Identification of Leishmania major UDP-Sugar Pyrophosphorylase Inhibitors Using Biosensor-Based Small Molecule Fragment Library Screening

Leishmaniasis is a neglected disease that is caused by different species of the protozoan parasite Leishmania, and it currently affects 12 million people worldwide. The antileishmanial therapeutic arsenal remains very limited in number and efficacy, and there is no vaccine for this parasitic disease. One pathway that has been genetically validated as an antileishmanial drug target is the biosynthesis of uridine diphosphate-glucose (UDP-Glc), and its direct derivative UDP-galactose (UDP-Gal). De novo biosynthesis of these two nucleotide sugars is controlled by the specific UDP-glucose pyrophosphorylase (UGP). Leishmania parasites additionally express a UDP-sugar pyrophosphorylase (USP) responsible for monosaccharides salvage that is able to generate both UDP-Gal and UDP-Glc. The inactivation of the two parasite pyrophosphorylases UGP and USP, results in parasite death. The present study reports on the identification of structurally diverse scaffolds for the development of USP inhibitors by fragment library screening. Based on this screening, we selected a small set of commercially available compounds, and identified molecules that inhibit both Leishmania major USP and UGP, with a half-maximal inhibitory concentration in the 100 µM range. The inhibitors were predicted to bind at allosteric regulation sites, which were validated by mutagenesis studies. This study sets the stage for the development of potent USP inhibitors

Decoding Allosteric Networks in Biocatalysts: Rational Approach to Therapies and Biotechnologies

Biocatalysts utilize allosteric mechanisms to control selectivity, catalytic activity, and the transport of reaction components. The allosteric control of catalysis has a high potential for the development of drugs and technologies. In particular, it opens the way to specific regulation of vital enzymes with conserved active sites. Using the central metabolic enzyme UDP-glucose pyrophosphorylase from the pathogen Leishmania major (LmUGP), we demonstrate how specific allosteric inhibition sites and their links to the catalytic center can be revealed rationally, through analysis of molecular interfaces along the enzymatic reaction cycle. Two previously unknown specific allosteric inhibition sites in LmUGP were rationally identified and experimentally verified. The molecular scaffold for allosteric inhibitor targeting the pathogen’s enzyme was developed. This led to the identification of murrayamine-I as an allosteric inhibitor that selectively blocks LmUGP. The presented approach opens up the possibility of using central metabolic enzymes with highly conserved active sites as allosteric drug targets, thus solving the cross-reactivity problem. In particular, it paves the ways to antimicrobial treatments

Structural and mechanistic basis of capsule O-acetylation in Neisseria meningitidis serogroup A

$O$-Acetylation of the capsular polysaccharide (CPS) of Neisseria meningitidis serogroup A (NmA) is critical for the induction of functional immune responses, making this modification mandatory for CPS-based anti-NmA vaccines. Using comprehensive NMR studies, we demonstrate that $O$-acetylation stabilizes the labile anomeric phosphodiester-linkages of the NmA-CPS and occurs in position C3 and C4 of the $N$-acetylmannosamine units due to enzymatic transfer and non-enzymatic ester migration, respectively. To shed light on the enzymatic transfer mechanism, we solved the crystal structure of the capsule $O$-acetyltransferase CsaC in its apo and acceptor-bound form and of the CsaC-H228A mutant as trapped acetyl-enzyme adduct in complex with CoA. Together with the results of a comprehensive mutagenesis study, the reported structures explain the strict regioselectivity of CsaC and provide insight into the catalytic mechanism, which relies on an unexpected Gln-extension of a classical Ser-His-Asp triad, embedded in an $α$/$β$-hydrolase fold