A mechanism-inspired UDP- N-acetylglucosamine pyrophosphorylase inhibitor

Abstract

UDP-N-acetylglucosamine pyrophosphorylase (UAP1) catalyses the last step in eukaryotic biosynthesis of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), converting UTP and GlcNAc-1P to the sugar nucleotide. Gene disruption studies have shown that this gene is essential in eukaryotes and a possible antifungal target, yet no inhibitors of fungal UAP1 have so far been reported. Here we describe the crystal structures of substrate/product complexes of UAP1 from Aspergillus fumigatus that together provide snapshots of catalysis. A structure with UDP-GlcNAc, pyrophosphate and Mg²⁺ provides the first Michaelis complex trapped for this class of enzyme, revealing the structural basis of the previously reported Mg²⁺ dependence and direct observation of pyrophosphorolysis. We also show that a highly conserved lysine mimics the role of a second metal observed in structures of bacterial orthologues. A mechanism-inspired UTP α,β-methylenebisphosphonate analogue (meUTP) was designed and synthesized and was shown to be a micromolar inhibitor of the enzyme. The mechanistic insights and inhibitor described here will facilitate future studies towards the discovery of small molecule inhibitors of this currently unexploited potential antifungal drug target.

Fig. 1. UDP-GlcNAc biosynthetic pathway. The four main enzymes of the pathway are coloured in red. Metabolites feeding the pathway are in blue and the final product of the pathway is coloured in pink. The various fates of UDP-GlcNAc are linked by the branching arrows.

Fig. 2. Ligand-induced conformational changes of AfUAP1. (A) Cartoon representation of AfUAP1, the N-terminal domain is coloured red, the C-terminal domain is coloured blue and the pyrophosphorylase domain is coloured in grey. Loops A, B, and C–D together with α* are coloured in yellow, cyan and pink, respectively. The flexible regions are indicated by red arrows. (B) Cartoon representation of the superposed “apo-like” structure with the different complexes showing a large area around the active site. While the colours for the mobile regions are maintained for the “apo-like” structure, the same loops and α* are coloured in orange in the different complexes. (C) The surface representation shows conformational changes around the active site in the various AfUAP1 complexes. The substrates and the products are shown as sticks, the Mg²⁺ ion is shown as a blue sphere.

Fig. 3. Comparison of AfUAP1 with other UAP1s. (A) Overall structure of CaUAP1. Coloured in orange, cyan and purple are the N-terminal, catalytic and C-terminal domains, respectively. (B) Overall structure of HsUAP1. Coloured in red, green and blue are the N-terminal, catalytic and C-terminal domains, respectively. (C) Active site of CaUAP1 with GlcNAc-1P bound. Active site residues are shown in sticks. Hydrogen bond interactions are shown as black dashes. (D) Active site of CaUAP1 with UDP-GlcNAc–SO₄²⁻ bound. Active site residues are shown in sticks. Hydrogen bond interactions are shown as black dashes. SO₄²⁻ is shown as sticks. (E) Stick representation of UDP-GlcNAc in the AfUAP1–UDP-GlcNAc–PPi–Mg²⁺ ternary complex, showing the observed dual conformation around the α-phosphate. (F) Superposition of UDP-GlcNAc from the CaUAP1–UDP-GlcNAc product complex (PDB: 2yqs, blue) with UDP-GlcNAc from the CaUAP1–UDP-GlcNAc reaction-completed product complex (PDB: 2yqj, green). This superposition recapitulates the dual conformation observed in the AfUAP1–UDP-GlcNAc–PPi–Mg²⁺ complex.

Fig. 4. Active site of AfUAP1 complexes and comparison with the MtGlmU ternary complex. Amino acids and ligands are shown as sticks with grey and green carbon atoms respectively. Water molecules, Mg²⁺ and Co²⁺ atoms are shown as spheres in red, blue and pink respectively. Hydrogen bonds and metal coordination are represented with dotted lines coloured black and orange respectively. The unbiased |F_o| − |F_c|φ_calc electron density maps for the ligands is shown in blue, contoured from 2.2 to 2.5σ depending on the ligand. The blue characters indicate the residues mutated in the mutagenesis assay.

Fig. 5. Magnesium coordination and AfUAP1 mechanism. (A) Magnesium coordination in the AfUAP1–UDP-GlcNAc–PPi–Mg²⁺ complex active site. A view of the active site of the AfUAP1–UDP-GlcNAc–PPi–Mg²⁺ and MtGlmU–UDP-GlcNAc–PPi–Mg²⁺–Co²⁺ complexes. The attack distance for pyrophosphorolysis is 2.2 and 2.8 Å and the attack angle is 166° and 169° for UAP1 and GlmU, respectively. The distance between the α-phosphates in both UDP-GlcNAc adopting the dual conformation is 1.7 Å. The Mg²⁺ is tetragonal bipyramidally coordinated (albeit with a disordered 6th ligand in AfUAP1) as suggested by coordination distances and angles. (B) Magnesium coordination in the MtGlmU–UDP-GlcNAc–PPi–Mg²⁺–Co²⁺ complex active site. (C) Schematic diagram of the proposed forward and reverse reaction mechanisms. The left panel indicates chemical bond formation/cleavage (cyan arrows) in the AfUAP1–PPi–UDP-GlcNAc ternary complex shown in Fig. 5A leading to the formation of GlcNAc-1P and UTP. The right panel using the same ternary complex structure as a template indicates chemical bond formation/cleavage in the reverse reaction leading to the formation of UDP-GlcNAc and PPi from GlcNAc-1P and UTP is proposed.

Fig. 6. Kinetic characterization of meUTP (compound 5). (A) Chemical structures of UTP and meUTP. The α,β-methylenebisphosphanate is indicated in red. (B) HPAEC chromatogram of AfUAP1 inhibition by meUTP, where [UTP] ≈ K_m and [GlcNAc-1-P] is in excess. The inhibitor concentration is indicated on each curve (0–200 µM). The reaction mixtures (20 µM UTP, 50 µM GlcNAc-1P, 1 mM DTT, 5 nM enzyme, 50 mM Tris–HCl pH 8.0, 10 mM MgCl₂ and 2% glycerol, total volume 100 µL) with and without varying concentrations of the inhibitor were incubated for 30 min and the reactions were terminated by the addition of 10 µL of 0.1 M NaOH (∼10 mM final concentration) prior to analysis on a CarboPac-PA-1 column (Dionex) equilibrated with a 80 : 20 mixture of 1 mM NaOH and a 1 : 1 mixture of 1 mM NaOH and 1 M sodium acetate. The eluent was monitored at 260 nm and peaks were identified by comparison to standards. (C) IC₅₀ curves of meUTP with fixed [UTP] and [GlcNAc-1P] and varying concentration of inhibitor.

Scheme 1. Synthesis of α,β-methylenebisphosphanate UTP analogue (meUTP). Reagents and conditions: (i) DIAD, PPh₃, trimethyl methylenediphosphonate, THF, 2 h, reflux, 92%; (ii) (a) TFA–H₂O (9 : 1), DCM, 2 h, RT, 85%, (b) (i-PrCO)₂O, Py, DMAP, 5 h, RT, 87%; (iii) (a) TMSBr, MeCN, 1 h, RT, (b) MeOH, MeONa, then Dowex50W8-Et₃NH⁺, quant; (iv) Im₂CO, DMF, then (Bu₃NH)₃PO₄, 16 h, RT, ion-exchange chromatography, 15%.