Biochemical analysis of leishmanial and human GDP-Mannose Pyrophosphorylases and selection of inhibitors as new leads

Abstract

Leishmaniases are an ensemble of diseases caused by the protozoan parasite of the genus Leishmania. Current antileishmanial treatments are limited and present main issues of toxicity and drug resistance emergence. Therefore, the generation of new inhibitors specifically directed against a leishmanial target is an attractive strategy to expand the chemotherapeutic arsenal. GDP-Mannose Pyrophosphorylase (GDP-MP) is a prominent therapeutic target involved in host-parasite recognition which has been described to be essential for parasite survival. In this work, we produced and purified GDP-MPs from L. mexicana (LmGDP-MP), L. donovani (LdGDP-MP), and human (hGDP-MP), and compared their enzymatic properties. From a rationale design of 100 potential inhibitors, four compounds were identified having a promising and specific inhibitory effect on parasite GDP-MP and antileishmanial activities, one of them exhibits a competitive inhibition on LdGDP-MP and belongs to the 2-substituted quinoline series.

GDP-Mannose Pyrophosphorylase: A Biologically Validated Target for Drug Development Against Leishmaniasis

“…Both human and leishmanial GDP-MP have been reported to display a high substrate specificity (Mao et al, 2017), in agreement with previous studies performed in bacterial, trypanosomal, and swine GDP-MP (Ning and Elbein, 2000; Denton et al, 2010; Pelissier et al, 2010). The investigation of the mechanism of reaction has shown a sequential ordered mechanism in most bacterial GDP-MP like in some other nucleotidyl-transferases, with GTP fixation prior to mannose-1-phosphate (Barton et al, 2001; Zuccotti et al, 2001; Asencion Diez et al, 2010; Pelissier et al, 2010; Boehlein et al, 2013).…”

“…Magnesium ion is represented by a green sphere. The amino acids that make contact with compound A,B in the catalytic sites are indicated in their one-letter code and number in the sequence (Adapted from Daligaux et al, 2016a; Mao et al, 2017).…”

“…In bacteria, GDP-MP are mostly dimeric, either mono- or bifunctional, the latter displaying both GDP-MP and PMI activities in separate domains of an individual enzyme (Shinabarger et al, 1991; May et al, 1994; Ning and Elbein, 1999; Wu et al, 2002; Asencion Diez et al, 2010; Pelissier et al, 2010; Akutsu et al, 2015). Unlike in other organisms, leishmanial GDP-MP has been shown to assemble as a hexamer of 240 kDa in several Leishmania species (Davis et al, 2004; Mao et al, 2017). As this hexamer can dissociate at low ionic strength conditions and at low protein concentration, a mixture of the three forms may be present in the reaction medium in vitro .…”

“…The investigation of the mechanism of reaction has shown a sequential ordered mechanism in most bacterial GDP-MP like in some other nucleotidyl-transferases, with GTP fixation prior to mannose-1-phosphate (Barton et al, 2001; Zuccotti et al, 2001; Asencion Diez et al, 2010; Pelissier et al, 2010; Boehlein et al, 2013). However, leishmanial and human GDP-MP have been characterized by a sequential random mechanism (Mao et al, 2017), in which the substrate binding order is not defined, in agreement with a mammalian nucleotidyl-transferase (Persat et al, 1983), suggesting that the GDP-MP mechanism of reaction differs from bacteria to Leishmania and human.…”

UDP-Sugar Producing Pyrophosphorylases: Distinct and Essential Enzymes With Overlapping Substrate Specificities, Providing de novo Precursors for Glycosylation Reactions

“…The pyrophosphorylases’ general role as providers of direct precursors for formation of essential glycosylated end-products makes them attractive targets for drug and pharmaceutical industries to develop efficient compounds which can selectively target pathogens. For instance, several novel inhibitors were identified for GDP-Man pyrophosphorylase from two different species of Leishmania (Mao et al., 2017). The inhibitors were rationally designed from docking analyses, and some of them exerted stronger effects on leishmanial GDP-Man pyrophosphorylases than on corresponding human enzyme, suggesting a pharmaceutical potential.…”