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1.  Screening-based discovery of Aspergillus fumigatus plant-type chitinase inhibitors 
Febs Letters  2014;588(17):3282-3290.
Highlights
•We performed a high-throughput screen of 60,000 compounds against A. fumigatus chitinase A1.•Novel low micromolar competitive inhibitors were identified.•These represent the most potent selective plant-type A. fumigatus chitinase inhibitors to date.•We provide new tools for probing chitinase inhibition in A. fumigatus and other fungi.
A limited therapeutic arsenal against increasing clinical disease due to Aspergillus spp. necessitates urgent characterisation of new antifungal targets. Here we describe the discovery of novel, low micromolar chemical inhibitors of Aspergillus fumigatus family 18 plant-type chitinase A1 (AfChiA1) by high-throughput screening (HTS). Analysis of the binding mode by X-ray crystallography confirmed competitive inhibition and kinetic studies revealed two compounds with selectivity towards fungal plant-type chitinases. These inhibitors provide new chemical tools to probe the effects of chitinase inhibition on A. fumigatus growth and virulence, presenting attractive starting points for the development of further potent drug-like molecules.
doi:10.1016/j.febslet.2014.07.015
PMCID: PMC4158421  PMID: 25063338
HTS, high-throughput screen/screening; GlcNAc, N-acetylglucosamine; AfChiA1, Aspergillus fumigatus chitinase A1; ScCTS1, Saccharomyces cerevisiae chitinase 1; AfChiB, Aspergillus fumigatus chitinase B1; AMCase, acidic mammalian chitinase; HsCHT, Homo sapiens chitotriosidase (chitinase 1); PI, percentage inhibition; Chitinases; Inhibitors; High-throughput screen (HTS); Aspergillus fumigatus
2.  O-GlcNAc transferase invokes nucleotide sugar pyrophosphate participation in catalysis 
Nature chemical biology  2012;8(12):969-974.
Protein O-GlcNAcylation is an essential post-translational modification on hundreds of intracellular proteins in metazoa, catalyzed by O-GlcNAc transferase using unknown mechanisms of transfer and substrate recognition. Through crystallographic snapshots and mechanism-inspired chemical probes, we define how human O-GlcNAc transferase recognizes the sugar donor and acceptor peptide and employs a novel catalytic mechanism of glycosyl transfer, involving the sugar donor α-phosphate as the catalytic base, as well as an essential lysine. This mechanism appears to be a unique evolutionary solution to the spatial constraints imposed by a bulky protein acceptor substrate, and explains the unexpected specificity of a recently reported metabolic O-GlcNAc transferase inhibitor.
doi:10.1038/nchembio.1108
PMCID: PMC3509171  PMID: 23103942
3.  Natural product-guided discovery of a fungal chitinase inhibitor 
Chemistry & biology  2010;17(12):1275-1281.
Summary
Natural products are often large, synthetically intractable molecules, yet frequently offer surprising inroads into previously unexplored chemical space for enzyme inhibitors. Argifin is a cyclic pentapeptide that was originally isolated as a fungal natural product. It competitively inhibits family 18 chitinases by mimicking the chitooligosaccharide substrate of these enzymes. Interestingly, argifin is a nanomolar inhibitor of the bacterial-type subfamily of fungal chitinases that possess an extensive chitin-binding groove, but does not inhibit the much smaller, plant-type enzymes from the same family that are involved in fungal cell division and are thought to be potential drug targets. Here we show that a small, highly efficient, argifin-derived nine-atom fragment is a micromolar inhibitor of the plant-type chitinase ChiA1 from the opportunistic pathogen Aspergillus fumigatus. Evaluation of the binding mode with the first crystal structure of an A. fumigatus plant-type chitinase reveals that the compound binds the catalytic machinery in the same manner as observed for argifin with the bacterial-type chitinases. The structure of the complex was used to guide synthesis of derivatives to explore a pocket near the catalytic machinery. This work provides synthetically tractable plant-type family 18 chitinase inhibitors from the repurposing of a natural product.
doi:10.1016/j.chembiol.2010.07.018
PMCID: PMC3518266  PMID: 21168763
4.  Structural and biochemical characterization of a trapped coenzyme A adduct of Caenorhabditis elegans glucosamine-6-phosphate N-acetyltransferase 1 
Glucosamine-6-phosphate N-acetyltransferase is an essential enzyme of the eukaryotic UDP-GlcNAc biosynthetic pathway. A crystal structure at 1.55 Å resolution revealed a highly unusual covalent product complex and biochemical studies investigated the function of a fully conserved active-site cysteine.
Glucosamine-6-phosphate N-acetyltransferase 1 (GNA1) produces GlcNAc-6-phosphate from GlcN-6-phosphate and acetyl coenzyme A. Early mercury-labelling experiments implicated a conserved cysteine in the reaction mechanism, whereas recent structural data appear to support a mechanism in which this cysteine plays no role. Here, two crystal structures of Caenorhabditis elegans GNA1 are reported, revealing an unusual covalent complex between this cysteine and the coenzyme A product. Mass-spectrometric and reduction studies showed that this inactive covalent complex can be reactivated through reduction, yet mutagenesis of the cysteine supports a previously reported bi-bi mechanism. The data unify the apparently contradictory earlier reports on the role of a cysteine in the GNA1 active site.
doi:10.1107/S0907444912019592
PMCID: PMC3413214  PMID: 22868768
carbohydrates; glycobiology; Caenorhabditis elegans; glucosamine-6-phosphate N-acetyltransferase; coenzyme A adduct; mechanism
5.  Acetazolamide-based fungal chitinase inhibitors 
Bioorganic & Medicinal Chemistry  2010;18(23):8334-8340.
Graphical abstract
Chitin is an essential structural component of the fungal cell wall. Chitinases are thought to be important for fungal cell wall remodelling, and inhibition of these enzymes has been proposed as a potential strategy for development of novel anti-fungals. The fungal pathogen Aspergillus fumigatus possesses two distinct multi-gene chitinase families. Here we explore acetazolamide as a chemical scaffold for the inhibition of an A. fumigatus ‘plant-type’ chitinase. A co-crystal structure of AfChiA1 with acetazolamide was used to guide synthesis and screening of acetazolamide analogues that yielded SAR in agreement with these structural data. Although acetazolamide and its analogues are weak inhibitors of the enzyme, they have a high ligand efficiency and as such are interesting leads for future inhibitor development.
doi:10.1016/j.bmc.2010.09.062
PMCID: PMC2997425  PMID: 21044846
Chitinase; Aspergillus fumigatus
6.  Substrate and product analogues as human O-GlcNAc transferase inhibitors 
Amino Acids  2010;40(3):781-792.
Protein glycosylation on serine/threonine residues with N-acetylglucosamine (O-GlcNAc) is a dynamic, inducible and abundant post-translational modification. It is thought to regulate many cellular processes and there are examples of interplay between O-GlcNAc and protein phosphorylation. In metazoa, a single, highly conserved and essential gene encodes the O-GlcNAc transferase (OGT) that transfers GlcNAc onto substrate proteins using UDP–GlcNAc as the sugar donor. Specific inhibitors of human OGT would be useful tools to probe the role of this post-translational modification in regulating processes in the living cell. Here, we describe the synthesis of novel UDP–GlcNAc/UDP analogues and evaluate their inhibitory properties and structural binding modes in vitro alongside alloxan, a previously reported weak OGT inhibitor. While the novel analogues are not active on living cells, they inhibit the enzyme in the micromolar range and together with the structural data provide useful templates for further optimisation.
Electronic supplementary material
The online version of this article (doi:10.1007/s00726-010-0688-y) contains supplementary material, which is available to authorized users.
doi:10.1007/s00726-010-0688-y
PMCID: PMC3040809  PMID: 20640461
O-GlcNAc; Post-translational modification; Inhibitor; Signalling; Crystal structure

Results 1-6 (6)