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

Abstract

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.

Global Analysis of UDP Glucose Pyrophosphorylase (UDPGP) Gene Family in Plants: Conserved Evolution Involved in Cell Death

“…In addition, these four amino acids were not located in any motif assigned by MEME. The only one study reported on the key amino acid of USP was from Leishmania major ( Prakash et al, 2019 ). V330, F383, and V199 were key sites for the activity of USP in LsUSP , corresponding to Q353, F405, and V239 in OsUSP ( Supplementary Table 4 and Supplementary Figure 8 ).…”

Targeting a critical step in fungal hexosamine biosynthesis

“…In an attempt to circumvent issues of high structural homology between human and fungal Gna1, we deployed a fragmentbased approach (40) to discover small molecules that target previously unidentified pockets that are selective for the fungal enzyme. Bio-layer interferometry (BLI) was used to identify Gna1 binders from a 650-compound in-house fragment library (41)(42)(43). This produced an initial hit rate of 5.7% (Fig.…”

“…To identify a potential fragment binder of AfGna1, the fragment library of the University of Dundee Drug Discovery Unit (DDU) comprising of 652 structurally diverse compounds was screened using BLI (41)(42)(43). BLI experiments were performed on an Octet Red 384 system (Forte Bio, U.S.A.) using super streptavidin (SSA) biosensors and biotinyated-AfGna1.…”

Targeting a critical step in fungal hexosamine biosynthesis

“…In an attempt to circumvent issues of high structural homology between human and fungal Gna1, we deployed a fragment-based approach 40 to discover small molecules that target previously unidentified pockets that are selective for the fungal enzyme. Bio-layer interferometry (BLI) was used to identify Gna1 binders from a 650 compound in-house fragment library [41][42][43] . This produced an initial hit rate of 5.7% ( Figure S2).…”

“…To identify a potential fragment binder of AfGna1, the fragment library of the University of Dundee Drug Discovery Unit (DDU) comprising of 652 structurally diverse compounds was screened using biolayer interferometry (BLI) [41][42][43] . BLI experiments were performed on an Octet Red 384 system (Forte Bio, USA) using super streptavidin (SSA) biosensors and biotinyated-AfGna1…”