Structural–Functional Studies of Burkholderia cenocepacia d-Glycero-β-d-manno-heptose 7-Phosphate Kinase (HldA) and Characterization of Inhibitors with Antibiotic Adjuvant and Antivirulence Properties

Abstract

As an essential constituent of the outer membrane of Gram-negative bacteria, lipopolysaccharide contributes significantly to virulence and antibiotic resistance. The lipopolysaccharide biosynthetic pathway therefore serves as a promising therapeutic target for antivirulence drugs and antibiotic adjuvants. Here we report the structural–functional studies of d-glycero-β-d-manno-heptose 7-phosphate kinase (HldA), an absolutely conserved enzyme in this pathway, from Burkholderia cenocepacia. HldA is structurally similar to members of the PfkB carbohydrate kinase family and appears to catalyze heptose phosphorylation via an in-line mechanism mediated mainly by a conserved aspartate, Asp270. Moreover, we report the structures of HldA in complex with two potent inhibitors in which both inhibitors adopt a folded conformation and occupy the nucleotide-binding sites. Together, these results provide important insight into the mechanism of HldA-catalyzed heptose phosphorylation and necessary information for further development of HldA inhibitors.

Unraveling the B. pseudomallei Heptokinase WcbL: From Structure to Drug Discovery

“…While two structures of other putative enzymes with the same predicted activity as WcbL have been reported (PDB: 3K85 and 4N3O ), neither of these structures provided any insight into the binding of substrates to the protein or the catalytic mechanism. WcbL represents an excellent target for novel antimicrobials: seven membered sugars are rarely used in eukaryotes, and humans have no equivalent to this pathway ( Durka et al., 2011 , Lee et al., 2013 , Taylor and Wright, 2008 ). Furthermore, kinases are among the best validated drug targets, representing approximately one-third of protein targets currently under investigation for drug development ( Fabbro et al., 2012 ), while none of the other enzymes in the pathway belong to classes that have previously had successful drugs discovered.…”

“…The biosynthetic pathways for heptoses have been proposed as an excellent source of novel antibiotics ( Durka et al., 2011 , Taylor and Wright, 2008 ): similar sugars are not found in mammals, and the sugars are widely used by bacteria, so any inhibitor would have a broad spectrum. Compounds active against the kinase activity of HldE at micromolar concentrations have been identified ( De Leon et al., 2006 , Lee et al., 2013 ). The development of compounds targeting the GDP-linked biosynthetic pathways has not had the same attention, partly because the kinases have not been as thoroughly characterized as HldE.…”

Aplicações Clínicas Dos Glicopeptídeos Nas Unidades De Terapia Intensivas

“…As polimixinas são antimicrobianos indicados para o tratamento de infecções causadas por bactérias Gram-negativas, devido à sua alta afinidade a camada de lipopolissacarídeo (LPS) presente nestes microrganismos. Embora algumas espécies bacterianas possam apresentar diferenças na camada de LPS, esta estrutura geralmente é composta por uma cadeia O específica, uma porção de oligossacarídeos (LOS) e pelo lipídeo A (Figura 2) (SILIPO et al, 2010;LEE et al, 2013;GALLARDO-GODOY et al, 2019). O LPS está localizado na face extracelular da membrana externa de Gramnegativas e é ancorada Lipídeo A, devido a interações hidrofóbicas e eletrostáticas derivadas da alta afinidade entre o grupo fosfato do lipídeo A e os cátions Mg 2+ e Ca 2+ presentes na estrutura da membrana, promovendo a estabilização do LPS (GALLARDO-GODOY et al, 2019;MOFFATT;HARPER;BOYCE, 2019;KINOSHITA et al, 2018).…”

“…A afinidade apresentada pelas polimixinas às bactérias Gram-negativas ocorre devido a presença de um grupo fosfato livre presente na estrutura do lipídeo A, assim, a presença de cargas positivas na estrutura química das polimixinas promove uma interação eletrostática entre os grupamentos Dab e grupos fosfato do lipídeo A que apresentam carga negativa. Observa-se também, interações hidrofóbicas entre os resíduos D-Phe/D-Leu e a cadeia de gordura acil do lipídio A (SILIPO et al, 2010;LEE et al, 2013;MOFFATT;HARPER;BOYCE, 2019;VELKOV;ROBERTS, 2019). A desestabilização da camada de LPS ocorre devido ao deslocamento de cátions Mg 2+ e Ca 2+ promovendo o aumento da permeabilidade e posterior ruptura da membrana, levando ao extravasamento do material intracelular que terá como consequência a morte da bactéria (Figura 4) ( GALLARDO-GODOY et al, 2019;MOFFATT;HARPER;BOYCE, 2019;TRIMBLE et al, 2016;KINOSHITA et al, 2018; VELKOV; ROBERTS, 2019).…”

Crystal structure of D‐glycero‐Β‐D‐manno‐heptose‐1‐phosphate adenylyltransferase fromBurkholderia pseudomallei

“…Therefore, intensive research for developing novel antibiotics and vaccines against melioidosis is required. The X‐ray crystal structures of GmhA, HldA, GmhB, and HldD were determined, but that of HldC has not yet been reported. In order to provide a structural template for the development of novel antibiotic agents and elucidate the structural and functional relationship of HldC, structure determination of HldC from B. pseudomallei ( Bp HldC) was carried out.…”