Antimicrobial combinations against Helicobacter pylori including benzoxadiazol-based flavodoxin inhibitors: in vitro characterization.

IMPORTANCE: The antimicrobial resistance of Helicobacter pylori (Hp) currently poses a threat to available treatment regimens. Developing antimicrobial drugs targeting new bacterial targets is crucial, and one such class of drugs includes Hp-flavodoxin (Hp-fld) inhibitors that target an essential metabolic pathway in Hp. Our study demonstrated that combining these new drugs with conventional antibiotics used for Hp infection treatment prevented the regrowth observed with drugs used alone. Hp-fld inhibitors show promise as new drugs to be incorporated into the treatment of Hp infection, potentially reducing the development of resistance and shortening the treatment duration.

α-Lactam Electrophiles for Covalent Chemical Biology.

Electrophilic groups are one of the key pillars of contemporary chemical biology and medicinal chemistry. For instance, 3-membered N-heterocyclic compounds-such as aziridines, azirines, and oxaziridines-possess unique electronic and structural properties which underlie their potential and applicability as covalent tools. The α-lactams are also members of this group of compounds, however, their utility within the field remains unexplored. Here, we demonstrate an α-lactam reagent (AM2) that is tolerant to aqueous buffers while being reactive towards biologically relevant nucleophiles. Interestingly, carboxylesterases 1 and 2 (CES1/2), both serine hydrolases with key roles in endo- and xenobiotic metabolism, were found as primary covalent targets for AM2 in HepG2 liver cancer cells. All in all, this study constitutes the starting point for the further development and exploration of α-lactam-based electrophilic probes in covalent chemical biology.

Alchemical Design of Pharmacological Chaperones with Higher Affinity for Phenylalanine Hydroxylase.

Phenylketonuria (PKU) is a rare metabolic disease caused by variations in a human gene, PAH, encoding phenylalanine hydroxylase (PAH), and the enzyme converting the essential amino acid phenylalanine into tyrosine. Many PKU-causing variations compromise the conformational stability of the encoded enzyme, decreasing or abolishing its catalytic activity, and leading to an elevated concentration of phenylalanine in the blood, which is neurotoxic. Several therapeutic approaches have been developed to treat the more severe manifestations of the disorder, but they are either not entirely effective or difficult to adhere to throughout life. In a search for novel pharmacological chaperones to treat PKU, a lead compound was discovered (compound IV) that exhibited promising in vitro and in vivo chaperoning activity on PAH. The structure of the PAH-IV complex has been reported. Here, using alchemical free energy calculations (AFEC) on the structure of the PAH-IV complex, we design a new generation of compound IV-analogues with a higher affinity for the enzyme. Seventeen novel analogues were synthesized, and thermal shift and isothermal titration calorimetry (ITC) assays were performed to experimentally evaluate their stabilizing effect and their affinity for the enzyme. Most of the new derivatives bind to PAH tighter than lead compound IV and induce a greater thermostabilization of the enzyme upon binding. Importantly, the correspondence between the calculated alchemical binding free energies and the experimentally determined ΔΔGb values is excellent, which supports the use of AFEC to design pharmacological chaperones to treat PKU using the X-ray structure of their complexes with the target PAH enzyme.

Design, synthesis and structure-activity evaluation of novel 2-pyridone-based inhibitors of α-synuclein aggregation with potentially improved BBB permeability.

The treatment of Parkinson's disease (PD), the second most common neurodegenerative human disorder, continues to be symptomatic. Development of drugs able to stop or at least slowdown PD progression would benefit several million people worldwide. SynuClean-D is a low molecular weight 2-pyridone-based promising drug candidate that inhibits the aggregation of α-synuclein in human cultured cells and prevents degeneration of dopaminergic neurons in a Caenorhabditis elegans model of PD. Improving SynuClean-D pharmacokinetic/pharmacodynamic properties, performing structure/activity studies and testing its efficacy in mammalian models of PD requires the use of gr-amounts of the compound. However, not enough compound is on sale, and no synthetic route has been reported until now, which hampers the molecule progress towards clinical trials. To circumvent those problems, we describe here an efficient and economical route that enables the synthesis of SynuClean-D with good yields as well as the synthesis of SynuClean-D derivatives. Structure-activity comparison of the new compounds with SynuClean-D reveals the functional groups of the molecule that can be disposed of without activity loss and those that are crucial to interfere with α-synuclein aggregation. Several of the derivatives obtained retain the parent's compound excellent in vitro anti-aggregative activity, without compromising its low toxicity. Computational predictions and preliminary testing indicate that the blood brain barrier (BBB) permeability of SynuClean-D is low. Importantly, several of the newly designed and obtained active derivatives are predicted to display good BBB permeability. The synthetic route developed here will facilitate their synthesis for BBB permeability determination and for efficacy testing in mammalian models of PD.

Selective Targeting of Human and Animal Pathogens of the Helicobacter Genus by Flavodoxin Inhibitors: Efficacy, Synergy, Resistance and Mechanistic Studies.

Antimicrobial resistant (AMR) bacteria constitute a global health concern. Helicobacter pylori is a Gram-negative bacterium that infects about half of the human population and is a major cause of peptic ulcer disease and gastric cancer. Increasing resistance to triple and quadruple H. pylori eradication therapies poses great challenges and urges the development of novel, ideally narrow spectrum, antimicrobials targeting H. pylori. Here, we describe the antimicrobial spectrum of a family of nitrobenzoxadiazol-based antimicrobials initially discovered as inhibitors of flavodoxin: an essential H. pylori protein. Two groups of inhibitors are described. One group is formed by narrow-spectrum compounds, highly specific for H. pylori, but ineffective against enterohepatic Helicobacter species and other Gram-negative or Gram-positive bacteria. The second group includes extended-spectrum antimicrobials additionally targeting Gram-positive bacteria, the Gram-negative Campylobacter jejuni, and most Helicobacter species, but not affecting other Gram-negative pathogens. To identify the binding site of the inhibitors in the flavodoxin structure, several H. pylori-flavodoxin variants have been engineered and tested using isothermal titration calorimetry. An initial study of the inhibitors capacity to generate resistances and of their synergism with antimicrobials commonly used in H. pylori eradication therapies is described. The narrow-spectrum inhibitors, which are expected to affect the microbiota less dramatically than current antimicrobial drugs, offer an opportunity to develop new and specific H. pylori eradication combinations to deal with AMR in H. pylori. On the other hand, the extended-spectrum inhibitors constitute a new family of promising antimicrobials, with a potential use against AMR Gram-positive bacterial pathogens.

Asymmetric synthesis of (1R,5S)-2-methyl-6,7-benzomorphan via Aza-Prins reaction.

(1R,5S)-2-Methyl-6,7-benzomorphan has been synthesised from (R)-(benzyloxy)(phenyl)acetaldehyde. On a 2-mmol scale Bi (OTf)3 promoted Aza-Prins reaction with N-tosylhomoallylamine afforded an 88/12 mixture of 6-oxa-2-azabicyclo[3.2.1]octanes. Major diastereoisomer was converted to enantiomerically pure (2S,4S)-2-benzyl-1- methylpiperidin-4-ol via a high-yielding sequence hydrogenolysis/N-detosylation/N-methylation. Acid-catalysed intramolecular Friedel-Crafts cyclisation of the piperidinol afforded (1R,5S)-2-methyl-6,7-benzomorphan in five steps with a yield of 25%.

Design, Synthesis, and Efficacy Testing of Nitroethylene- and 7-Nitrobenzoxadiazol-Based Flavodoxin Inhibitors against Helicobacter pylori Drug-Resistant Clinical Strains and in Helicobacter pylori-Infected Mice.

Helicobacter pylori (Hp) infection is the main cause of peptic ulcer and gastric cancer. Hp eradication rates have fallen due to increasing bacterial resistance to currently used broad-spectrum antimicrobials. We have designed, synthesized, and tested redox variants of nitroethylene- and 7-nitrobenzoxadiazole-based inhibitors of the essential Hp protein flavodoxin. Derivatives of the 7-nitrobenzoxadiazole lead, carrying reduced forms of the nitro group and/or oxidized forms of a sulfur atom, display high therapeutic indexes against several reference Hp strains. These inhibitors are effective against metronidazole-, clarithromycin-, and rifampicin-resistant Hp clinical isolates. Their toxicity for mice after oral administration is low, and, when administered individually at single daily doses for 8 days in a mice model of Hp infection, they decrease significantly Hp gastric colonization rates and are able to eradicate the infection in up to 60% of the mice. These flavodoxin inhibitors constitute a novel family of Hp-specific antimicrobials that may help fight the constant increase of Hp antimicrobial-resistant strains.

A pyrene-inhibitor fluorescent probe with large Stokes shift for the staining of Aß1-42, α-synuclein, and amylin amyloid fibrils as well as amyloid-containing Staphylococcus aureus biofilms.

Amyloid fibrils formed by a variety of peptides are biological markers of different human diseases, such as Alzheimer's disease, Parkinson's disease, and type II diabetes, and are structural constituents of bacterial biofilms. Novel fluorescent probes offering improved sensitivity or specificity toward that diversity of amyloid fibrils or providing alternative spectral windows are needed to improve the detection or the identification of amyloid structures. One potential source for such new probes is offered by molecules known to interact with fibrils, such as the inhibitors of amyloid aggregation found in drug discovery projects. Here we show the feasibility of the approach by designing, synthesizing, and testing several pyrene-based fluorescent derivatives of a previously discovered inhibitor of the aggregation of the Aß1-42 peptide. All the derivatives tested retain the interaction with the amyloid architecture and allow its staining. The most soluble derivative, N-acetyl-2-(2-methyl-4-oxo-5,6,7,8-tetrahydro-4H-benzo[4,5]thieno[2,3-d][1,3]oxazin-7-yl)-N-(pyren-1-ylmethyl)acetamide (compound 1D), stains similarly well amyloid fibrils formed by Aß1-42, α-synuclein, or amylin, provides a sensitivity only slightly lower than that of thioflavin T, displays a large Stokes shift, allows efficient excitation in the UV spectral region, and is not cytotoxic. Compound 1D can also stain amyloid fibrils formed by staphylococcal peptides present in biofilm matrices and can be used to distinguish, by direct staining, Staphylococcus aureus biofilms containing amyloid-forming phenol-soluble modulins from those lacking them. Graphical abstract ᅟ.

Diastereoselective Construction of the 6-Oxa-2-azabicyclo[3.2.1]octane Scaffold from Chiral α-Hydroxyaldehyde Derivatives by the Aza-Prins Reaction.

(R)-2,3-Di-O-benzylglyceraldehyde and N-tosyl homoallylamine undergo aza-Prins cyclization to afford (1R,5S,7S)-7-[(benzyloxy)methyl]-2-tosyl-6-oxa-2-azabicyclo[3.2.1]octane in a highly diastereoselective manner through an unexpected intramolecular nucleophilic attack. Our work has opened a new route toward the asymmetric synthesis of 7-(alkyl or aryl)-6-oxa-2-azabicyclo[3.2.1]octane derivatives from chiral α-hydroxyaldehyde derivatives in one step.