The Search for Antibacterial Inhibitors Targeting Cell Division Protein FtsZ at Its Nucleotide and Allosteric Binding Sites.

The global spread of bacterial antimicrobial resistance is associated to millions of deaths from bacterial infections per year, many of which were previously treatable. This, combined with slow antibiotic deployment, has created an urgent need for developing new antibiotics. A still clinically unexploited mode of action consists in suppressing bacterial cell division. FtsZ, an assembling GTPase, is the key protein organizing division in most bacteria and an attractive target for antibiotic discovery. Nevertheless, developing effective antibacterial inhibitors targeting FtsZ has proven challenging. Here we review our decade-long multidisciplinary research on small molecule inhibitors of bacterial division, in the context of global efforts to discover FtsZ-targeting antibiotics. We focus on methods to characterize synthetic inhibitors that either replace bound GTP from the FtsZ nucleotide binding pocket conserved across diverse bacteria or selectively bind into the allosteric site at the interdomain cleft of FtsZ from Bacillus subtilis and the pathogen Staphylococcus aureus. These approaches include phenotype screening combined with fluorescence polarization screens for ligands binding into each site, followed by detailed cytological profiling, and biochemical and structural studies. The results are analyzed to design an optimized workflow to identify effective FtsZ inhibitors, and new approaches for the discovery of FtsZ-targeting antibiotics are discussed.

New Trends in Aging Drug Discovery.

Aging is considered the main risk factor for many chronic diseases that frequently appear at advanced ages. However, the inevitability of this process is being questioned by recent research that suggests that senescent cells have specific features that differentiate them from younger cells and that removal of these cells ameliorates senescent phenotype and associated diseases. This opens the door to the design of tailored therapeutic interventions aimed at reducing and delaying the impact of senescence in life, that is, extending healthspan and treating aging as another chronic disease. Although these ideas are still far from reaching the bedside, it is conceivable that they will revolutionize the way we understand aging in the next decades. In this review, we analyze the main and well-validated cellular pathways and targets related to senescence as well as their implication in aging-associated diseases. In addition, the most relevant small molecules with senotherapeutic potential, with a special emphasis on their mechanism of action, ongoing clinical trials, and potential limitations, are discussed. Finally, a brief overview of alternative strategies that go beyond the small molecule field, together with our perspectives for the future of the field, is provided.

Cannabinoid WIN55212-2 impairs peanut-allergic sensitization and promotes the generation of allergen-specific regulatory T cells.

BACKGROUND: Cannabinoids are lipid-derived mediators with anti-inflammatory properties in different diseases. WIN55212-2, a non-selective synthetic cannabinoid, reduces immediate anaphylactic reactions in a mouse model of peanut allergy, but its capacity to prevent peanut-allergic sensitization and the underlying mechanisms remains largely unknown. OBJECTIVE: To investigate the capacity of WIN55212-2 to immunomodulate peanut-stimulated human dendritic cells (DCs) and peanut-allergic sensitization in mice. METHODS: Surface markers and cytokines were quantified by flow cytometry, ELISA and qPCR in human monocyte-derived DCs (hmoDCs) and T-cell cocultures after stimulation with peanut alone or in the presence of WIN55212-2. Mice were epicutaneously sensitized with peanut alone or peanut/WIN55212-2. After peanut challenge, drop in body temperature, haematocrit, clinical symptoms, peanut-specific antibodies in serum and FOXP3+ regulatory (Treg) cells in spleen and lymph nodes were quantified. Splenocytes were stimulated in vitro with peanut to analyse allergen-specific T-cell responses. RESULTS: WIN55212-2 reduced peanut-induced hmoDC activation and promoted the generation of CD4+ CD127- CD25+ FOXP3+ Treg cells, while reducing the induction of IL-5-producing T cells. In vivo, WIN55212-2 impaired the peanut-induced migration of DCs to lymph nodes and their maturation. WIN55212-2 significantly reduced the induction of peanut-specific IgE and IgG1 antibodies in serum during epicutaneous peanut sensitization, reduced the clinical symptoms score upon peanut challenge and promoted the generation of allergen-specific FOXP3+ Treg cells. CONCLUSIONS: The synthetic cannabinoid WIN55212-2 interferes with peanut sensitization and promotes tolerogenic responses, which might well pave the way for the development of novel prophylactic and therapeutic strategies for peanut allergy.

Cannabinoids induce functional Tregs by promoting tolerogenic DCs via autophagy and metabolic reprograming.

The generation of functional regulatory T cells (Tregs) is essential to keep tissue homeostasis and restore healthy immune responses in many biological and inflammatory contexts. Cannabinoids have been pointed out as potential therapeutic tools for several diseases. Dendritic cells (DCs) express the endocannabinoid system, including the cannabinoid receptors CB1 and CB2. However, how cannabinoids might regulate functional properties of DCs is not completely understood. We uncover that the triggering of cannabinoid receptors promote human tolerogenic DCs that are able to prime functional FOXP3+ Tregs in the context of different inflammatory diseases. Mechanistically, cannabinoids imprint tolerogenicity in human DCs by inhibiting NF-κB, MAPK and mTOR signalling pathways while inducing AMPK and functional autophagy flux via CB1- and PPARα-mediated activation, which drives metabolic rewiring towards increased mitochondrial activity and oxidative phosphorylation. Cannabinoids exhibit in vivo protective and anti-inflammatory effects in LPS-induced sepsis and also promote the generation of FOXP3+ Tregs. In addition, immediate anaphylactic reactions are decreased in peanut allergic mice and the generation of allergen-specific FOXP3+ Tregs are promoted, demonstrating that these immunomodulatory effects take place in both type 1- and type 2-mediated inflammatory diseases. Our findings might open new avenues for novel cannabinoid-based interventions in different inflammatory and immune-mediated diseases.

Isoprenylcysteine Carboxylmethyltransferase-Based Therapy for Hutchinson-Gilford Progeria Syndrome.

Hutchinson-Gilford progeria syndrome (HGPS, progeria) is a rare genetic disease characterized by premature aging and death in childhood for which there were no approved drugs for its treatment until last November, when lonafarnib obtained long-sought FDA approval. However, the benefits of lonafarnib in patients are limited, highlighting the need for new therapeutic strategies. Here, we validate the enzyme isoprenylcysteine carboxylmethyltransferase (ICMT) as a new therapeutic target for progeria with the development of a new series of potent inhibitors of this enzyme that exhibit an excellent antiprogeroid profile. Among them, compound UCM-13207 significantly improved the main hallmarks of progeria. Specifically, treatment of fibroblasts from progeroid mice with UCM-13207 delocalized progerin from the nuclear membrane, diminished its total protein levels, resulting in decreased DNA damage, and increased cellular viability. Importantly, these effects were also observed in patient-derived cells. Using the Lmna G609G/G609G progeroid mouse model, UCM-13207 showed an excellent in vivo efficacy by increasing body weight, enhancing grip strength, extending lifespan by 20%, and decreasing tissue senescence in multiple organs. Furthermore, UCM-13207 treatment led to an improvement of key cardiovascular hallmarks such as reduced progerin levels in aortic and endocardial tissue and increased number of vascular smooth muscle cells (VSMCs). The beneficial effects go well beyond the effects induced by other therapeutic strategies previously reported in the field, thus supporting the use of UCM-13207 as a new treatment for progeria.

Targeting the FtsZ Allosteric Binding Site with a Novel Fluorescence Polarization Screen, Cytological and Structural Approaches for Antibacterial Discovery.

Bacterial resistance to antibiotics makes previously manageable infections again disabling and lethal, highlighting the need for new antibacterial strategies. In this regard, inhibition of the bacterial division process by targeting key protein FtsZ has been recognized as an attractive approach for discovering new antibiotics. Binding of small molecules to the cleft between the N-terminal guanosine triphosphate (GTP)-binding and the C-terminal subdomains allosterically impairs the FtsZ function, eventually inhibiting bacterial division. Nonetheless, the lack of appropriate chemical tools to develop a binding screen against this site has hampered the discovery of FtsZ antibacterial inhibitors. Herein, we describe the first competitive binding assay to identify FtsZ allosteric ligands interacting with the interdomain cleft, based on the use of specific high-affinity fluorescent probes. This novel assay, together with phenotypic profiling and X-ray crystallographic insights, enables the identification and characterization of FtsZ inhibitors of bacterial division aiming at the discovery of more effective antibacterials.

A Potent Isoprenylcysteine Carboxylmethyltransferase (ICMT) Inhibitor Improves Survival in Ras-Driven Acute Myeloid Leukemia.

Blockade of Ras activity by inhibiting its post-translational methylation catalyzed by isoprenylcysteine carboxylmethyltransferase (ICMT) has been suggested as a promising antitumor strategy. However, the paucity of inhibitors has precluded the clinical validation of this approach. In this work we report a potent ICMT inhibitor, compound 3 [UCM-1336, IC50 = 2 µM], which is selective against the other enzymes involved in the post-translational modifications of Ras. Compound 3 significantly impairs the membrane association of the four Ras isoforms, leading to a decrease of Ras activity and to inhibition of Ras downstream signaling pathways. In addition, it induces cell death in a variety of Ras-mutated tumor cell lines and increases survival in an in vivo model of acute myeloid leukemia. Because ICMT inhibition impairs the activity of the four Ras isoforms regardless of its activating mutation, compound 3 surmounts many of the common limitations of available Ras inhibitors described so far. In addition, these results validate ICMT as a valuable target for the treatment of Ras-driven tumors.

Deregulation of the endocannabinoid system and therapeutic potential of ABHD6 blockade in the cuprizone model of demyelination.

Multiple sclerosis (MS) is a chronic demyelinating disease of unknown etiology in which tissue pathology suggests both immune-dependent attacks to oligodendroglia and primary oligodendrocyte demise. The endocannabinoid system has been crucially involved in the control of autoimmune demyelination and cannabinoid-based therapies exhibit therapeutic potential, but also limitations, in MS patients. In this context, growing evidence suggests that targeting the hydrolysis of the main endocannabinoid 2-arachidonoylglycerol (2-AG) may offer a more favorable benefit-to-risk balance in MS than existing cannabinoid medicines. Here we evaluated the modulation of endocannabinoid signaling and the therapeutic potential of targeting the 2-AG hydrolytic enzyme alpha/beta-hydrolase domain-containing 6 (ABHD6) in the cuprizone model of non-immune dependent demyelination. The concentrations of N-arachidonoylethanolamine (anandamide, AEA) and its congener N-palmitoylethanolamine (PEA) were reduced following 6 weeks of cuprizone feeding. Deregulation of AEA and PEA levels was not due to differences in the expression of the hydrolytic and biosynthetic enzymes fatty acid amide hydrolase and N-acylphosphatidylethanolamine-phospholipase D, respectively. Conversely, we measured elevated transcript levels of 2-AG hydrolytic enzymes monoacylglycerol lipase, ABHD6 and ABHD12 without changes in bulk 2-AG concentration. Upregulated CB1 and CB2 receptors expression, ascribed in part to microglia, was also detected in the brain of cuprizone-treated mice. Administration of an ABHD6 inhibitor partially attenuated myelin damage, astrogliosis and microglia/macrophage reactivity associated to cuprizone feeding. However, ABHD6 blockade was ineffective at engaging protective or differentiation promoting effects in oligodendrocyte cultures. These results show specific alterations of the endocannabinoid system and modest beneficial effects resulting from ABHD6 inactivation in a relevant model of primary demyelination.

Monoacylglycerol lipase (MAGL) as a promising therapeutic target.

Monoacylglycerol lipase (MAGL) has been characterized as the main enzyme responsible for the inactivation of the most abundant brain endocannabinoid, 2-arachidonoylglycerol (2-AG). Besides this role, MAGL has progressively acquired a growing importance as an integrative metabolic hub that controls not only the in vivo levels of 2-AG but also of other monoacylglycerides and, indirectly, the levels of free fatty acids derived from their hydrolysis as well as other lipids with pro-inflammatory or pro-tumorigenic effects, coming from the further metabolism of fatty acids. All these functions have only started to be elucidated in the last years due to the progress made in the knowledge of the structure of MAGL and in the development of genetic and chemical tools. In this review we report the advances made in the field with a special focus on the last decade and how MAGL has become a promising therapeutic target for the treatment of several diseases that currently lack appropriate therapies.

Development of a Fluorescent Bodipy Probe for Visualization of the Serotonin 5-HT1A Receptor in Native Cells of the Immune System.

Serotonin (5-HT) modulates key aspects of the immune system. However, its precise function and the receptors involved in the observed effects have remained elusive. Among the different serotonin receptors, 5-HT1A plays an important role in the immune system given its presence in cells involved in both the innate and adaptive immune responses, but its actual levels of expression under different conditions have not been comprehensively studied due to the lack of suitable tools. To further clarify the role of 5-HT1A receptor in the immune system, we have developed a fluorescent small molecule probe that enables the direct study of the receptor levels in native cells. This probe allows direct profiling of the receptor expression in immune cells using flow cytometry. Our results show that important subsets of immune cells including human monocytes and dendritic cells express functional 5-HT1A and that its activation is associated with anti-inflammatory signaling. Furthermore, application of the probe to the experimental autoimmune encephalomyelitis model of multiple sclerosis demonstrates its potential to detect the specific overexpression of the 5-HT1A receptor in CD4+ T cells. Accordingly, the probe reported herein represents a useful tool whose use can be extended to study the levels of 5-HT1A receptor in ex vivo samples of different immune system conditions.

Unique pharmacological properties of serotoninergic G-protein coupled receptors from cestodes.

BACKGROUND: Cestodes are a diverse group of parasites, some of them being agents of neglected diseases. In cestodes, little is known about the functional properties of G protein coupled receptors (GPCRs) which have proved to be highly druggable targets in other organisms. Notably, serotoninergic G-protein coupled receptors (5-HT GPCRs) play major roles in key functions like movement, development and reproduction in parasites. METHODOLOGY/PRINCIPAL FINDINGS: Three 5-HT GPCRs from Echinococcus granulosus and Mesocestoides corti were cloned, sequenced, bioinformatically analyzed and functionally characterized. Multiple sequence alignment with other GPCRs showed the presence of seven transmembrane segments and conserved motifs but interesting differences were also observed. Phylogenetic analysis grouped these new sequences within the 5-HT7 clade of GPCRs. Molecular modeling showed a striking resemblance in the spatial localization of key residues with their mammalian counterparts. Expression analysis using available RNAseq data showed that both E. granulosus sequences are expressed in larval and adult stages. Localization studies performed in E. granulosus larvae with a fluorescent probe produced a punctiform pattern concentrated in suckers. E. granulosus and M. corti larvae showed an increase in motility in response to serotonin. Heterologous expression revealed elevated levels of cAMP production in response to 5-HT and two of the GPCRs showed extremely high sensitivity to 5-HT (picomolar range). While each of these GPCRs was activated by 5-HT, they exhibit distinct pharmacological properties (5-HT sensitivity, differential responsiveness to ligands). CONCLUSIONS/SIGNIFICANCE: These data provide the first functional report of GPCRs in parasitic cestodes. The serotoninergic GPCRs characterized here may represent novel druggable targets for antiparasitic intervention.

A Fluorescent Probe to Unravel Functional Features of Cannabinoid Receptor CB1 in Human Blood and Tonsil Immune System Cells.

The human endogenous cannabinoid system (ECS) regulates key physiological processes and alterations in its signaling pathways, and endocannabinoid levels are associated with diseases such as neurological and neuropsychiatric conditions, cancer, pain and inflammation, obesity, and metabolic and different immune related disorders. Immune system cells express the G-protein coupled cannabinoid receptor 1 (CB1), but its functional role has not been fully understood, likely due to the lack of appropriate tools. The availability of novel tools to investigate the role of CB1 in immune regulation might contribute to identify CB1 as a potential novel therapeutic target or biomarker for many diseases. Herein, we report the development and validation of the first fluorescent small molecule probe to directly visualize and quantify CB1 in blood and tonsil immune cells by flow cytometry and confocal microscopy. We coupled the cannabinoid agonist HU210 to the fluorescent tag Alexa Fluor 488, generating a fluorescent probe with high affinity for CB1 and selectivity over CB2. We validate HU210-Alexa488 for the rapid, simultaneous, and reproducible identification of CB1 in human monocytes, T cells, and B cells by multiplexed flow cytometry. This probe is also suitable for the direct visualization of CB1 in tonsil tissues, allowing the in vivo identification of tonsil CB1-expressing T and B cells. This study provides the first fluorescent chemical tool to investigate CB1 expression and function in human blood and tonsil immune cells, which might well pave the way to unravel essential features of CB1 in different immune and ECS-related diseases.

The structural assembly switch of cell division protein FtsZ probed with fluorescent allosteric inhibitors.

FtsZ is a widely conserved tubulin-like GTPase that directs bacterial cell division and a new target for antibiotic discovery. This protein assembly machine cooperatively polymerizes forming single-stranded filaments, by means of self-switching between inactive and actively associating monomer conformations. The structural switch mechanism was proposed to involve a movement of the C-terminal and N-terminal FtsZ domains, opening a cleft between them, allosterically coupled to the formation of a tight association interface between consecutive subunits along the filament. The effective antibacterial benzamide PC190723 binds into the open interdomain cleft and stabilizes FtsZ filaments, thus impairing correct formation of the FtsZ ring for cell division. We have designed fluorescent analogs of PC190723 to probe the FtsZ structural assembly switch. Among them, nitrobenzoxadiazole probes specifically bind to assembled FtsZ rather than to monomers. Probes with several spacer lengths between the fluorophore and benzamide moieties suggest a binding site extension along the interdomain cleft. These probes label FtsZ rings of live Bacillus subtilis and Staphylococcus aureus, without apparently modifying normal cell morphology and growth, but at high concentrations they induce impaired bacterial division phenotypes typical of benzamide antibacterials. During the FtsZ assembly-disassembly process, the fluorescence anisotropy of the probes changes upon binding and dissociating from FtsZ, thus reporting open and closed FtsZ interdomain clefts. Our results demonstrate the structural mechanism of the FtsZ assembly switch, and suggest that the probes bind into the open clefts in cellular FtsZ polymers preferably to unassembled FtsZ in the bacterial cytosol.

Mechanisms of immune regulation in allergic diseases: the role of regulatory T and B cells.

Allergy is a major public health problem with a high socio-economic impact. The number of allergic patients is expected to reach to four billion within two decades when the World's population reaches to 10 billion. Our knowledge on the molecular mechanisms underlying allergic diseases and allergen tolerance induction had significant advances during the last years. Nowadays, it is well accepted that the generation and maintenance of allergen-specific regulatory T cells (Tregs) and regulatory B cells (Bregs) and the involvement of their suppressive cytokines and surface molecules are essential for the induction of allergen tolerance. These mechanisms play essential roles for the restoration of healthy immune responses to allergens in allergen-specific immunotherapy (AIT) and healthy immune response during high-dose antigen exposure in beekeepers and cat owners. AIT remains as the only disease-modifying and curative treatment for allergic diseases and represents a perfect model to investigate the antigen-specific immune responses in humans. A large number of clinical trials demonstrated AIT as an effective treatment in many patients, but it still faces several drawbacks in relation to efficacy, safety, long duration, and patient adherence. Novel strategies to overcome these inconveniences, such as the development of novel adjuvants and alternative routes of administration are being developed. The better understanding of the molecular mechanism governing the generation of Treg and Breg cells during allergen tolerance might well open new avenues for alternative therapeutic interventions in allergic diseases and help better understanding of other immune-tolerance-related diseases.

A new serotonin 5-HT6 receptor antagonist with procognitive activity - Importance of a halogen bond interaction to stabilize the binding.

Serotonin 5-HT6 receptor has been proposed as a promising therapeutic target for cognition enhancement though the development of new antagonists is still needed to validate these molecules as a drug class for the treatment of Alzheimer's disease and other pathologies associated with memory deficiency. As part of our efforts to target the 5-HT6 receptor, new benzimidazole-based compounds have been designed and synthesized. Site-directed mutagenesis and homology models show the importance of a halogen bond interaction between a chlorine atom of the new class of 5-HT6 receptor antagonists identified herein and a backbone carbonyl group in transmembrane domain 4. In vitro pharmacological characterization of 5-HT6 receptor antagonist 7 indicates high affinity and selectivity over a panel of receptors including 5-HT2B subtype and hERG channel, which suggests no major cardiac issues. Compound 7 exhibited in vivo procognitive activity (1 mg/kg, ip) in the novel object recognition task as a model of memory deficit.

Development of a Nucleotide Exchange Inhibitor That Impairs Ras Oncogenic Signaling.

Despite more than three decades of intense effort, no anti-Ras therapies have reached clinical application. Contributing to this failure has been an underestimation of Ras complexity and a dearth of structural information. In this regard, recent studies have revealed the highly dynamic character of the Ras surface and the existence of transient pockets suitable for small-molecule binding, opening up new possibilities for the development of Ras modulators. Herein, a novel Ras inhibitor (compound 12) is described that selectively impairs mutated Ras activity in a reversible manner without significantly affecting wild-type Ras, reduces the Ras-guanosine triphosphate (GTP) levels, inhibits the activation of the mitogen-activated protein kinase (MAPK) pathway, and exhibits remarkable cytotoxic activity in Ras-driven cellular models. The use of molecular dynamics simulations and NMR spectroscopy experiments has enabled the molecular bases responsible for the interactions between compound 12 and Ras protein to be explored. The new Ras inhibitor binds partially to the GTP-binding region and extends into the adjacent hydrophobic pocket delimited by switch II. Hence, Ras inhibitor 12 could represent a new compound for the development of more efficacious drugs to target Ras-driven cancers; a currently unmet clinical need.

Chemoproteomic Approach to Explore the Target Profile of GPCR ligands: Application to 5-HT1A and 5-HT6 Receptors.

Determination of the targets of a compound remains an essential aspect in drug discovery. A complete understanding of all binding interactions is critical to recognize in advance both therapeutic effects and undesired consequences. However, the complete polypharmacology of many drugs currently in clinical development is still unknown, especially in the case of G protein-coupled receptor (GPCR) ligands. In this work we have developed a chemoproteomic platform based on the use of chemical probes to explore the target profile of a compound in biological systems. As proof of concept, this methodology has been applied to selected ligands of the therapeutically relevant serotonin 5-HT1A and 5-HT6 receptors, and we have identified and validated some of their off-targets. This approach could be extended to other drugs of interest to study the targeted proteome in disease-relevant systems.

Auranofin efficacy against MDR Streptococcus pneumoniae and Staphylococcus aureus infections.

BACKGROUND: Auranofin is an FDA-approved, gold-containing compound in clinical use for the oral treatment of rheumatoid arthritis and has been recently granted by the regulatory authorities due to its antiprotozoal properties. METHODS: A reprofiling strategy was performed with a Streptococcus pneumoniae phenotypic screen and a proprietary library of compounds, consisting of both FDA-approved and unapproved bioactive compounds. Two different multiresistant S. pneumoniae strains were employed in a sepsis mouse model of infection. In addition, an MRSA strain was tested using both the thigh model and a mesh-associated biofilm infection in mice. RESULTS: The repurposing approach showed the high potency of auranofin against multiresistant clinical isolates of S. pneumoniae and Staphylococcus aureus in vitro and in vivo. Efficacy in the S. pneumoniae sepsis model was obtained using auranofin by the oral route in the dose ranges used for the treatment of rheumatoid arthritis. Thioglucose replacement by alkyl chains showed that this moiety was not essential for the antibacterial activity and led to the discovery of a new gold derivative (MH05) with remarkable activity in vitro and in vivo. CONCLUSIONS: Auranofin and the new gold derivative MH05 showed encouraging in vivo activity against multiresistant clinical isolates of S. pneumoniae and S. aureus. The clinical management of auranofin, alone or in combination with other antibiotics, deserves further exploration before use in patients presenting therapeutic failure caused by infections with multiresistant Gram-positive pathogens. Decades of clinical use mean that this compound is safe to use and may accelerate its evaluation in humans.