Hydroxylamine derivatives as a new paradigm in the search of antibacterial agents

Altres ajuts: Catalan and Spanish cystic fibrosis federation, the EIT Health, and Obra Social "La Caixa"Serious infections caused by bacteria that are resistant to commonly used antibiotics have become a major global healthcare problem in the 21st century. Multidrug-resistant bacteria causing severe infections mainly grow in complex bacterial communities known as biofilms, in which bacterial resistance to antibacterial agents and to the host immune system is strengthened. As drug resistance is becoming a threatening problem, it is necessary to develop new antimicrobial agents with novel mechanisms of action. Here, we designed and synthesized a small library of N -substituted hydroxylamine (N-HA) compounds with antibacterial activity. These compounds, acting as radical scavengers, inhibit the bacterial ribonucleotide reductase (RNR) enzyme. RNR enzyme is essential for bacterial proliferation during infection, as it provides the building blocks for DNA synthesis and repair. We demonstrate the broad antimicrobial effect of several drug candidates against a variety of Gram-positive and Gram-negative bacteria, together with low toxicity toward eukaryotic cells. Furthermore, the most promising compounds can reduce the biomass of an established biofilm on Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli. This study settles the starting point to develop new N -hydroxylamine compounds as potential effective antibacterial agents to fight against drug-resistant pathogenic bacteria

Hydroxylamine Derivatives as a New Paradigm in the Search of Antibacterial Agents

Serious infections caused by bacteria that are resistant to commonly used antibiotics have become a major global healthcare problem in the 21st century. Multidrug-resistant bacteria causing severe infections mainly grow in complex bacterial communities known as biofilms, in which bacterial resistance to antibacterial agents and to the host immune system is strengthened. As drug resistance is becoming a threatening problem, it is necessary to develop new antimicrobial agents with novel mechanisms of action. Here, we designed and synthesized a small library of N-substituted hydroxylamine (N-HA) compounds with antibacterial activity. These compounds, acting as radical scavengers, inhibit the bacterial ribonucleotide reductase (RNR) enzyme. RNR enzyme is essential for bacterial proliferation during infection, as it provides the building blocks for DNA synthesis and repair. We demonstrate the broad antimicrobial effect of several drug candidates against a variety of Gram-positive and Gram-negative bacteria, together with low toxicity toward eukaryotic cells. Furthermore, the most promising compounds can reduce the biomass of an established biofilm on Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli. This study settles the starting point to develop new N-hydroxylamine compounds as potential effective antibacterial agents to fight against drug-resistant pathogenic bacteria

Acid‐Resistant BODIPY Amino Acids for Peptide‐based Fluorescence Imaging of GPR54 Receptors in Pancreatic Islets

The G protein-coupled kisspeptin receptor (GPR54 or KISS1R) is an important mediator in reproduction, metabolism and cancer biology; however, there are limited fluorescent probes or antibodies for direct imaging of these receptors in cells and intact tissues, which can help to interrogate their multiple biological roles. Herein, we describe the rational design and characterization of a new acid-resistant BODIPY-based amino acid (Trp-BODIPY PLUS), and its implementation for solid-phase synthesis of fluorescent bioactive peptides. Trp-BODIPY PLUS retains the binding capabilities of both short linear and cyclic peptides and displays notable turn-on fluorescence emission upon target binding for wash-free imaging. Finally, we employed Trp-BODIPY PLUS to prepare some of the first fluorogenic kisspeptin-based probes and visualized the expression and localization of GPR54 receptors in human cells and in whole mouse pancreatic islets by fluorescence imaging

Small molecules for modulating biological targets

[cat] CAPÍTOL 1: Les malalties respiratòries infeccioses com per exemple la pneumònia, la bronquitis, la fibrosis quística i la malaltia pulmonar obstructiva crònica són les principals causes de morbidesa i mortalitat a tot el món. Aquestes infeccions són causades per un nombre creixent de bacteris multi resistents als antibiòtics que existeixen actualment. Uns enzims indispensables pels bacteris són els enzims ribonucleòtids reductases (RNR), els quals produeixen els desoxiribonucleòtids per la síntesi d’ADN. Una molècula capaç d’inhibir l’enzim RNR pararia el creixement del bacteri, esdevenint una estratègia terapèutica en el tractament de les malalties respiratòries infeccioses. La hidroxiurea i els derivats de la N-hidroxilamina són inhibidors de l’enzim RNR. Per tant, vam decidir sintetitzar una llibreria d’hidroxilamines, a partir d’aldehids per aminació reductiva. Els compostos van ser avaluats en quatre línies diferents de bacteris: Pseudomonas aeruginosa, Burkholderia cepacia, Staphylococcus aureus i Bacillus anthracis, que causen infeccions oportunistes en pacients que pateixen malalties respiratòries. CAPÍTOL 2: La mitofusina 2 és una proteïna mitocondrial que participa en la fusió mitocondrial i en regula el metabolisme. A més, Mfn2 està disminuïda en el múscul de pacients que pateixen obesitat o diabetis de tipus 2. Per tant, activadors de l’expressió de Mfn2 es podrien utilitzar com a tractament terapèutic. Per trobar activadors de Mfn2 es va portar a terme un cribratge d’alt rendiment amb la llibreria de compostos Prestwick utilitzant les cèl•lules HeLa que expressen de forma estable la proteïna luciferasa sota el promotor humà de Mfn2. El compost LM1, que va ser identificat, augmenta l’expressió de les proteïnes de fusió Mfn2, Mfn1 i OPA1, i disminueix la proteïna de fissió Drp1 en cèl•lules, produint una elongació de la xarxa mitocondrial. El compost LM1 i el LM2, que inhibeixen l’enzim DHODH directa o indirectament, causen la disminució de la síntesi de pirimidines que produeix un estrès cel•lular i l’activació i acumulació de p53. p53 produeix una parada de cicle cel•lular i modula les proteïnes involucrades en dinàmica mitocondrial promovent l’elongació mitocondrial. L’allargament de la xarxa mitocondrial proporciona més resistència a les cèl•lules que pateixen un estrès cel•lular.[eng] CHAPTER 1: Respiratory Infectious Diseases (RID) are diseases affecting the air passages, the bronchi and the lungs. They range from acute infections, such as pneumonia and bronchitis, to chronic conditions such as asthma, cystic fibrosis and chronic obstructive pulmonary disease. Respiratory lung infections are leading causes of morbidity and mortality worldwide with a considerable human, social and financial burden. RID can be caused by an increasing number of multi resistant bacteria to the existing antibiotics. Bacteria, which normally inhabit the mucus, grow out of control and consequently colonize and infect the lungs. The altered mucus leads to formation of bacterial microenvironments known as biofilms, which are difficult for antibiotics and inmunitary cells to penetrate. A potential key enzyme of bacteria, associated with chronic lung infections, to be targeted by antiproliferative drugs are ribonucleotide reductase enzymes (RNR). RNR enzyme provides deoxyribonucleotides for DNA synthesis which are needed for growth and spore germination of the pathogen. Inhibition of RNR enzymes could be a valuable therapeutic strategy for the treatment of RID. In the literature, it has been suggested that hydroxyurea (HU) and N-hydroxylamine derivatives can be used as antiproliferative drugs against bacterial lung infections. Therefore, a library of these compounds was synthesized and evaluated against four different bacteria lines: Pseudomonas aeruginosa, Staphylococcus aureus, Burkholderia cenocepacia and Bacillus anthracis; all of them are opportunistic infectious pathogens affecting respiratory system. CHAPTER 2: Mitofusin-2 (Mfn2) participates in mitochondrial fusion and moreover, regulates mitochondrial metabolism. We have previously reported that Mfn2 is down regulated in muscle from obese or type 2 diabetic patients, and recently we have demonstrated that Mfn2 deficiency in liver or muscle leads to glucose intolerance and insulin resistance in mice. Therefore, activators of Mfn2 expression could be used as a valuable potential therapeutic strategy for the treatment of type 2 diabetes and obesity. For this purpose, we decided to search activators of mitofusin-2 expression by High Throughput Screening using a FDA-approved library. HeLa cells stably expressing luciferase under the control of 2 kb of human Mfn2 promoter were incubated with the library and compound LM1 was identified as a potent activator of Mfn2 transcriptional activity. Importantly, LM1 increased Mfn2 expression in HeLa and C2C12 cells. Furthermore, LM1 also induced the expression of the mitochondrial fusion protein Mfn1 and repressed the mitochondrial fission protein Drp1 in both cell lines. LM1 modulates the mitochondrial proteins involved in mitochondrial dynamics producing an elongation of the mitochondrial network. In addition, LM1 was able to increase Mfn2, Mfn1 and OPA1 protein levels and promoted mitochondrial elongation in MEFwt, MEF Mfn2-/- and MEF Mfn1-/- cells. However, mitochondrial elongation is more dependent of Mfn1 than Mfn2 due to the higher mitochondrial elongation of MEF Mfn2-/- compared to MEF Mfn1-/- cells. LM1 decreases the synthesis of pyrimidines by inhibiting dihydroorotate dehydrogenase (DHODH), which causes cell stress and p53 activation by phosphorylation at Ser15. p53 triggers Mfns up-regulation promoting mitochondrial elongation. The addition of external uridine, which reverses the deficiency in pyrimidines synthesis, prevents p53 and Mfns increase in C2C12 cells. Moreover, LM2, an inhibitor of complex III that indirectly inhibit DHODH, and consequently pyrimidine synthesis, also up-regulates p53 producing an increase in Mfns protein expression. LM2 also promotes mitochondrial elongation in HeLa, C2C12 and MEFwt cells. We can conclude that the depletion of pyrimidine pools, by complex III or DHODH inhibitors, cause cell stress, and trigger p53 accumulation and activation by p53 phosphorylation at Ser15, which causes cell cycle arrest. p53 up-regulates Mfns and down-regulates Drp1 promoting mitochondrial elongation that confer stress resistance on cells. Therefore, mitochondrial elongation represents an adaptive response against cell stress caused by LM1 and LM2

Cell surface biotinylation using furan cross-linking chemistry

A detailed study of the cellular surfaceome poses major challenges for mass spectrometry analysis. Surface proteins are low abundant compared to intracellular proteins, and their inefficient extraction in aqueous medium leads to their aggregation and precipitation. To tackle such problems, surface biotinylation is frequently used to tag surface proteins with biotin, allowing for their enrichment, leading to a more sensitive mapping of surface proteomes. We here detail a new surface biotinylation protocol based on furan-biotin affinity purification to enrich plasma membrane proteins for proteomics. This protocol involves biotinylation of cell surface membrane proteins on viable cells, followed by affinity enrichment using streptavidin beads, trypsin digestion, peptide cleanup, and LC-MS/MS analysis

Equipping coiled-coil peptide dimers with furan warheads reveals novel cross-link partners

Using a coiled-coil peptide dimer as a model system to explore furan reactivity, we describe novel cross-link partners of furan warheads for site-specific cross-linking. We demonstrate that replacement of weak interhelical ionic contacts with a furan moiety and its potential cross-link partner affords covalently connected coiled-coil motifs upon furan activation. We describe for the first time the reaction of the activated furan warhead with cysteine and tyrosine, besides the previously reported lysine, thus enhancing the versatility of the furan cross-link methodology by the possibility to target different amino acids. The present in vitro validation of “furan-armed” α-helices provides further grounds for exploiting furan technology in the development of furan-modified ligands/proteins to target proteins in a covalent way through various amino acid side chains

Photo-induced crosslinking uncovers an antiparallel strand orientation in heterodimeric (EIAALEK)3/(KIAALKE)3 and (EIAALEK)3/(RIAALRE)3 coiled-coil systems

We describe for the first time the co-existence of the parallel and antiparallel conformation of the heterodimeric E3/K3 and E3/R3 coiled-coil systems in solution. The introduction of a furanylated amino acid in the (EIAALEK)(3) sequence allowed, upon photo-induced covalent crosslinking, freezing of the respective coiled-coil complexes present in solution. The occurrence of both parallel and antiparallel conformations in solution was supported by computational simulations and further confirmed by fluorescence experiments based on pyrene-pyrene stacking

Hydroxylamine Derivatives as a New Paradigm in the Search of Antibacterial Agents

Serious infections caused by bacteria that are resistant to commonly used antibiotics have become a major global healthcare problem in the 21st century. Multidrug-resistant bacteria causing severe infections mainly grow in complex bacterial communities known as biofilms, in which bacterial resistance to antibacterial agents and to the host immune system is strengthened. As drug resistance is becoming a threatening problem, it is necessary to develop new antimicrobial agents with novel mechanisms of action. Here, we designed and synthesized a small library of N-substituted hydroxylamine (N-HA) compounds with antibacterial activity. These compounds, acting as radical scavengers, inhibit the bacterial ribonucleotide reductase (RNR) enzyme. RNR enzyme is essential for bacterial proliferation during infection, as it provides the building blocks for DNA synthesis and repair. We demonstrate the broad antimicrobial effect of several drug candidates against a variety of Gram-positive and Gram-negative bacteria, together with low toxicity toward eukaryotic cells. Furthermore, the most promising compounds can reduce the biomass of an established biofilm on Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli. This study settles the starting point to develop new N-hydroxylamine compounds as potential effective antibacterial agents to fight against drug-resistant pathogenic bacteria

Furan warheads for covalent trapping of weak protein-protein interactions : cross-linking of thymosin β4 to actin

We describe furan as a triggerable 'warhead' for site-specific cross-linking using the actin and thymosin beta 4 (T beta 4)-complex as model of a weak and dynamic protein-protein interaction (PPI) with known 3D structure and with application potential in disease contexts. The identified cross-linked residues demonstrate that lysine is a target for the furan warhead. The presented in vitro validation of covalently acting 'furan-armed' T beta 4-variants provides initial proof to further exploit furan-technology for covalent drug design targeting lysines