Rapid Discovery of Aspartyl Protease Inhibitors Using an Anchoring Approach

Pharmacophore searches that include anchors, fragments contributing above average to receptor binding, combined with one-step syntheses are a powerful approach for the fast discovery of novel bioactive molecules. Here, we are presenting a pipeline for the rapid and efficient discovery of aspartyl protease inhibitors. First, we hypothesized that hydrazine could be a multi-valent warhead to interact with the active site Asp carboxylic acids. We incorporated the hydrazine anchor in a multicomponent reaction and created a large virtual library of hydrazine derivatives synthetically accessible in one-step. Next, we performed anchor-based pharmacophore screening of the libraries and resynthesized top-ranked compounds. The inhibitory potency of the molecules was finally assessed by an enzyme activity assay and the binding mode confirmed by several soaked crystal structures supporting the validity of the hypothesis and approach. The herein reported pipeline of tools will be of general value for the rapid generation of receptor binders beyond Asp proteases.     

An Atomistic Understanding of Allosteric Inhibition of Glutamate Racemase: a Dampening of Native Activation Dynamics

Glutamate racemases (GR) are members of the family of bacterial enzymes known as cofactor-independent racemases and epimerases and catalyze the stereoinversion of glutamate. D-amino acids are universally important for the proper construction of viable bacterial cell walls, and thus have been repeatedly validated as attractive targets for novel antimicrobial drug design. Significant aspects of the mechanism of this challenging stereoinversion remain unknown. The current study employs a combination of MD and QM/MM computational approaches to show that the GR from H. pylori must proceed via a pre-activation step, which is dependent on the enzyme's flexibility. This mechanism is starkly different from previously proposed mechanisms. These findings have immediate pharmaceutical relevance, as the H. pylori GR enzyme is a very attractive allosteric drug target. The results presented in this study offer a distinctly novel understanding of how AstraZeneca's lead series of inhibitors cripple the H. pylori GR's native motions, via prevention of this critical chemical pre-activation step. Our experimental studies, using SPR, fluorescence and NMR WaterLOGSY, show that H. pylori GR is not inhibited by the uncompetitive mechanism originally put forward by Lundqvist et al.. The current study supports a deep connection between native enzyme motions and chemical reactivity, which has strong relevance to the field of allosteric drug discovery.     

Studies on the O/C-Arylation of Cyclic β-Diketones with activated aryl fluorides

The reaction of nitrosubstituted aryl fluorides 1 with cyclic β-diketones 2 proceeds at 20–80°C in the presence of bases, such as KOH, KF or NaOEt, leading to the aryl ethers 3a – m . Depending on the base the reaction of dimedone 2a or 1,3-cyclohexanedione 2b with 2,4-dinitrofluorobenzene 1a or 4-fluoro-3-nitrobenzonitrile 1b furnishes C- and/or O-arylated products. Upon heating of 3g , e and 3m at 40–100°C in DMF/K₂CO₃, the C-arylated ketones 4a – c are formed in good yields. Starting from 3a we obtained the chromenedione 5 under the same conditions.     

Zur heterogenkatalytischen Oxydation und Ammoxydation von Isobuten. IV. Über die Beeinflussung der heterogenkatalytischen Oxydation von Isobuten zu Methacrolein durch n-Buten

On the Heterogeneous-Catalytic Oxidation and Ammoxidation of Isobutene. IV. Influence of n-Butene on the Heterogeneous-Catalytic Oxidation of Isobutene to Methacroleine The influence of n-butenes 2 on the oxidation of isobutenes 1 to methacroleine 3 has been investigated in presence of a catalyst containing Bi, Mo, P, Fe, Mg, Mn, Si and O. Addition of 2 to the gas mixture increase the selectivity and yield of 3 . This fact can be correlated by decrease of oxygen partial pressure in consequence of the dehydrogenation of 2 to butadiene 4 .     

Alkylation of Staurosporine to Derive a Kinase Probe for Fluorescence Applications

The natural product staurosporine is a high‐affinity inhibitor of nearly all mammalian protein kinases. The labelling of staurosporine has proven effective as a means of generating protein kinase research tools. Most tools have been generated by acylation of the 4′‐methylamine of the sugar moiety of staurosporine. Herein we describe the alkylation of this group as a first step to generate a fluorescently labelled staurosporine. Following alkylation, a polyethylene glycol linker was installed, allowing subsequent attachment of fluorescein. We report that this fluorescein–staurosporine conjugate binds to cAMP‐dependent protein kinase in the nanomolar range. Furthermore, its binding can be antagonised with unmodified staurosporine as well as ATP, indicating it targets the ATP binding site in a similar fashion to native staurosporine. This reagent has potential application as a screening tool for protein kinases of interest.     

A Boronic Acid Assay for the Detection of Mucin‐1 Glycoprotein from Cancer Cells

Cell surface glycoproteins are commonly aberrant in disease and act as biomarkers that facilitate diagnostics. Mucin‐1 (MUC1) is a prominent example, exhibiting truncated glycosylation in cancer. We present herein a boronic acid microplate assay for sensitive and high‐throughput detection of such glycoproteins. The immobilization of biotin–boronic acid 1 onto streptavidin plates generated a multivalent surface for glycoprotein recruitment and detection. We first validated the binding properties of 1 in solution through titrations with alizarin dye. Next, the microplate assay was explored through horseradish peroxidase (HRP) analysis as a proof‐of‐concept glycoprotein with chemiluminescence detection. Finally, this platform was applied for the detection of MUC1 directly from MCF‐7 human breast cancer cell lysates by using an HRP‐tagged antibody that targets the cancerous form of this glycoprotein. Sensitive, dose‐dependent detection of MUC1 was observed, showcasing the efficacy of this platform for detecting disease‐associated glycoproteins.     

Spaltung von 1,2,4-Thiadiazol-3-onen mit Triphenylphosphan: Triphenylphosphonio-thioimidazolate und ihre Folgereaktionen

Fission of 1,2,4-Thiadiazol-3-ones by Triphenylphosphane: gewidinet Triphenylphosphonio Thioimidazolates and Their Consecutive Reactions Treatment of benzimidazo[1,2-d][1,2,4]thiadiazol-3(2H)-ones ( 1 ) and imidazo[1,2-d]-[1,2,4]thiadiazol-3(2H)-ones ( 17 ) with triphenylphosphan leads to the liberation of isocyanate and the formation of triphenylphosphonio-thiobenzimidazolat ( 4 ) and triphenylphosphonio-thioimidazolat ( 18 ), respectively. 2,4-Diphenyl-5-phenylimino-1,2,4-thiadiazol-3-one ( 23 ) is desulfurized with Ph₃P and decomposed into phenyl isocyanate and diphenylcarbodiimide whereas the N-unsubstituted imino-thiadiazol-3-one ( 25 ) is attacked at the exocyclic imino group by Ph₃P to give the N-phosphoranylidene thiourea ( 26 ). The structure of 4 can be rationalized as a dynamic system between polar and apolar valence isomeres. Reactions of 1 and 17 with cyanates, isocyanates, carbon disulfide, acetic anhydride, amines, benzyl chloride, grignard compounds, and CH acidic compounds are described.     

Expanding the Spectrum of Antibiotics Capable of Killing Multidrug-Resistant Staphylococcus aureus and Pseudomonas aeruginosa

Infections from antibiotic-resistant Staphylococcus aureus and Pseudomonas aeruginosa are a serious threat because reduced antibiotic efficacy complicates treatment decisions and prolongs the disease state in many patients. To expand the arsenal of treatments against antimicrobial-resistant (AMR) pathogens, 600-Da branched polyethylenimine (BPEI) can overcome antibiotic resistance mechanisms and potentiate β-lactam antibiotics against Gram-positive bacteria. BPEI binds cell-wall teichoic acids and disables resistance factors from penicillin binding proteins PBP2a and PBP4. This study describes a new mechanism of action for BPEI potentiation of antibiotics generally regarded as agents effective against Gram-positive pathogens but not Gram-negative bacteria. 600-Da BPEI is able to reduce the barriers to drug influx and facilitate the uptake of a non-β-lactam co-drug, erythromycin, which targets the intracellular machinery. Also, BPEI can suppress production of the cytokine interleukin IL-8 by human epithelial keratinocytes. This enables BPEI to function as a broad-spectrum antibiotic potentiator, and expands the opportunities to improve drug design, antibiotic development, and therapeutic approaches against pathogenic bacteria, especially for wound care.