Optical Modulation of Antibiotic Resistance by Photoswitchable Cystobactamids.

The rise of antibiotic resistance causes a serious health care problem, and its counterfeit demands novel, innovative concepts. The combination of photopharmacology, enabling a light-controlled reversible modulation of drug activity, with antibiotic drug design has led to first photoswitchable antibiotic compounds derived from established scaffolds. In this study, we converted cystobactamids, gyrase-inhibiting natural products with an oligoaryl scaffold and highly potent antibacterial activities, into photoswitchable agents by inserting azobenzene in the N-terminal part and/or an acylhydrazone moiety near the C-terminus, yielding twenty analogs that contain mono- as well as double-switches. Antibiotic and gyrase inhibition properties could be modulated 3.4-fold and 5-fold by light, respectively. Notably, the sensitivity of photoswitchable cystobactamids towards two known resistance factors, the peptidase AlbD and the scavenger protein AlbA, was light-dependent. While irradiation of an analog with an N-terminal azobenzene with 365 nm light led to less degradation by AlbD, the AlbA-mediated inactivation was induced. This provides a proof-of-principle that resistance towards photoswitchable antibiotics can be optically controlled.

Effects on weight loss and glycemic control with SAR441255, a potent unimolecular peptide GLP-1/GIP/GCG receptor triagonist.

Unimolecular triple incretins, combining the activity of glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), and glucagon (GCG), have demonstrated reduction in body weight and improved glucose control in rodent models. We developed SAR441255, a synthetic peptide agonist of the GLP-1, GCG, and GIP receptors, structurally based on the exendin-4 sequence. SAR441255 displays high potency with balanced activation of all three target receptors. In animal models, metabolic outcomes were superior to results with a dual GLP-1/GCG receptor agonist. Preclinical in vivo positron emission tomography imaging demonstrated SAR441255 binding to GLP-1 and GCG receptors. In healthy subjects, SAR441255 improved glycemic control during a mixed-meal tolerance test and impacted biomarkers for GCG and GIP receptor activation. Single doses of SAR441255 were well tolerated. The results demonstrate that integrating GIP activity into dual GLP-1 and GCG receptor agonism provides improved effects on weight loss and glycemic control while buffering the diabetogenic risk of chronic GCG receptor agonism.

Liver-Specific Knockdown of Class IIa HDACs Has Limited Efficacy on Glucose Metabolism but Entails Severe Organ Side Effects in Mice.

Histone deacetylases (HDACs) are important regulators of epigenetic gene modification that are involved in the transcriptional control of metabolism. In particular class IIa HDACs have been shown to affect hepatic gluconeogenesis and previous approaches revealed that their inhibition reduces blood glucose in type 2 diabetic mice. In the present study, we aimed to evaluate the potential of class IIa HDAC inhibition as a therapeutic opportunity for the treatment +of metabolic diseases. For that, siRNAs selectively targeting HDAC4, 5 and 7 were selected and used to achieve a combinatorial knockdown of these three class IIa HDAC isoforms. Subsequently, the hepatocellular effects as well as the impact on glucose and lipid metabolism were analyzed in vitro and in vivo. The triple knockdown resulted in a statistically significant decrease of gluconeogenic gene expression in murine and human hepatocyte cell models. A similar HDAC-induced downregulation of hepatic gluconeogenesis genes could be achieved in mice using a liver-specific lipid nanoparticle siRNA formulation. However, the efficacy on whole body glucose metabolism assessed by pyruvate-tolerance tests were only limited and did not outweigh the safety findings observed by histopathological analysis in spleen and kidney. Mechanistically, Affymetrix gene expression studies provide evidence that class IIa HDACs directly target other key factors beyond the described forkhead box (FOXP) transcription regulators, such as hepatocyte nuclear factor 4 alpha (HNF4a). Downstream of these factors several additional pathways were regulated not merely including glucose and lipid metabolism and transport. In conclusion, the liver-directed combinatorial knockdown of HDAC4, 5 and 7 by therapeutic siRNAs affected multiple pathways in vitro, leading in vivo to the downregulation of genes involved in gluconeogenesis. However, the effects on gene expression level were not paralleled by a significant reduction of gluconeogenesis in mice. Combined knockdown of HDAC isoforms was associated with severe adverse effects in vivo, challenging this approach as a treatment option for chronic metabolic disorders like type 2 diabetes.

Labyrinthopeptins as virolytic inhibitors of respiratory syncytial virus cell entry.

Acute lower respiratory tract infections (ALRI) caused by respiratory syncytial virus (RSV) are associated with a severe disease burden among infants and elderly patients. Treatment options are limited. While numerous drug candidates with different viral targets are under development, the utility of RSV entry inhibitors is challenged by a low resistance barrier and by single mutations causing cross-resistance against a wide spectrum of fusion inhibitor chemotypes. We developed a cell-based screening assay for discovery of compounds inhibiting infection with primary RSV isolates. Using this system, we identified labyrinthopeptin A1 and A2 (Laby A1/A2), lantibiotics isolated from Actinomadura namibiensis, as effective RSV cell entry inhibitors with IC50s of 0.39 µM and 4.97 µM, respectively, and with favourable therapeutic index (>200 and > 20, respectively). Both molecules were active against multiple RSV strains including primary isolates and their antiviral activity against RSV was confirmed in primary human airway cells ex vivo and a murine model in vivo. Laby A1/A2 were antiviral in prophylactic and therapeutic treatment regimens and displayed synergistic activity when applied in combination with each other. Mechanistic studies showed that Laby A1/A2 exert virolytic activity likely by binding to phosphatidylethanolamine moieties within the viral membrane and by disrupting virus particle membrane integrity. Probably due to its specific mode of action, Laby A1/A2 antiviral activity was not affected by common resistance mutations to known RSV entry inhibitors. Taken together, Laby A1/A2 represent promising candidates for development as RSV inhibitors. Moreover, the cell-based screening system with primary RSV isolates described here should be useful to identify further antiviral agents.

Labyrinthopeptins Exert Broad-Spectrum Antiviral Activity through Lipid-Binding-Mediated Virolysis.

To counteract the serious health threat posed by known and novel viral pathogens, drugs that target a variety of viruses through a common mechanism have attracted recent attention due to their potential in treating (re)emerging infections, for which direct-acting antivirals are not available. We found that labyrinthopeptins A1 and A2, the prototype congeners of carbacyclic lanthipeptides, inhibit the proliferation of diverse enveloped viruses, including dengue virus, Zika virus, West Nile virus, hepatitis C virus, chikungunya virus, Kaposi's sarcoma-associated herpesvirus, cytomegalovirus, and herpes simplex virus, in the low micromolar to nanomolar range. Mechanistic studies on viral particles revealed that labyrinthopeptins induce a virolytic effect through binding to the viral membrane lipid phosphatidylethanolamine (PE). These effects are enhanced by a combined equimolar application of both labyrinthopeptins, and a clear synergism was observed across a concentration range corresponding to 10% to 90% inhibitory concentrations of the compounds. Time-resolved experiments with large unilamellar vesicles (LUVs) reveal that membrane lipid raft compositions (phosphatidylcholine [PC]/PE/cholesterol/sphingomyelin at 17:10:33:40) are particularly sensitive to labyrinthopeptins in comparison to PC/PE (90:10) LUVs, even though the overall PE amount remains constant. Labyrinthopeptins exhibited low cytotoxicity and had favorable pharmacokinetic properties in mice (half-life [t1/2] = 10.0 h), which designates them promising antiviral compounds acting by an unusual viral lipid targeting mechanism.IMPORTANCE For many viral infections, current treatment options are insufficient. Because the development of each antiviral drug is time-consuming and expensive, the prospect of finding broad-spectrum antivirals that can fight multiple, diverse viruses-well-known viruses as well as (re)emerging species-has gained attention, especially for the treatment of viral coinfections. While most known broad-spectrum agents address processes in the host cell, we found that targeting lipids of the free virus outside the host cell with the natural products labyrinthopeptin A1 and A2 is a viable strategy to inhibit the proliferation of a broad range of viruses from different families, including chikungunya virus, dengue virus, Zika virus, Kaposi's sarcoma-associated herpesvirus, and cytomegalovirus. Labyrinthopeptins bind to viral phosphatidylethanolamine and induce virolysis without exerting cytotoxicity on host cells. This represents a novel and unusual mechanism to tackle medically relevant viral infections.

Synthetic studies of cystobactamids as antibiotics and bacterial imaging carriers lead to compounds with high in vivo efficacy.

There is an alarming scarcity of novel chemical matter with bioactivity against multidrug-resistant Gram-negative bacterial pathogens. Cystobactamids, recently discovered natural products from myxobacteria, are an exception to this trend. Their unusual chemical structure, composed of oligomeric para-aminobenzoic acid moieties, is associated with a high antibiotic activity through the inhibition of gyrase. In this study, structural determinants of cystobactamid's antibacterial potency were defined at five positions, which were varied using three different synthetic routes to the cystobactamid scaffold. The potency against Acinetobacter baumannii could be increased ten-fold to an MIC (minimum inhibitory concentration) of 0.06 µg mL-1, and the previously identified spectrum gap of Klebsiella pneumoniae could be closed compared to the natural products (MIC of 0.5 µg mL-1). Proteolytic degradation of cystobactamids by the resistance factor AlbD was prevented by an amide-triazole replacement. Conjugation of cystobactamid's N-terminal tetrapeptide to a Bodipy moiety induced the selective localization of the fluorophore for bacterial imaging purposes. Finally, a first in vivo proof of concept was obtained in an E. coli infection mouse model, where derivative 22 led to the reduction of bacterial loads (cfu, colony-forming units) in muscle, lung and kidneys by five orders of magnitude compared to vehicle-treated mice. These findings qualify cystobactamids as highly promising lead structures against infections caused by Gram-positive and Gram-negative bacterial pathogens.

Subcellular Quantification of Uptake in Gram-Negative Bacteria.

Infections by Gram-negative pathogens represent a major health care issue of growing concern due to a striking lack of novel antibacterial agents over the course of the last decades. The main scientific problem behind the rational optimization of novel antibiotics is our limited understanding of small molecule translocation into, and their export from, the target compartments of Gram-negative species. To address this issue, a versatile, label-free assay to determine the intracellular localization and concentration of a given compound has been developed for Escherichia coli and its efflux-impaired ΔTolC mutant. The assay applies a fractionation procedure to antibiotic-treated bacterial cells to obtain periplasm, cytoplasm, and membrane fractions of high purity, as demonstrated by Western Blots of compartment-specific marker proteins. This is followed by an LC-MS/MS-based quantification of antibiotic content in each compartment. Antibiotic amounts could be converted to antibiotic concentrations by assuming that an E. coli cell is a cylinder flanked by two half spheres and calculating the volumes of bacterial compartments. The quantification of antibiotics from different classes, namely ciprofloxacin, tetracycline, trimethoprim, and erythromycin, demonstrated pronounced differences in uptake quantities and distribution patterns across the compartments. For example, in the case of ciprofloxacin, a higher amount of compound was located in the cytoplasm than in the periplasm (592 ± 50 pg vs 277 ± 13 pg per 3.9 × 109 cells), but owing to the smaller volume of the periplasmic compartment, its concentration in the cytoplasm was much lower (37 ± 3 vs 221 ± 10 pg/µL for the periplasm). For erythromycin and tetracycline, differences in MICs between WT and ΔTolC mutant strains were not reflected by equal differences in uptake, illustrating that additional experimental data are needed to predict antibiotic efficacy. We believe that our assay, providing the antibiotic concentration at the compartment in which the drug target is expressed, constitutes an essential piece of information for a more rational optimization of novel antibiotics against Gram-negative infections.

Exposure of vital cells to necrotic cell lysates induce the IRE1α branch of the unfolded protein response and cell proliferation.

Necrosis is a form of cell death that is detrimental to the affected tissue because the cell ruptures and releases its content (reactive oxygen species among others) into the extracellular space. Clusterin (CLU), a cytoprotective extracellular chaperone has been shown to be upregulated in the face of necrosis. We here show that in addition to CLU upregulation, necrotic cell lysates induce JNK/SAPK signaling, the IRE1α branch of the unfolded protein response (UPR), the MAPK/ERK1/2, and the mTOR signaling pathways and results in an enhanced proliferation of the vital surrounding cells. We name this novel response mechanism: Necrosis-induced Proliferation (NiP).

Multivalent Siderophore-DOTAM Conjugates as Theranostics for Imaging and Treatment of Bacterial Infections.

There is a strong need to better diagnose infections at deep body sites through noninvasive molecular imaging methods. Herein, we describe the synthesis and characterization of probes based on siderophore conjugates with catechol moieties and a central DOTAM scaffold. The probes can accommodate a metal ion as well as an antibiotic moiety and are therefore suited for theranostic purposes. The translocation of the conjugates across the outer and inner cell membranes of E. coli was confirmed by growth recovery experiments with enterobactin-deficient strains, by the antibacterial activity of ampicillin conjugates, and by confocal imaging using a fluorogen-activating protein-malachite green system adapted to E. coli. The suitability of the probes for in vivo imaging was demonstrated with a Cy5.5 conjugate in mice infected with P. aeruginosa.

The CLU-files: disentanglement of a mystery.

The multifaceted protein clusterin (CLU) has been challenging researchers for more than 35 years. The characterization of CLU as a molecular chaperone was one of the major breakthroughs in CLU research. Today, secretory clusterin (sCLU), also known as apolipoprotein J (apoJ), is considered one of the most important extracellular chaperones ever found. It is involved in a broad range of physiological and pathophysiological functions, where it exerts a cytoprotective role. Descriptions of various forms of intracellular CLU have led to further and even contradictory functions. To untangle the current state of knowledge of CLU, this review will combine old views in the field, with new discoveries to highlight the nature and function of this fascinating protein(s). In this review, we further describe the expression and subcellular location of various CLU forms. Moreover, we discuss recent insights into the structure of CLU and assess how structural properties as well as the redox environment determine the chaperone activity of CLU. Eventually, the review connects the biochemistry and molecular cell biology of CLU with medical aspects, to formulate a hypothesis of a CLU function in health and disease.

The chaperone activity of clusterin is dependent on glycosylation and redox environment.

BACKGROUND/AIMS: Clusterin (CLU), also known as Apolipoprotein J (ApoJ) is a highly glycosylated extracellular chaperone. In humans it is expressed from a broad spectrum of tissues and related to a plethora of physiological and pathophysiological processes, such as Alzheimer's disease, atherosclerosis and cancer. In its dominant form it is expressed as a secretory protein (secreted CLU, sCLU). During its maturation, the sCLU-precursor is N-glycosylated and cleaved into an α- and a ß-chain, which are connected by five symmetrical disulfide bonds. Recently, it has been demonstrated that besides the predominant sCLU, rare intracellular CLU forms are expressed in stressed cells. Since these forms do not enter or complete the secretory pathway, they are not proteolytically modified and show either no or only core glycosylation. Due to their sparsity, these intracellular forms are functionally poorly characterized. To evaluate the function(s) of these stress-related intracellular forms, we investigate for the first time the impact of proteolytic cleavage, differential glycosylation and the influence of the redox environment on the chaperone activity of CLU. METHODS: Non-cleavable sCLU was generated by expression from a mutant construct of sCLU, in which the furin-like proprotein convertase (PC) recognition site was modified. After purification of recombinant uncleaved sCLU from the medium of over-expressing cells, we performed chaperone activity assays to compare the activities of wild-type (cleaved) and uncleaved mutant sCLU. Additionally, this approach enabled us to investigate the role of carbohydrates, the proteolytic maturation and reducing conditions on CLU chaperone activity. Further, we characterized the differentially treated CLU forms by using MALDI-TOF, CD-spectroscopy and Western blotting in addition to the functional assay. RESULTS: We show that the PC-cleavage is dispensable for sCLU chaperone activity. Moreover, our data demonstrate that while fully deglycosylated sCLU lacks chaperone activity, partially deglycosylated sCLU is still capable of solubilizing target proteins. Most importantly, we here demonstrate for the first time that uncleaved sCLU is highly sensitive towards reducing conditions. CONCLUSIONS: Our study provides evidence that unglycosylated intracellular CLU forms cannot exhibit a chaperone activity compared to sCLU. Additionally, we support recent postulates that glycosylated intracellular CLU forms may act as a redox sensor under oxidative stress conditions. Furthermore, we conclude that the proteolytic cleavage of sCLU is important to maintain full chaperone activity, i.e. in the presence of necrosis.

Non-secreted clusterin isoforms are translated in rare amounts from distinct human mRNA variants and do not affect Bax-mediated apoptosis or the NF-κB signaling pathway.

Clusterin, also known as apolipoprotein J, is expressed from a variety of tissues and implicated in pathological disorders such as neurodegenerative diseases, ischemia and cancer. In contrast to secretory clusterin (sCLU), which acts as an extracellular chaperone, the synthesis, subcellular localization and function(s) of intracellular CLU isoforms is currently a matter of intense discussion. By investigating human CLU mRNAs we here unravel mechanisms leading to the synthesis of distinct CLU protein isoforms and analyze their subcellular localization and their impact on apoptosis and on NF-κB-activity. Quantitative PCR-analyses revealed the expression of four different stress-inducible CLU mRNA variants in non-cancer and cancer cell lines. In all cell lines variant 1 represents the most abundant mRNA, whereas all other variants collectively account for no more than 0.34% of total CLU mRNA, even under stressed conditions. Overexpression of CLU cDNAs combined with in vitro mutagenesis revealed distinct translational start sites including a so far uncharacterized non-canonical CUG start codon. We show that all exon 2-containing mRNAs encode sCLU and at least three non-glycosylated intracellular isoforms, CLU1­449, CLU21­449 and CLU34­449, which all reside in the cytosol of unstressed and stressed HEK­293 cells. The latter is the only form expressed from an alternatively spliced mRNA variant lacking exon 2. Functional analysis revealed that none of these cytosolic CLU forms modulate caspase-mediated intrinsic apoptosis or significantly affects TNF-α-induced NF-κB-activity. Therefore our data challenge some of the current ideas regarding the physiological functions of CLU isoforms in pathologies.