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Here, we report the design, synthesis, and evaluation of a series of inhibitors of Staphylococcus aureus BPL (SaBPL), where the central acyl phosphate of the natural intermediate biotinyl-5′-AMP (1) is replaced by a sulfonamide isostere. Acylsulfamide (6) and amino sulfonylurea (7) showed potent in vitro inhibitory activity (Ki = 0.007 ± 0.003 and 0.065 ± 0.03 μM, respectively) and antibacterial activity against S. aureus ATCC49775 with minimum inhibitory concentrations of 0.25 and 4 μg/mL, respectively. Additionally, the bimolecular interactions between the BPL and inhibitors 6 and 7 were defined by X-ray crystallography and molecular dynamics simulations. The high acidity of the sulfonamide linkers of 6 and 7 likely contributes to the enhanced in vitro inhibitory activities by promoting interaction with SaBPL Lys187. Analogues with alkylsulfamide (8), β-ketosulfonamide (9), and β-hydroxysulfonamide (10) isosteres were devoid of significant activity. Binding free energy estimation using computational methods suggests deprotonated 6 and 7 to be the best binders, which is consistent with enzyme assay results. Compound 6 was unstable in whole blood, leading to poor pharmacokinetics. Importantly, 7 has a vastly improved pharmacokinetic profile compared to that of 6 presumably due to the enhanced metabolic stability of the sulfonamide linker moiety. Refereed/Peer-reviewed

Cytosolic thiolase (CT) catalyzes the reversible Claisen condensation of two molecules of acetyl-CoA to produce acetoacetyl-CoA. The reaction cycle proceeds via a ping-pong mechanism involving an acetylated enzyme intermediate and two separate oxyanion holes which stabilize negatively charged reaction intermediates. This is the initial step in the synthesis of ergosterol in the prominent fungal pathogen Aspergillus fumigatus and is essential for the growth and survival of the organism. Here, we present crystal structures of A. fumigatus CT in liganded and apo forms and in a complex with different monovalent cations. Careful observation of the electron density at the active sites of two different afCT structures crystallized in the presence of acetyl-CoA shows that our crystals have trapped various stages of the thiolase catalytic cycle, including two tetrahedral reaction intermediates that have previously eluded structural characterization. Unexpectedly, we have also shown that afCT is activated by monova...

Aspergillus fumigatus infections are associated with high mortality rates and high treatment costs. Limited available antifungals and increasing antifungal resistance highlight an urgent need for new antifungals. Thioredoxin reductase (TrxR) is essential for maintaining redox homeostasis and presents as a promising target for novel antifungals. We show that ebselen [2-phenyl-1,2-benzoselenazol-3(2H)-one] is an inhibitor of A. fumigatus TrxR (K i 0.22 M) and inhibits growth of Aspergillus spp., with in vitro MIC values of 16 to 64 g/ml. Mass spectrometry analysis demonstrates that ebselen interacts covalently with a catalytic cysteine of TrxR, Cys148. We also present the X-ray crystal structure of A. fumigatus TrxR and use in silico modeling of the enzyme-inhibitor complex to outline key molecular interactions. This provides a scaffold for future design of potent and selective antifungal drugs that target TrxR, improving the potency of ebselen toward inhbition of A. fumigatus growth Refereed/Peer-reviewed

Summary: Peroxisome proliferator activated receptor γ (PPARγ) is a nuclear receptor and target for antidiabetics that increase insulin sensitivity. Owing to the side effects of PPARγ full agonists, research has recently focused on non-activating ligands of PPARγ, which increase insulin sensitivity with decreased side effects. Here, we present the crystal structures of inverse agonist SR10171 and a chemically related antagonist SR11023 bound to the PPARγ ligand-binding domain, revealing an allosteric switch in the activation helix, helix 12 (H12), forming an antagonist conformation in the receptor. H12 interacts with the antagonists to become fixed in an alternative location. Native mass spectrometry indicates that this prevents contacts with coactivator peptides and allows binding of corepressor peptides. Antagonists of related nuclear receptors act to sterically prevent the active configuration of H12, whereas these antagonists of PPARγ alternatively trap H12 in an inactive configuration, which we have termed the tumble and trap mechanism. : Biological Sciences; Endocrinology; Structural Biology Subject Areas: Biological Sciences, Endocrinology, Structural Biology

The fungal pathogen Aspergillus fumigatus has been implicated in a drastic increase in life-threatening infections over the past decade. However, compared to other microbial pathogens, little is known about the essential molecular processes of this organism. One such fundamental process is DNA replication. The protein responsible for ensuring processive DNA replication is PCNA (proliferating cell nuclear antigen, also known as the sliding clamp), which clamps the replicative polymerase to DNA. Here we present the first crystal structure of a sliding clamp from a pathogenic fungus (A. fumigatus), at 2.6A. Surprisingly, the structure bears more similarity to the human sliding clamp than other available fungal sliding clamps. Reflecting this, fluorescence polarization experiments demonstrated that AfumPCNA interacts with the PCNA-interacting protein (PIP-box) motif of human p21 with an affinity (Kd) of 3.1 μm. Molecular dynamics simulations were carried out to better understand how AfumPCNA interacts with human p21. These simulations revealed that the PIP-box bound to AfuPCNA forms a secondary structure similar to that observed in the human complex, with a central 310 helix contacting the hydrophobic surface pocket of AfumPCNA as well as a β-strand that forms an antiparallel sheet with the AfumPCNA surface. Differences in the 310 helix interaction with PCNA, attributed to residue Thr131 of AfumPCNA, and a less stable β-strand formation, attributed to residues Gln123 and His125 of AfumPCNA, are likely causes of the over 10-fold lower affinity of the p21 PIP-box for AfumPCNA as compared to hPCNA. Database The atomic coordinates and structure factors for the Aspergillus fumigatus sliding clamp can be found in the RCSB Protein Data Bank (http://www.rcsb.org) under the accession code 5TUP.