Synthesis of C6-modified mannose 1-phosphates and evaluation of derived sugar nucleotides against GDP-mannose dehydrogenase

Abstract

Sufferers of cystic fibrosis are at significant risk of contracting chronic bacterial lung infections. The dominant pathogen in these cases is mucoid Pseudomonas aeruginosa. Such infections are characterised by overproduction of the exopolysaccharide alginate. We present herein the design and chemoenzymatic synthesis of sugar nucleotide tools to probe a critical enzyme within alginate biosynthesis, GDP-mannose dehydrogenase (GMD). We first synthesise C6-modified glycosyl 1-phosphates, incorporating 6-amino, 6-chloro and 6-sulfhydryl groups, followed by their evaluation as substrates for enzymatic pyrophosphorylative coupling. The development of this methodology enables access to GDP 6-chloro-6-deoxy-ᴅ-mannose and its evaluation against GMD.

Virtual screening, identification and in vitro validation of small molecule GDP-mannose dehydrogenase inhibitors

“…We recently disclosed the first targeted sugar nucleotide probes for GMD. [9][10][11][12][13] Utilising a chemoenzymatic approach enabled synthetic pyranose modification of the GDP-sugar, [14][15][16] and delivered a C6-Me homologue 3(which was oxidised by GMD), and amide 4 as the first inhibitor (IC 50 = 112 mM). 10 Despite the value in access to such probes, molecular architectures better amenable to crossing a Gram-negative cell envelope to target the GMD active site are required.…”

“…Hits from this screening are verified experimentally using our previously established assay for GMD inhibition. 10,12,13…”

The GDP-Mannose Dehydrogenase of Pseudomonas aeruginosa: An Old and New Target to Fight against Antibiotics Resistance of Mucoid Strains

“…Recently, two English teams have resumed synthesizing P. aeruginosa GMD inhibitors by manufacturing 6-thio, 6-Chloro and 6-amino-mannose derivatives of GDP-mannose. These molecules, as well as C6-methyl-GDP mannose, tend to behave like competitive inhibitors [49][50][51]. As it seems extremely difficult to eradicate persistent bacteria such as P. aeruginosa, which is capable of inducing chronic infections in many hospitalized patients, a strategy involving several targets, including the inhibition of alginate biosynthesis in mucoid strains, is more likely to work to reduce the proliferation of these pathogenic strains, hence the interest in the synthesis of new molecules capable of acting in vivo on specific targets.…”

Bacterial Alginate Biosynthesis and Metabolism

“…It is known that bacterial alginate is initially synthesized as a linear poly-Dmannuronic acid homopolysaccharide before undergoing chemical modifications and epimerization in the periplasmic space, which implies in mannuronic acid (ManA) being the main precursor molecule specifically involved in the biosynthesis of bacterial alginate. However, prior to its utilization to form the poly-D-ManA chain that initiates alginate biosynthesis, ManA must first be synthesized from fructose-6-phosphate (F6P), which is derived from the cell's central carbon metabolism, and then activated with a high-energy bond to yield the sugar nucleotide GDP-ManA [109]. All steps leading to the conversion of F6P to GDP-ManA, the basic building block of alginate, occur in the cytosol and have been extensively analyzed and reported in the past, and a summary of this process is shown in Figure 3.…”