Escherichia coli Topoisomerase IV E Subunit and an Inhibitor Binding Mode Revealed by NMR Spectroscopy.

Bacterial topoisomerases are attractive antibacterial drug targets because of their importance in bacterial growth and low homology with other human topoisomerases. Structure-based drug design has been a proven approach of efficiently developing new antibiotics against these targets. Past studies have focused on developing lead compounds against the ATP binding pockets of both DNA gyrase and topoisomerase IV. A detailed understanding of the interactions between ligand and target in a solution state will provide valuable information for further developing drugs against topoisomerase IV targets. Here we describe a detailed characterization of a known potent inhibitor containing a 9H-pyrimido[4,5-b]indole scaffold against the N-terminal domain of the topoisomerase IV E subunit from Escherichia coli (eParE). Using a series of biophysical and biochemical experiments, it has been demonstrated that this inhibitor forms a tight complex with eParE. NMR studies revealed the exact protein residues responsible for inhibitor binding. Through comparative studies of two inhibitors of markedly varied potencies, it is hypothesized that gaining molecular interactions with residues in the α4 and residues close to the loop of ß1-α2 and residues in the loop of ß3-ß4 might improve the inhibitor potency.

Biophysical Studies of Bacterial Topoisomerases Substantiate Their Binding Modes to an Inhibitor.

Bacterial DNA topoisomerases are essential for bacterial growth and are attractive, important targets for developing antibacterial drugs. Consequently, different potent inhibitors that target bacterial topoisomerases have been developed. However, the development of potent broad-spectrum inhibitors against both Gram-positive (G(+)) and Gram-negative (G(-)) bacteria has proven challenging. In this study, we carried out biophysical studies to better understand the molecular interactions between a potent bis-pyridylurea inhibitor and the active domains of the E-subunits of topoisomerase IV (ParE) from a G(+) strain (Streptococcus pneumoniae (sParE)) and a G(-) strain (Pseudomonas aeruginosa (pParE)). NMR results demonstrated that the inhibitor forms a tight complex with ParEs and the resulting complexes adopt structural conformations similar to those observed for free ParEs in solution. Further chemical-shift perturbation experiments and NOE analyses indicated that there are four regions in ParE that are important for inhibitor binding, namely, α2, the loop between ß2 and α3, and the ß2 and ß6 strands. Surface plasmon resonance showed that this inhibitor binds to sParE with a higher KD than pParE. Point mutations in α2 of ParE, such as A52S (sParE), affected its binding affinity with the inhibitor. Taken together, these results provide a better understanding of the development of broad-spectrum antibacterial agents.

Characterization of the interaction between Escherichia coli topoisomerase IV E subunit and an ATP competitive inhibitor.

Bacterial topoisomerase IV (ParE) is essential for DNA replication and serves as an attractive target for antibacterial drug development. The X-ray structure of the N-terminal 24 kDa ParE, responsible for ATP binding has been solved. Due to the accessibility of structural information of ParE, many potent ParE inhibitors have been discovered. In this study, a pyridylurea lead molecule against ParE of Escherichia coli (eParE) was characterized with a series of biochemical and biophysical techniques. More importantly, solution NMR analysis of compound binding to eParE provides better understanding of the molecular interactions between the inhibitor and eParE.

Fragment-based Discovery of a Small-Molecule Protein Kinase C-iota Inhibitor Binding Post-kinase Domain Residues.

The atypical protein kinase C-iota (PKC-ι) enzyme is implicated in various cancers and has been put forward as an attractive target for developing anticancer therapy. A high concentration biochemical screen identified pyridine fragment weakly inhibiting PKC-ι with IC50 = 424 µM. Driven by structure-activity relationships and guided by docking hypothesis, the weakly bound fragment was eventually optimized into a potent inhibitor of PKC-ι (IC50= 270 nM). Through the course of the optimization, an intermediate compound was crystallized with the protein, and careful analysis of the X-ray crystal structure revealed a unique binding mode involving the post-kinase domain (C-terminal tail) of PKC-ι.

Discovery of dual GyrB/ParE inhibitors active against Gram-negative bacteria.

Even though many GyrB and ParE inhibitors have been reported in the literature, few possess activity against Gram-negative bacteria. This is primarily due to limited permeability across Gram-negative bacterial membrane as well as bacterial efflux mechanisms. Permeability of compounds across Gram-negative bacterial membranes depends on many factors including physicochemical properties of the inhibitors. Herein, we show the optimization of pyridylureas leading to compounds with potent activity against Gram-negative bacterial species such as P.aeruginosa, E.coli and A.baumannii.

Fragment-Based Drug Discovery of Potent Protein Kinase C Iota Inhibitors.

Protein kinase C iota (PKC-ι) is an atypical kinase implicated in the promotion of different cancer types. A biochemical screen of a fragment library has identified several hits from which an azaindole-based scaffold was chosen for optimization. Driven by a structure-activity relationship and supported by molecular modeling, a weakly bound fragment was systematically grown into a potent and selective inhibitor against PKC-ι.

Small Molecules Targeting the Inactive Form of the Mnk1/2 Kinases.

Overexpression of the eukaryotic initiation factor 4E (eIF4E) is linked to a variety of cancers. Both mitogen-activated protein kinases-interacting kinases 1 and 2 (Mnk1/2) activate the oncogene eIF4E through posttranslational modification (phosphorylating it at the conserved Ser209). Inhibition of Mnk prevents eIF4E phosphorylation, making the Mnk-eIF4E axis a potential therapeutic target for oncology. Recently, the design and synthesis of a series of novel potent compounds inhibiting the Mnk1/2 kinases were carried out in-house. Here, we describe computational models of the interactions between Mnk1/2 kinases and these inhibitors. Molecular modeling combined with free energy calculations show that these compounds bind to the inactive forms of the kinases. All compounds adopt similar conformations in the catalytic sites of both kinases, stabilized by hydrogen bonds with the hinge regions and with the catalytic Lys78 (Mnk1) and Lys113 (Mnk2). These hydrogen bond interactions clearly play a critical role in determining the conformational stability and potency of the compounds. We also find that van der Waals interactions with an allosteric pocket are key to their binding and potency. Two distinct hydration sites that appear to further stabilize the ligand binding/interactions were observed. Critically, the inclusion of explicit water molecules in the calculations results in improving the agreement between calculated and experimental binding free energies.

Structure-Activity Relationship Studies of Mitogen Activated Protein Kinase Interacting Kinase (MNK) 1 and 2 and BCR-ABL1 Inhibitors Targeting Chronic Myeloid Leukemic Cells.

Clinically used BCR-ABL1 inhibitors for the treatment of chronic myeloid leukemia do not eliminate leukemic stem cells (LSC). It has been shown that MNK1 and 2 inhibitors prevent phosphorylation of eIF4E and eliminate the self-renewal capacity of LSCs. Herein, we describe the identification of novel dual MNK1 and 2 and BCR-ABL1 inhibitors, starting from the known kinase inhibitor 2. Initial structure-activity relationship studies resulted in compound 27 with loss of BCR-ABL1 inhibition. Further modification led to orally bioavailable dual MNK1 and 2 and BCR-ABL1 inhibitors 53 and 54, which are efficacious in a mouse xenograft model and also reduce the level of phosphorylated eukaryotic translation initiation factor 4E in the tumor tissues. Kinase selectivity of these compounds is also presented.

NMR structural characterization of the N-terminal active domain of the gyrase B subunit from Pseudomonas aeruginosa and its complex with an inhibitor.

The N-terminal ATP binding domain of the DNA gyrase B subunit is a validated drug target for antibacterial drug discovery. Structural information for this domain (pGyrB) from Pseudomonas aeruginosa is still missing. In this study, the interaction between pGyrB and a bis-pyridylurea inhibitor was characterized using several biophysical methods. We further carried out structural analysis of pGyrB using NMR spectroscopy. The secondary structures of free and inhibitor bound pGyrB were obtained based on backbone chemical shift assignment. Chemical shift perturbation and NOE experiments demonstrated that the inhibitor binds to the ATP binding pocket. The results of this study will be helpful for drug development targeting P. aeruginosa.