-Arap), and UDP-l-arabinofuranose (UDP-l-Araf) using the corresponding nucleotide sugar synthases of plant and microbial origins. We also expressed genes encoding the salvage pathway to directly activate free sugars to achieve the biosynthesis of UDP-l-Arap and UDP-l-Araf. We observed strong inhibition of UDP-d-Glc 6-dehydrogenase (UGD) by the downstream product UDP-d-Xyl, which we circumvented using an induction system (Tet-On) to delay the production of UDP-d-Xyl to maintain the upstream UDP-sugar pool. Finally, we performed a time-course study using strains containing the biosynthetic pathways to produce five non-native UDP-sugars to elucidate their time-dependent interconversion and the role of UDP-d-Xyl in regulating UDP-sugar metabolism. These engineered yeast strains are a robust platform to (i) functionally characterize sugar synthases in vivo, (ii) biosynthesize a diverse selection of UDP-sugars, (iii) examine the regulation of intracellular UDP-sugar interconversions, and (iv) produce glycosylated secondary metabolites and proteins

Reactivity of Diphenylpropynone Derivatives Toward Metal-Associated Amyloid‑β Species

In Alzheimer’s disease (AD), metal-associated amyloid-β (metal–Aβ) species have been suggested to be involved in neurotoxicity; however, their role in disease development is still unclear. To elucidate this aspect, chemical reagents have been developed as valuable tools for targeting metal–Aβ species, modulating the interaction between the metal and Aβ, and subsequently altering metal–Aβ reactivity. Herein, we report the design, preparation, characterization, and reactivity of two diphenylpropynone derivatives (<b>DPP1</b> and <b>DPP2</b>) composed of structural moieties for metal chelation and Aβ interaction (bifunctionality). The interactions of these compounds with metal ions and Aβ species were confirmed by UV–vis, NMR, mass spectrometry, and docking studies. The effects of these bifunctional molecules on the control of in vitro metal-free and metal-induced Aβ aggregation were investigated and monitored by gel electrophoresis and transmission electron microscopy (TEM). Both <b>DPP1</b> and <b>DPP2</b> showed reactivity toward metal–Aβ species over metal-free Aβ species to different extents. In particular, <b>DPP2</b>, which contains a dimethylamino group, exhibited greater reactivity with metal–Aβ species than <b>DPP1</b>, suggesting a structure-reactivity relationship. Overall, our studies present a new bifunctional scaffold that could be utilized to develop chemical reagents for investigating metal–Aβ species in AD