A Method for Determining the Kinetics of Small-Molecule-Induced Ubiquitination.

Recent advances in targeted protein degradation have enabled chemical hijacking of the ubiquitin-proteasome system to treat disease. The catalytic rate of cereblon (CRBN)-dependent bifunctional degradation activating compounds (BiDAC), which recruit CRBN to a chosen target protein, resulting in its ubiquitination and proteasomal degradation, is an important parameter to consider during the drug discovery process. In this work, an in vitro system was developed to measure the kinetics of BRD4 bromodomain 1 (BD1) ubiquitination by fitting an essential activator kinetic model to these data. The affinities between BiDACs, BD1, and CRBN in the binary complex, ternary complex, and full ubiquitination complex were characterized. Together, this work provides a new tool for understanding and optimizing the catalytic and thermodynamic properties of BiDACs.

Antibacterial Spiropyrimidinetriones with N-Linked Azole Substituents on a Benzisoxazole Scaffold Targeting DNA Gyrase.

Herein, we report spiropyrimidinetriones (SPTs) incorporating N-linked azole substituents on a benzisoxazole scaffold with improved Gram-positive antibacterial activity relative to previously described analogues. SPTs have an unusual spirocyclic architecture and represent a new antibacterial class of bacterial DNA gyrase and topoisomerase IV inhibitors. They are not cross-resistant to fluoroquinolones and other DNA gyrase/topoisomerase IV inhibitors used clinically. The activity of the SPTs was assessed for DNA gyrase inhibition, and the antibacterial activity across Gram-positive and Gram-negative pathogens with N-linked 1,2,4-triazoles substituted on the 5-position provides the most worthwhile profile. Directed nucleophilic and electrophilic chemistry was developed to vary this 5-position with carbon, nitrogen, or oxygen substituents and explore structure-activity relationships including those around a target binding model. Compounds with favorable pharmacokinetic parameters were identified, and two compounds demonstrated cidality in a mouse model of Staphylococcus aureus infection.

Dynamics of human protein kinase Aurora A linked to drug selectivity.

Protein kinases are major drug targets, but the development of highly-selective inhibitors has been challenging due to the similarity of their active sites. The observation of distinct structural states of the fully-conserved Asp-Phe-Gly (DFG) loop has put the concept of conformational selection for the DFG-state at the center of kinase drug discovery. Recently, it was shown that Gleevec selectivity for the Tyr-kinase Abl was instead rooted in conformational changes after drug binding. Here, we investigate whether protein dynamics after binding is a more general paradigm for drug selectivity by characterizing the binding of several approved drugs to the Ser/Thr-kinase Aurora A. Using a combination of biophysical techniques, we propose a universal drug-binding mechanism, that rationalizes selectivity, affinity and long on-target residence time for kinase inhibitors. These new concepts, where protein dynamics in the drug-bound state plays the crucial role, can be applied to inhibitor design of targets outside the kinome.

Responding to the challenge of untreatable gonorrhea: ETX0914, a first-in-class agent with a distinct mechanism-of-action against bacterial Type II topoisomerases.

With the diminishing effectiveness of current antibacterial therapies, it is critically important to discover agents that operate by a mechanism that circumvents existing resistance. ETX0914, the first of a new class of antibacterial agent targeted for the treatment of gonorrhea, operates by a novel mode-of-inhibition against bacterial type II topoisomerases. Incorporating an oxazolidinone on the scaffold mitigated toxicological issues often seen with topoisomerase inhibitors. Organisms resistant to other topoisomerase inhibitors were not cross-resistant with ETX0914 nor were spontaneous resistant mutants to ETX0914 cross-resistant with other topoisomerase inhibitor classes, including the widely used fluoroquinolone class. Preclinical evaluation of ETX0914 pharmacokinetics and pharmacodynamics showed distribution into vascular tissues and efficacy in a murine Staphylococcus aureus infection model that served as a surrogate for predicting efficacious exposures for the treatment of Neisseria gonorrhoeae infections. A wide safety margin to the efficacious exposure in toxicological evaluations supported progression to Phase 1. Dosing ETX0914 in human volunteers showed sufficient exposure and minimal adverse effects to expect a highly efficacious anti-gonorrhea therapy.

Inhibition of Neisseria gonorrhoeae Type II Topoisomerases by the Novel Spiropyrimidinetrione AZD0914.

We characterized the inhibition of Neisseria gonorrhoeae type II topoisomerases gyrase and topoisomerase IV by AZD0914 (AZD0914 will be henceforth known as ETX0914 (Entasis Therapeutics)), a novel spiropyrimidinetrione antibacterial compound that is currently in clinical trials for treatment of drug-resistant gonorrhea. AZD0914 has potent bactericidal activity against N. gonorrhoeae, including multidrug-resistant strains and key Gram-positive, fastidious Gram-negative, atypical, and anaerobic bacterial species (Huband, M. D., Bradford, P. A., Otterson, L. G., Basrab, G. S., Giacobe, R. A., Patey, S. A., Kutschke, A. C., Johnstone, M. R., Potter, M. E., Miller, P. F., and Mueller, J. P. (2014) In Vitro Antibacterial Activity of AZD0914: A New Spiropyrimidinetrione DNA Gyrase/Topoisomerase Inhibitor with Potent Activity against Gram-positive, Fastidious Gram-negative, and Atypical Bacteria. Antimicrob. Agents Chemother. 59, 467-474). AZD0914 inhibited DNA biosynthesis preferentially to other macromolecules in Escherichia coli and induced the SOS response to DNA damage in E. coli. AZD0914 stabilized the enzyme-DNA cleaved complex for N. gonorrhoeae gyrase and topoisomerase IV. The potency of AZD0914 for inhibition of supercoiling and the stabilization of cleaved complex by N. gonorrhoeae gyrase increased in a fluoroquinolone-resistant mutant enzyme. When a mutation, conferring mild resistance to AZD0914, was present in the fluoroquinolone-resistant mutant, the potency of ciprofloxacin for inhibition of supercoiling and stabilization of cleaved complex was increased greater than 20-fold. In contrast to ciprofloxacin, religation of the cleaved DNA did not occur in the presence of AZD0914 upon removal of magnesium from the DNA-gyrase-inhibitor complex. AZD0914 had relatively low potency for inhibition of human type II topoisomerases α and ß.

Discovery of Novel DNA Gyrase Inhibiting Spiropyrimidinetriones: Benzisoxazole Fusion with N-Linked Oxazolidinone Substituents Leading to a Clinical Candidate (ETX0914).

A novel class of bacterial type-II topoisomerase inhibitor displaying a spiropyrimidinetrione architecture fused to a benzisoxazole scaffold shows potent activity against Gram-positive and fastidious Gram-negative bacteria. Here, we describe a series of N-linked oxazolidinone substituents on the benzisoxazole that improve upon the antibacterial activity of initially described compounds of the class, show favorable PK properties, and demonstrate efficacy in an in vivo Staphylococcus aureus infection model. Inhibition of the topoisomerases DNA gyrase and topoisomerase IV from both Gram-positive and a Gram-negative organisms was demonstrated. Compounds showed a clean in vitro toxicity profile, including no genotoxicity and no bone marrow toxicity at the highest evaluated concentrations or other issues that have been problematic for some fluoroquinolones. Compound 1u was identified for advancement into human clinical trials for treatment of uncomplicated gonorrhea based on a variety of beneficial attributes including the potent activity and the favorable safety profile.

Novel DNA gyrase inhibiting spiropyrimidinetriones with a benzisoxazole scaffold: SAR and in vivo characterization.

The compounds described herein with a spirocyclic architecture fused to a benzisoxazole ring represent a new class of antibacterial agents that operate by inhibition of DNA gyrase as corroborated in an enzyme assay and by the inhibition of precursor thymidine into DNA during cell growth. Activity resided in the configurationally lowest energy (2S,4R,4aR) diastereomer. Highly active compounds against Staphylococcus aureus had sufficiently high solubility, high plasma protein free fraction, and favorable pharmacokinetics to suggest that in vivo efficacy could be demonstrated, which was realized with compound (-)-1 in S. aureus mouse infection models. A high drug exposure NOEL on oral dosing in the rat suggested that a high therapeutic margin could be achieved. Importantly, (-)-1 was not cross-resistant with other DNA gyrase inhibitors such as fluoroquinolone and aminocoumarin antibacterials. Hence, this class shows considerable promise for the treatment of infections caused by multidrug resistant bacteria, including S. aureus.

Fragment-based lead discovery and design.

Fragment-based lead discovery and design has and continues to show increasing promise in drug discovery. In this article, the current state of the art in terms of hot-spot characterization, fragment screening techniques, and fragment-based design is discussed. Three overall fragment-based lead generation strategies are explored and involve the chemical biology characterization of biological targets via fragment screening, fragment screening as a complementary approach to high-throughput screening of drug-like compounds, and direct fragment-based drug discovery, respectively. The evolution and development of fragment libraries is described. With an emphasis on computational approaches and the strategies applied at AstraZeneca, the review illustrates how integration of data from one regime can inform the design of experiments in the other, ultimately leading to the discovery of high quality chemical matter.

Early prediction of polymyxin-induced nephrotoxicity with next-generation urinary kidney injury biomarkers.

Despite six decades of clinical experience with the polymyxin class of antibiotics, their dose-limiting nephrotoxicity remains difficult to predict due to a paucity of sensitive biomarkers. Here, we evaluate the performance of standard of care and next-generation biomarkers of renal injury in the detection and monitoring of polymyxin-induced acute kidney injury in male Han Wistar rats using colistin (polymyxin E) and a polymyxin B (PMB) derivative with reduced nephrotoxicity, PMB nonapeptide (PMBN). This study provides the first histopathological and biomarker analysis of PMBN, an important test of the hypothesis that fatty acid modifications and charge reductions in polymyxins can reduce their nephrotoxicity. The results indicate that alterations in a panel of urinary kidney injury biomarkers can be used to monitor histopathological injury, with Kim-1 and α-GST emerging as the most sensitive biomarkers outperforming clinical standards of care, serum or plasma creatinine and blood urea nitrogen. To enable the prediction of polymyxin-induced nephrotoxicity, an in vitro cytotoxicity assay was employed using human proximal tubule epithelial cells (HK-2). Cytotoxicity data in these HK-2 cells correlated with the renal toxicity detected via safety biomarker data and histopathological evaluation, suggesting that in vitro and in vivo methods can be incorporated within a screening cascade to prioritize polymyxin class analogs with more favorable renal toxicity profiles.

A high-throughput-compatible fluorescence anisotropy-based assay for competitive inhibitors of Escherichia coli UDP-N-acetylglucosamine acyltransferase (LpxA).

LpxA, the first enzyme in the biosynthetic pathway for the Lipid A component of the outer membrane lipopolysaccharide in Gram-negative bacteria, is a potential target for novel antibacterial drug discovery. A fluorescence polarization assay was developed to facilitate high-throughput screening for competitive inhibitors of LpxA. The assay detects displacement of a fluorescently labeled peptide inhibitor, based on the previously reported inhibitor peptide 920, by active site ligands. The affinity of the fluorescent ligand was increased ~10-fold by acyl carrier protein (ACP). Competition with peptide binding was observed with UDP-N-acetylglucosamine (IC(50) ~6 mM), UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine (IC(50) ~200 nM), and DL-3-hydroxymyristic acid (IC(50) ~50 µM) and peptide 920 (IC(50) ~600 nM). The IC(50)s were not significantly affected by the presence of ACP.

Avibactam is a covalent, reversible, non-ß-lactam ß-lactamase inhibitor.

Avibactam is a ß-lactamase inhibitor that is in clinical development, combined with ß-lactam partners, for the treatment of bacterial infections comprising gram-negative organisms. Avibactam is a structural class of inhibitor that does not contain a ß-lactam core but maintains the capacity to covalently acylate its ß-lactamase targets. Using the TEM-1 enzyme, we characterized avibactam inhibition by measuring the on-rate for acylation and the off-rate for deacylation. The deacylation off-rate was 0.045 min(-1), which allowed investigation of the deacylation route from TEM-1. Using NMR and MS, we showed that deacylation proceeds through regeneration of intact avibactam and not hydrolysis. Other than TEM-1, four additional clinically relevant ß-lactamases were shown to release intact avibactam after being acylated. We showed that avibactam is a covalent, slowly reversible inhibitor, which is a unique mechanism of inhibition among ß-lactamase inhibitors.

Applications of biophysical tools to target-based discovery of novel antibacterial leads.

New antibacterial drugs are urgently needed to combat the growing problem of multidrug resistant bacterial infections. Major advances in bacterial genomics have uncovered many unexploited targets, leading to the possibility of discovering new antibacterials with novel mechanisms that would circumvent resistance. Many of these targets are soluble enzymes that vary in their degrees of mechanistic complexity. Protein crystallography as well as solution based biophysical methods are playing an increasingly important role in selecting, characterizing and validating promising targets as well as identifying and optimizing lead compounds that inhibit their functions. Advances made in recent years in sensitivity, resolution and throughput of biophysical tools are allowing multiple approaches to screening for hits and rational design of leads based on a deeper understanding of structure-activity relationships. However, the path from a lead compound to a safe and efficacious antibacterial drug still remains challenging. Structural and biophysical approaches have had less of an impact on this later phase of discovery than on the lead generation phase.

Aroma WaterLOGSY: a fast and sensitive screening tool for drug discovery.

One-dimensional NMR spectroscopy has proven to be a powerful technique for screening compound libraries in drug discovery. We report a novel water ligand-observed gradient spectroscopy (WaterLOGSY) pulse sequence, named Aroma WaterLOGSY, that selectively detects aromatic WaterLOGSY signals from compounds or ligands. In the Aroma WaterLOGSY, water magnetization is untouched after water excitation and utilizes the whole period of the remaining pulse sequence to relax back to the +z direction. Due to the phase cycling design, the water magnetization is allowed to relax for the period of two full scans before it gets inverted again. Therefore, the recycle delay can be significantly shortened. Within similar experimental time, Aroma WaterLOGSY shows approximately two times higher sensitivity than the standard scheme. This method also allows the use of non-deuterated reagents, thereby accelerating experimental set-up time for ligand-binding studies.

Exiguaquinol: a novel pentacyclic hydroquinone from Neopetrosia exigua that inhibits Helicobacter pylori MurI.

Bioassay-guided fractionation of the methanol extract of the Australian sponge Neopetrosia exigua led to the isolation of exiguaquinol (2), a new pentacyclic hydroquinone that inhibited Helicobacter pylori glutamate racemase (MurI) with an IC(50) of 4.4 microM. Its structure and relative configuration were assigned on the basis of spectroscopic data. Exiguaquinol (2), bearing a novel pentacyclic ring skeleton, is the first natural product to show inhibition of H. pylori MurI. Its protein-ligand modeling is also discussed.

Novel substituted (pyridin-3-yl)phenyloxazolidinones: antibacterial agents with reduced activity against monoamine oxidase A and increased solubility.

Oxazolidinones represent a new and promising class of antibacterial agents. Current research in this area is mainly concentrated on improving the safety profile and the antibacterial spectrum. Oxazolidinones bearing a (pyridin-3-yl)phenyl moiety (e.g., 3) generally show improved antibacterial activity compared to linezolid but suffer from potent monoamine oxidase A (MAO-A) inhibition and low solubility. We now disclose the finding that new analogues of 3 with acyclic substituents on the pyridyl moiety exhibit excellent activity against Gram-positive pathogens, including linezolid-resistant Streptococcus pneumoniae. Generally, more bulky substituents yielded significantly reduced MAO-A inhibition relative to the unsubstituted compound 3. The MAO-A SAR can be rationalized on the basis of docking studies using a MAO-A/MAO-B homology model. Solubility was enhanced with incorporation of polar groups. One optimized analogue, compound 13, showed low clearance in the rat and efficacy against S. pneumoniae in a mouse pneumonia model.

Exploitation of structural and regulatory diversity in glutamate racemases.

Glutamate racemase is an enzyme essential to the bacterial cell wall biosynthesis pathway, and has therefore been considered as a target for antibacterial drug discovery. We characterized the glutamate racemases of several pathogenic bacteria using structural and biochemical approaches. Here we describe three distinct mechanisms of regulation for the family of glutamate racemases: allosteric activation by metabolic precursors, kinetic regulation through substrate inhibition, and D-glutamate recycling using a d-amino acid transaminase. In a search for selective inhibitors, we identified a series of uncompetitive inhibitors specifically targeting Helicobacter pylori glutamate racemase that bind to a cryptic allosteric site, and used these inhibitors to probe the mechanistic and dynamic features of the enzyme. These structural, kinetic and mutational studies provide insight into the physiological regulation of these essential enzymes and provide a basis for designing narrow-spectrum antimicrobial agents.

Identification of 4-substituted 1,2,3-triazoles as novel oxazolidinone antibacterial agents with reduced activity against monoamine oxidase A.

Oxazolidinones represent a new and promising class of antibacterial agents. Current research in this area is mainly concentrated on improving the safety profile and the antibacterial spectrum. Many oxazolidinones, including linezolid (marketed as Zyvox), are inhibitors of monoamine oxidase A (MAO-A), which presents an undesired side effect. Recently, it was found that the 1,2,3-triazole is a good replacement for the conventional acetamide functionality found in oxazolidinones. We now disclose the finding that 1,2,3-triazoles bearing a substituent like methyl, small substituted methyl, bromo, or a linear (sp-hybridized) group at the 4 position (compounds such as 5, 16, 19, and 21) are good antibacterials with reduced or no activity, within the detection limit of the assay, against MAO-A. The results are especially promising for the development of oxazolidinones with an improved safety profile. The MAO-A SAR can be rationalized on the basis of docking studies to a MAO-A/MAO-B homology model.

Folding and stability of different oligomeric states of thiamin diphosphate dependent homomeric pyruvate decarboxylase.

The folding and stability of recombinant homomeric (alpha-only) pyruvate decarboxylase from yeast was investigated. Different oligomeric states (tetramers, dimers and monomers) of the enzyme occur under defined conditions. The enzymatic activity is used as a sensitive probe for structural differences between the active and inactive form (mis-assembled forms, aggregates) of the folded protein. Unfolding kinetics starting from the native protein comprise both the dissociation of the oligomers into monomers and their subsequent denaturation, which could be monitored by stopped-flow kinetics. In the course of unfolding, the tetramers do not directly dissociate into monomers, but via a stable dimeric state. Starting from the unfolded state, a reactivation of homomeric pyruvate decarboxylase requires both refolding to monomers and their correct association to enzymatically active dimers or tetramers. The reactivation yield under the in vitro conditions used follows an optimum behavior.

Structural properties of the histidine-containing loop in HIV-1 RNase H.

The isolated HIV-1 RNase H domain is inactive. This inactivity has been linked to the lack of structure in the C-terminus of the isolated domain. Thermodynamic stability experiments on the RNase H domain as well as a deletion mutant lacking the C-terminal helix have implied that this region is structured. His539 residing in a loop preceding the C-terminal helix was studied by NMR to determine the stability and conformational properties of this region. The stability of the structural environment of His539 matches that of the entire RNase H domain. Furthermore, His539 is locked into a defined tautometric state in the folded protein and its pK(a) is shifted compared to a freely accessible His, suggesting that this region is structured. The data support the view that the overall dynamics rather than the lack of structure in a small portion of the protein render activity of the isolated HIV-1 RNase H.