Sanne H. Olesen

Acetyl-lysine Binding Site of Bromodomain-Containing Protein 4 (BRD4) Interacts with Diverse Kinase Inhibitors.

Members of the bromodomain and extra terminal (BET) family of proteins are essential for the recognition of acetylated lysine (KAc) residues in histones and have emerged as promising drug targets in cancer, inflammation, and contraception research. In co-crystallization screening campaigns using the first bromodomain of BRD4 (BRD4-1) against kinase inhibitor libraries, we identified and characterized 14 kinase inhibitors (10 distinct chemical scaffolds) as ligands of the KAc binding site. Among these, the PLK1 inhibitor BI2536 and the JAK2 inhibitor TG101209 displayed strongest inhibitory potential against BRD4 (IC50 = 25 nM and 130 nM, respectively) and high selectivity for BET bromodomains. Comparative structural analysis revealed markedly different binding modes of kinase hinge-binding scaffolds in the KAc binding site, suggesting that BET proteins are potential off-targets of diverse kinase inhibitors. Combined, these findings provide a new structural framework for the rational design of next-generation BET-selective and dual-activity BET-kinase inhibitors.

Structural basis of glyphosate resistance resulting from the double mutation Thr97 -> Ile and Pro101 -> Ser in 5-enolpyruvylshikimate-3-phosphate synthase from Escherichia coli.

The shikimate pathway enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is the target of the broad spectrum herbicide glyphosate. The genetic engineering of EPSPS led to the introduction of glyphosate-resistant crops worldwide. The genetically engineered corn lines NK603 and GA21 carry distinct EPSPS enzymes. CP4 EPSPS, expressed in NK603 corn and transgenic soybean, cotton, and canola, belongs to class II EPSPS, glyphosate-insensitive variants of this enzyme isolated from certain Gram-positive bacteria. GA21 corn, on the other hand, was created by point mutations of class I EPSPS, such as the enzymes from Zea mays or Escherichia coli, which are sensitive to low glyphosate concentrations. The structural basis of the glyphosate resistance resulting from these point mutations has remained obscure. We studied the kinetic and structural effects of the T97I/P101S double mutation, the molecular basis for GA21 corn, using EPSPS from E. coli. The T97I/P101S enzyme is essentially insensitive to glyphosate (Ki = 2.4 mm) but maintains high affinity for the substrate phosphoenolpyruvate (PEP) (Km = 0.1 mm). The crystal structure at 1.7-Å resolution revealed that the dual mutation causes a shift of residue Gly96 toward the glyphosate binding site, impairing efficient binding of glyphosate, while the side chain of Ile97 points away from the substrate binding site, facilitating PEP utilization. The single site T97I mutation renders the enzyme sensitive to glyphosate and causes a substantial decrease in the affinity for PEP. Thus, only the concomitant mutations of Thr97 and Pro101 induce the conformational changes necessary to produce catalytically efficient, glyphosate-resistant class I EPSPS.

The Fungal Product Terreic Acid Is a Covalent Inhibitor of the Bacterial Cell Wall Biosynthetic Enzyme UDP-N-Acetylglucosamine 1-Carboxyvinyltransferase (MurA),

Terreic acid is a metabolite with antibiotic properties produced by the fungus Aspergillus terreus. We found that terreic acid is a covalent inhibitor of the bacterial cell wall biosynthetic enzyme MurA from Enterobacter cloacae and Escherichia coli in vitro. The crystal structure of the MurA dead-end complex with terreic acid revealed that the quinine ring is covalently attached to the thiol group of Cys115, the molecular target of the antibiotic fosfomycin. Kinetic characterization established that the inactivation requires the presence of substrate UNAG (UDP-N-acetylglucosamine), proceeding with an inactivation rate constant k(inact) of 130 M(-1) s(-1). Although the mechanisms of inactivation are similar, fosfomycin is approximately 50 times more potent than terreic acid, and the structural consequences of covalent modification by these two inhibitors are fundamentally different. The MurA-fosfomycin complex exists in the closed enzyme conformation, with the Cys115-fosfomycin adduct buried in the active site. In contrast, the dead-end complex with terreic acid is open, is free of UNAG, and has the Cys115-terreic acid adduct solvent-exposed. It appears that terreic acid reacts with Cys115 in the closed, binary state of the enzyme, but that the resulting Cys115-terreic acid adduct imposes steric clashes in the active site. As a consequence, the loop containing Cys115 rearranges, the enzyme opens, and UNAG is released. The differential kinetic and structural characteristics of MurA inactivation by terreic acid and fosfomycin reflect the importance of noncovalent binding potential, even for covalent inhibitors, in ensuring inactivation efficiency and specificity.

Functional Consequence of Covalent Reaction of Phosphoenolpyruvate with UDP-N-acetylglucosamine 1-Carboxyvinyltransferase (MurA)

Background: MurA is critical for the biosynthesis of the bacterial cell wall. Results: The covalent MurA-phosphoenolpyruvate adduct was captured in different reaction states. Conclusion: The covalent adduct primes phosphoenolpyruvate for catalysis and enables feedback inhibition by UDP-N-acetylmuramic acid, the product of MurB. Significance: Cellular MurA exists in a previously unrecognized and tightly locked complex, which presumably accounts for the failure of drug discovery efforts. The enzyme MurA has been an established antibiotic target since the discovery of fosfomycin, which specifically inhibits MurA by covalent modification of the active site residue Cys-115. Early biochemical studies established that Cys-115 also covalently reacts with substrate phosphoenolpyruvate (PEP) to yield a phospholactoyl adduct, but the structural and functional consequences of this reaction remained obscure. We captured and depicted the Cys-115-PEP adduct of Enterobacter cloacae MurA in various reaction states by X-ray crystallography. The data suggest that cellular MurA predominantly exists in a tightly locked complex with UDP-N-acetylmuramic acid (UNAM), the product of the MurB reaction, with PEP covalently attached to Cys-115. The uniqueness and rigidity of this “dormant” complex was previously not recognized and presumably accounts for the failure of drug discovery efforts toward the identification of novel and effective MurA inhibitors. We demonstrate that recently published crystal structures of MurA from various organisms determined by different laboratories were indeed misinterpreted and actually contain UNAM and covalently bound PEP. The Cys-115-PEP adduct was also captured in vitro during the reaction of free MurA and substrate UDP-N-acetylglucosamine or isomer UDP-N-acetylgalactosamine. The now available series of crystal structures allows a comprehensive view of the reaction cycle of MurA. It appears that the covalent reaction of MurA with PEP fulfills dual functions by tightening the complex with UNAM for the efficient feedback regulation of murein biosynthesis and by priming the PEP molecule for instantaneous reaction with substrate UDP-N-acetylglucosamine.

Differential antibacterial properties of the MurA inhibitors terreic acid and fosfomycin.

Terreic acid is a metabolite with antibiotic properties produced by the fungus Aspergillus terreus, but its cellular target remains unknown. We recently reported that terreic acid inactivates the bacterial cell wall biosynthetic enzyme MurA in vitro by covalent reaction with residue Cys115 in a similar manner as the MurA‐specific antibiotic fosfomycin. To address if terreic acid also targets MurA in vivo, we conducted antibacterial studies using selected E. coli strains in parallel with fosfomycin. While overexpression of MurA conferred resistance to fosfomycin, it did not protect cells treated with terreic acid. Furthermore, flow cytometry revealed that the antibiotic action of terreic acid appears to be primarily bacteriostatic, as opposed to the bactericidal action observed for fosfomycin. Combined, the data suggest that MurA is not the molecular target of terreic acid and that the antibiotic activity of terreic acid proceeds through a different mechanism of action. The methodology applied here provides a reliable and convenient tool to rapidly assess the potential of newly discovered in vitro inhibitors to target residue Cys115 of MurA in the cell.

MurA as a primary target of tulipalin B and 6-tuliposide B.

(−)-Tulipalin B and (+)-6-tuliposide B were confirmed to inhibit MurA in vitro. However, contrary to fosfomycin, these compounds showed potent inhibitory activities against MurA overexpressing Escherichia coli, especially in the presence of UDP-GlcNAc. These observations suggest that these compounds induced bacterial cell death not through a MurA malfunction, but in such a MurA-mediated indirect manner as the inhibition of other Mur enzymes.

Structural Basis of Wee Kinases Functionality and Inactivation by Diverse Small Molecule Inhibitors

Members of the Wee family of kinases negatively regulate the cell cycle via phosphorylation of CDK1 and are considered potential drug targets. Herein, we investigated the structure-function relationship of human Wee1, Wee2, and Myt1 (PKMYT1). Purified recombinant full-length proteins and kinase domain constructs differed substantially in phosphorylation states and catalytic competency, suggesting complex mechanisms of activation. A series of crystal structures reveal unique features that distinguish Wee1 and Wee2 from Myt1 and establish the structural basis of differential inhibition by the widely used Wee1 inhibitor MK-1775. Kinome profiling and cellular studies demonstrate that, in addition to Wee1 and Wee2, MK-1775 is an equally potent inhibitor of the polo-like kinase PLK1. Several previously unrecognized inhibitors of Wee kinases were discovered and characterized. Combined, the data provide a comprehensive view on the catalytic and structural properties of Wee kinases and a framework for the rational design of novel inhibitors thereof.

Stability of the Human Hsp90-p50Cdc37 Chaperone Complex against Nucleotides and Hsp90 Inhibitors, and the Influence of Phosphorylation by Casein Kinase 2

The molecular chaperone Hsp90 is regulated by co-chaperones such as p50Cdc37, which recruits a wide selection of client protein kinases. Targeted disruption of the Hsp90-p50Cdc37 complex by protein–protein interaction (PPI) inhibitors has emerged as an alternative strategy to treat diseases characterized by aberrant Hsp90 activity. Using isothermal microcalorimetry, ELISA and GST-pull down assays we evaluated reported Hsp90 inhibitors and nucleotides for their ability to inhibit formation of the human Hsp90β-p50Cdc37 complex, reconstituted in vitro from full-length proteins. Hsp90 inhibitors, including the proposed PPI inhibitors gedunin and H2-gamendazole, did not affect the interaction of Hsp90 with p50Cdc37 in vitro. Phosphorylation of Hsp90 and p50Cdc37 by casein kinase 2 (CK2) did not alter the thermodynamic signature of complex formation. However, the phosphorylated complex was vulnerable to disruption by ADP (IC50 = 32 µM), while ATP, AMPPNP and Hsp90 inhibitors remained largely ineffective. The differential inhibitory activity of ADP suggests that phosphorylation by CK2 primes the complex for dissociation in response to a drop in ATP/ADP levels. The approach applied herein provides robust assays for a comprehensive biochemical evaluation of potential effectors of the Hsp90-p50Cdc37 complex, such as phosphorylation by a kinase or the interaction with small molecule ligands.

Discovery of Diverse Small-Molecule Inhibitors of Mammalian Sterile20-like Kinase 3 (MST3).

Increasing evidence suggests key roles for members of the mammalian Sterile20‐like (MST) family of kinases in many aspects of biology. MST3 is a member of the STRIPAK complex, the deregulation of which has recently been associated with cancer cell migration and metastasis. Targeting MST3 with small‐molecule inhibitors may be beneficial for the treatment of certain cancers, but little information exists on the potential of kinase inhibitor scaffolds to engage with MST3. In this study we screened MST3 against a library of 277 kinase inhibitors using differential scanning fluorimetry and confirmed 14 previously unknown MST3 inhibitors by X‐ray crystallography. These compounds, of which eight are in clinical trials or FDA approved, comprise nine distinct chemical scaffolds that inhibit MST3 enzymatic activity with IC50 values between 0.003 and 23 μm. The structure–activity relationships explain the differential inhibitory activity of these compounds against MST3 and the structural basis for high binding potential, the information of which may serve as a framework for the rational design of MST3‐selective inhibitors as potential therapeutics and to interrogate the function of this enzyme in diseased cells.