Facile Solid-Phase Synthesis and Assessment of Nucleoside Analogs as Inhibitors of Bacterial UDP-Sugar Processing Enzymes

Abstract

The privileged uptake of nucleosides into cells has generated interest in the development of nucleoside-analog libraries for mining new inhibitors. Of particular interest are applications in the discovery of substrate mimetic inhibitors for the growing number of identified glycan-processing enzymes in bacterial pathogens. However, the high polarity and the need for appropriate protecting group strategies for nucleosides challenges the development of synthetic approaches. Here, we report an accessible, user-friendly synthesis that branches from a common solid phase-immobilized uridinyl-amine intermediate, which can be used as a starting point for diversity-oriented synthesis. We demonstrate the generation of five series of uridinyl nucleoside analogs for investigating inhibitor structure-activity relationships. This library was screened for inhibition of representative enzymes from three functional families including a phosphoglycosyl transferase, a UDP-aminosugar acetyltransferase, and a glycosyltransferase. These candidates were taken from the Gram-negative bacteria Campylobacter concisus and Campylobacter jejuni and the Gram-positive bacterium Clostridium difficile, respectively. Inhibition studies show that specific compound series preferentially inhibit selected enzymes, with IC values ranging from 35 ± 7 μM to 174 ± 21 μM. Insights from the screen provide a strong foundation for further structural elaboration, to improve potency, which will be enabled by the same synthetic strategy. The solid-phase strategy was also used to synthesize pseudouridine analogs of lead compounds. Finally, the compounds were found to be nontoxic to mammalian cells, further supporting the opportunities for future development.

Metabolic inhibitors of bacterial glycan biosynthesis

“…Although systematic efforts to screen for inhibitors of glycan biosynthesis have been undertaken in mammalian systems, [71][72][73][74] there is a relative paucity of bacterial glycosylation inhibitors. The progress that has been made in this area has relied on indepth characterization of glycosyltransferases, [75][76][77][78] information that is oen a rate-limiting step in inhibitor development. As an alternative to direct screening of glycosyltransferases, Vocadlo, 42 Paulson 44 and others 21,45 have demonstrated the potential of unnatural monosaccharides or glycosides bearing key structural features (e.g.…”

Squaramides as Bioisosteres in Contemporary Drug Design

“…Nevertheless, the best performing squaramide (i.e., 142) showed 78% residual activity of a DPMS recombinant protein, as well as good in vivo inhibition of the bloodstream form of T. brucei (ED 50 0.7 mM). 244 Recently, Madec et al 246 disclosed a collection of inhibitors in which the phosphoester linkage in uridine diphosphate (UDP) was replaced by an amide or squaramide linkage, with varying terminal groups (e.g., 143−145). These were designed as inhibitors of bacterial UDP-sugar processing enzymes; specifically, a phosphoglycosyl transferase (PglC), a UDP-aminosugar acetyltransferase (PglD), and a glycosyltransferase (TcdB-GTD).…”

Uridine natural products: Challenging targets and inspiration for novel small molecule inhibitors

“…In parallel to the previously mentioned efforts, the Imperiali laboratory developed a solid phase approach to minimize in-solution manipulations of the uridine analogs (Scheme 5). 62 This approach was inspired by a previously designed uridine linker, attaching uridinyl ribose through a similar acetal linkage. 59 Starting from the anisole with a para-substituted ketone (56), the hydroxyl group was alkylated with methyl 5-bromovalerate.…”

“…To install the C5' amine on the ribose (61), a Mitsunobu reaction was performed with pentachlorophthalimide (60), followed by deprotection. Deprotection poised the compound (61) for further solid phase-based manipulations to access a uridine library on solid phase (62). The final compounds (63 to 68a-e) were isolated after resin cleavage, global deprotection, and HPLC purification.…”