Lead discovery strategies for identification of Chlamydia pneumoniae inhibitors

Throughout its known history, the gram-negative bacterium Chlamydia pneumoniae has remained a challenging target for antibacterial chemotherapy and drug discovery. Owing to its well-known propensity for persistence and recent reports on antimicrobial resistence within closely related species, new approaches for targeting this ubiquitous human pathogen are urgently needed. In this review, we describe the strategies that have been successfully applied for the identification of nonconventional antichlamydial agents, including target-based and ligand-based virtual screening, ethnopharmacological approach and pharmacophore-based design of antimicrobial peptide-mimicking compounds. Among the antichlamydial agents identified via these strategies, most translational work has been carried out with plant phenolics. Thus, currently available data on their properties as antichlamydial agents are described, highlighting their potential mechanisms of action. In this context, the role of mitogen-activated protein kinase activation in the intracellular growth and survival of C. pneumoniae is discussed. Owing to the complex and often complementary pathways applied by C. pneumoniae in the different stages of its life cycle, multitargeted therapy approaches are expected to provide better tools for antichlamydial therapy than agents with a single molecular target.Peer reviewe

The role and host-directed targeting of long non-coding RNAs in macrophage polarization during Mycobacterium tuberculosis infection

In 2020, the World Health Organization (WHO) reported 1.5 million tuberculosis (TB)- associated deaths and an incidence of 10 million new cases. The causative, Mycobacterium tuberculosis (Mtb), evades host immune responses by skewing macrophage polarization towards a less microbicidal alternative state to avoid classical effector killing functions. However, the molecular details underlying these evasion mechanisms remain incomplete and current therapy is challenged with drug resistance. Host-directed therapy (HDT) has recently gained attention, with long non-coding RNAs (lncRNAs) as potential targets due to their emerging roles in pathogenic immune responses. We previously performed cap analysis gene expression (CAGE) transcriptomics on IFN-γ stimulated (classically activated) and IL-4/IL-13 stimulated (alternatively activated) mouse macrophages, identifying 151 differentially expressed lncRNAs following Mtb infection. We validated the top 11 differentially expressed lncRNAs and two were chosen for this study, lncRNA-125, whose expression was regulated at different levels unstimulated and in response to IFN-γ and IL-4/IL-13, and lncRNA-612 whose expression was only induced by IFN-γ stimulation. Interestingly, the expression of lncRNA125 and lncRNA-612 was downregulated following Mtb infection. Therefore, this study aimed at functionally validating these lncRNAs in unstimulated, IFN-γ and IL-4/IL-13 stimulated and/or Mtb-infected mouse and human macrophages by a loss-of-function approach using chemically engineered antisense oligonucleotides (gapmeRs). Knockdown of lncRNA-125 by gapmeRs reduced Mtb growth and anti-inflammatory cytokine production mediated by increased apoptosis, nitrite and pro-inflammatory cytokine production in IL-4/IL-13 prestimulated mouse macrophages. Whereas knockdown of lncRNA-125 in IFN-γ pre-stimulated mouse macrophages favoured Mtb growth and anti-inflammatory cytokine production, with reduction of apoptosis, nitrite and pro-inflammatory cytokine production. Therefore, indicating that lncRNA-125 regulates macrophage polarization during Mtb infection. Knockdown of lncRNA-125 in human macrophages resulted in reduced Mtb growth and increased proinflammatory cytokine production in unstimulated, IFN-γ and IL-4/IL-13 pre-stimulated BMDMs infected with Mtb. Comparatively, gapmeR knockdown of lncRNA-612 reduced Mtb growth and increased pro-inflammatory cytokine production in IFN-γ pre-stimulated mouse and human macrophages. In mouse macrophages, these responses were mediated by increased apoptosis and nitrite production, with reduced anti-inflammatory cytokine production. Overall, these findings highlight lncRNAs as novel host factors to be further investigated as targets for TB diagnostics and adjunctive HDTs

Blinding the CYCLOPS - Neuroblastoma vulnerabilities unveiled by 1p loss

Neuroblastoma is the most common solid tumor in infants arising during embryonal development or early post-natal life. A frequently recurrent event is the deletion of chromosome arm 1p, which accounts for ~35% of all high stage cases. In the past years research focused on identification of potential 1p tumor suppressor genes but therapeutic targeting of these was shown to be difficult. With tumor suppressor gene deletion also a wide range of passenger genes get lost. As some of these are cell essential, hemizygous loss and associated reduced expression renders cells vulnerable to further impairment. Genes fulfilling these requirements are referred to as CYCLOPS (copy number alterations yielding cancer liabilities owing to partial loss) genes and may open a new therapeutic window. In this study we aimed at identifying CYCLOPS genes on chromosome arm 1p in neuroblastoma. After detailed characterization of the 1p status in 35 neuroblastoma cell lines, we selected five cell lines with and five without 1p-deletion. For candidate gene identification, an siRNA screen for 184 druggable genes mapping to the distal end of 1p was done. Six candidates which showed high dependency in 1p-deleted but not in 1p non-deleted cells and differentially expression (1pdel < 1pnorm) were selected for further validation. In the end one gene met our requirements, EPHB2. This gene is especially important for embryonic cells and the developing nervous system. In 1p-deleted cell lines, EphB2 knock-down induced cell cycle arrest in G1/G0 and impaired cell survival. A small proportion of cells remained alive after activating HGF-induced c-MET signaling and MAPK/Akt pathway-mediated survival mechanisms. Induced EphB2 overexpression rescued the cells from cell death upon knock-down, supporting that EPHB2 expression is at a minimum level for survival in 1p-deleted cell lines. In the control group of 1p non-deleted cell lines the impact on viability and gene expression after EphB2 knock-down was minimal. We observed also G1/G0 arrest but viability was not impaired. Taking together, this study revealed EPHB2 as a promising 1p CYCLOPS candidate in neuroblastoma. As 1p is deleted in ~35% of all high-risk cases, a much wider range of patients may benefit from therapy approaches compared to strategies targeting other neuroblastoma-specific aberrations. Side effects of such approaches may be reduced as 1p non-deleted cells are not affected negatively. In general, this is a proof-of-principle for new drug target identification and is expandable to all cancers carrying frequent 1p-deletions

Engineered biomaterials as extracellular microenvironments for guiding cell programming and reprogramming

The interface between cells and materials is a dynamic and complex environment where cells in contact with materials can sense their properties such as stiffness, matrix protein, and geometry and respond to these cues in multiple ways including through mechanical forces exerted on the matrix by the cells. Cells incorporate these cues via signal propagation through integrins, and translate this information through intracellular signal transduction cascades to regulate gene expression and cell fate decisions. Advances in biomaterials to direct stem cell lineage decisions have focused on designing biomimetic materials that realize the ‘‘in vivo” microenvironments’ ability to interact with cells. However, not only is designing tailored biomaterials that present multiple signals challenging, but the precise roles of physical and biochemical cues in coordinating cellular processes such as migration, proliferation, and differentiation remains difficult to dissect. After a short introduction we explore using model polyacrylamide hydrogel systems in Chapter 2-5 to study the effects of biophysical (elasticity and geometry) and chemical (matrix protein) cues on mesenchymal stem cell (MSC) fate decisions, showing these cues can play a large role in differentiation. In Chapter 6 we explore how switching the biophysical microenvironment (matrix stiffness and cell shape) can be used to understand the plasticity of MSC lineage specification. Finally, in Chapter 7-9, we demonstrate how geometric cues at the interface of tissue, where interfacial energy and curvature can be modulated in vitro, will dictate cancer cell tumorigenicity, metastatic potential, and the regulation of tumorangiogenesis. Moreover, we reveal a mechanism where perimeter features initiate α5β1 adhesion and epithelial-to-mesenchymal transition, Mitogen Activated Protein Kinase (MAPK) and Signal Transducer and Activator of Transcription (STAT) pathways, and regulation of distinct histone marks, to guide gene expression underlying the phenotypic alterations of malignant melanoma. Overall, we believe the work presented here demonstrates the importance and utility of extracellular properties in modulating cell programming and reprogramming, and should aid in the development of biomaterials for more efficiently directing distinct cellular states for the development of synthetic model systems that more accurately recapitulate the in vivo microenvironment

Monocyte and endothelial cell interaction during sepsis

PhD ThesisSepsis complicated by coagulopathy is associated with a higher mortality rate. The cell surface expression of tissue factor (TF) may be important in its development. There at least three subsets of monocytes – classical, intermediate and non-classical – that vary in proportion during sepsis. This project used three approaches to investigate how the monocyte subsets and their endothelial interaction could contribute to sepsis-associated coagulopathy: the in vitro stimulation of healthy monocytes with lipopolysaccharide (LPS) and endothelial co-culture; the use of a human model of endotoxaemia; and the collection of blood samples from individuals with sepsis. LPS stimulation of healthy monocytes demonstrated that the monocyte subsets express cell surface TF to different extents. Classical and intermediate monocytes express the highest proportion of TF in response to LPS. An in vitro monocyte-endothelial co-culture model demonstrated that monocytes could promote coagulation through the increased cell surface expression of TF, independent of LPS, and influence the endothelial fibrinolytic response. Blood samples were taken at a range of time-points following an injection of LPS into a healthy volunteer. An increase in cell surface TF occurred within 90 minutes of exposure to LPS and was associated with an increase in markers of coagulation. Individuals varied in their response to LPS, with two groups identified: high and low responders. The response of individuals was consistent between the subsets. Samples from individuals with sepsis expressed a higher level of cell surface TF compared to individuals who were critically ill but did not have sepsis. The surface expression of TF increased further when measured following recovery from sepsis. This work demonstrates that the cell surface expression of TF varies between the monocyte subsets, that individuals may increase monocyte TF expression to different levels, and that individuals with sepsis express higher levels of TF both at the time of sepsis and following recovery

Experimental study on the role of lysophosphatidic acid in mediating cytokine/chemokine production and on the signaling pathways involved in inflammatory arthritis

La polyarthrite rhumatoïde (PR) est l’une des maladies auto-immunes qui atteint les articulations. Les symptômes principaux de la PR incluent une inflammation chronique de la synoviale dépendante de diverses cytokines et chimiokines inflammatoires, ainsi que de nombreux médiateurs de caractère lipidique, sécrétés par les cellules immunitaires et par les synoviocytes. L’acide lysophosphatidique (LPA) et son enzyme productrice, l’autotaxine (ATX) ont été détectés dans le liquide synovial. Les niveaux des ARNm de deux des six récepteurs du LPA, soit LPA1 et LPA3, sont aussi plus élevés dans les fibroblastes synoviaux des patients PR comparé à ceux d’arthrose ou suite avec une incubation avec le TNFα. Le LPA est aussi reconnu pour sa capacité à induire une production de cytokines et de chimiokines par des synoviocytes de type B (RAFLS) provenant de patients PR, ainsi que dans le modèle murin de poche d’air. D’ailleurs, la pré-incubation avec le facteur de nécrose tumorale alpha (TNFα) in vitro ou bien in vivo rehaussa cette sécrétion de cytokines et de chimiokines induite par la LPA. Dans le cadre de cette étude, nous avons démontré qu’une chimiokine appelée CXCL13 est impliquée dans le recrutement lymphocytaire induit par le LPA après une pré-incubation au TNFα, dans le modèle de poche d’air. Le mécanisme par lequel le LPA peut faire sécréter des cytokines et des chimiokines et par lequel la TNFα peut contribuer à une superproduction de cytokines et de chimiokines dépendante du LPA a aussi été investigué. Nos résultats indiquent que la p38MAPK, la cascade signalétique ERK-MSK-CREB, la Rho kinase, et la PI3K sont toutes des voies de signalisation stimulées par le LPA qui modulent une sécrétion de l’interleukine-8 (IL-8) chez les synoviocytes de type B. En revanche, nous avons remarqué que la sécrétion de l’IL-8 par les synoviocytes stimulés par le LPA devenait insensible aux inhibiteurs des dites kinases lorsque traités au préalable au TNFα. Il n’en demeure pas moins qu’une inhibition simultanée de la p38MAPK et d’ ERK a mené à une diminution efficace de la sécrétion de l’IL-6 et de l’IL-8. Cette étude a permis de mieux comprendre le mécanisme de la réponse inflammatoire déclenchée par le LPA et d’identifier les principales voies de signalisation intracellulaire contribuant à la sécrétion de cytokines et de chimiokines en présence ou en absence de TNFα. Le travail pourrait avoir des retombées majeures en ce qui concerne les stratégies pour développer des drogues ciblant l’axe ATX-LPA et les kinases mentionnées ci-dessus pour le traitement de la polyarthrite rhumatoïde.Rheumatoid Arthritis (RA) is one of the most severe inflammatory arthritides. The main features of RA include chronic inflammation in the synovium mediated by inflammatory cytokines and chemokines as well as lipid mediators, secreted by immune cells and synoviocytes. Lysophosphatidic acid (LPA), a monoacyl phospholipid mediator, and its producing enzyme autotaxin (ATX) were detected in synovial fluid. LPA1 and LPA3 receptor mRNA levels were also higher in synovium from RA patients than from normal individuals. LPA was previously reported to induce cytokine and chemokine production in RA fibroblast-like synoviocytes (RAFLS) and in a mouse air pouch model. In addition, TNFα pretreatment in vivo or in vitro enhanced this cytokine and chemokine secretion induced by LPA. In this study, we demonstrated that an LPA-induced chemokine named CXCL13 is also involved in LPA-induced leukocyte recruitment in the mouse air pouch model after TNFα pretreatment. The mechanism whereby LPA induces cytokine/chemokine secretion and whereby TNFα pretreatment induces cytokine/chemokine super-production mediated by LPA was also investigated in this study. Our data demonstrated that p38MAPK, the ERK-MSK-CREB axis, Rho kinase, and PI3K were all involved in the LPA signaling resulting in IL-8 secretion in RAFLS. However, we found that, after pretreatment with TNFα, LPA-induced IL-8 secretion became insensitive to inhibitors of those kinases mentioned above. Notwithstanding, blocking both the p38MAPK and ERK pathways could effectively decrease IL-8 and IL-6 secretion. This study allowed a deeper understanding of the mechanism of the LPA-induced inflammatory response, including the signal pathways regulating cytokine/chemokine secretion with or without the exacerbating effect of TNFα. The study may have important implications for the development of drugs targeting ATX-LPA axis and the aforementioned signaling pathways for the treatment of RA

Marine Compounds and Cancer 2020

The very first marine-derived anticancer drug, Cytarabine (aka Ara-C, Cytosar-U®), was approved by the FDA in 1969 for the treatment of leukemia. At the beginning of 2021, the list of approved marine-derived anticancer drugs consists of nine substances, five of which received approval within the last two years, demonstrating the rapid evolution of the field. The current book is a collection of scientific articles related to the exponentially growing field of anticancer marine compounds. These articles cover the whole field, from agents with cancer-preventive activity, to novel and previously characterized compounds with anticancer activity, both in vitro and in vivo, as well as the latest status of compounds under clinical development

The Role of MAP Kinase Cascade in Neonatal Brain Response to Hypoxia-Ischemic Insult

Babies that are born more than 8 weeks premature or those deprived of Oxygen during the perinatal period are susceptible to brain injury, particularly in conjunction with maternal or fetal infection, leading to neurological deficits later in life. Multiple studies have shown that even brief exposure to hypoxic conditions will cause rapid and selective increase in specific mitogen-activated protein kinases including extracellular signal - related kinase 1 and 2 (ERK1/2) and C-Jun N-terminus Kinases 1 to 3 (JNK1-3) as well as the JNK downstream effector: C-Jun. To gain insight into the in vivo function of some of these intracellular pathways that contribute to neuronal damage in ischemic postnatal brains, we examined the effects of global or cell type specific as single or combined deletions of ERK 1 and 2 and both cell type specific or functional deletions of C-Jun. In addition, ERK phosphorylation was prevented by the administration of the selective MEK inhibitor SL327. This was observed with both pre- and post-injury application. Lastly, to explore the effect of endotoxin as a sensitising agent prior to neonatal hypoxia ischemia: lipopolysaccharide given to neonatal mutant mice, with sensitising effects noted at a dosage of 0.6mg/kg of LPS and a time interval between endotoxin administration and hypoxia-ischemia of 12hr. The Rice-Vannucci mouse model is a well-established experimental paradigm for hypoxic ischemic injury, providing insights into molecular signals that determine both white and grey matter tissue loss. Normal pERK is detectable in periventricular white matter axons (15-45min post HI), followed by white and grey matter glia and cortical neurons (1-4h post HI), returning to normal by 8hr. Mice with double mutation of global ERK1 and neuronal ERK2 deletion showed a lack of pERK expression through the forebrain following HI. In LPS-sensitised HI, we observed a strong decrease in infarct size, histological brain injury and microglial activation in cortex, striatum, and thalamus. A more discreet effect was seen in subcortical white matter and hippocampus. ERK1 deletion attenuated the effect of neuronal ERK2 deletion. These results were reproduced following severe HI insult alone. Astrocytic ERK mutation exhibited a polar response with a 3-4fold increase in microglia activation and the number of dying cells within grey matter regions. Global inactivation of ERK, through pharmacological ERK inhibitor SL327 significantly reduced cell death, and associated microglial activation in both grey and white matter at 48h following HI insult. Application of SL327 even as late as 1hr post insult significantly reduced brain damage induced by mild HI exposure. Systemic administration 1hr after severe HI dramatically increased the survival rate of pups at 48hr post insult by 80% compared to sham-treated controls. Deletion of C-Jun in all neural-epithelial lineage cells resulted in a strong increase in injury following severe and LPS-sensitised insult. In contrast, neuron-specific deletion of C-Jun resulted in neuroprotection. Tunel positive cell death was significantly reduced compared to control groups, in white matter, cortex and thalamus. Microglial activation, and infarct volume loss was more discreetly decreased with a notable effect in cortex. C-Jun expression in astrocytes is not a major contributor to ischemic damage response, with very mild reduction in markers for cell death and microglia recruitment following severe hypoxia, and no change observed between mutants and controls after endotoxin-sensitised HI. Replacement of the four JNK-dependent C-Jun phosphorylation sites (jun4A) resulted in a mild decrease in cell death and microglial activation compared to littermate controls, which did not reach the level of statistical significance. Overall, the data points to an important role of neuronal ERK and C-Jun in both a cellular and biochemical response to HI in the neonatal cerebral brain, but also argues against the involvement of JNK-dependent C-Jun phosphorylation in mediating neural damage. In addition, inhibition of ERK via pharmaceutical agents shows promise in decreasing morbidity and mortality caused by mild to moderate HI exposure. Lastly, neuroepithelial C-Jun expression appears to be a critical factor in normal cortical development and employment of endogenous neuroprotective mechanisms against postnatal insult

Natural Products for Cancer Prevention and Therapy

ca. 200 words; this text will present the book in all promotional forms (e.g. flyers). Please describe the book in straightforward and consumer-friendly terms. [This Special Issue book, “Natural Products for Cancer Prevention and Therapy”, is based on recent advances in natural products for cancer prevention and therapy. For this purpose, the authors of this book have been organizing a biennial international conference series. The first meeting (First International Conference on Natural Products for Cancer Prevention and Therapy) was held in Istanbul between 31 August and 2 September 2015, with the support and contribution of many valuable researchers in this field. The abstracts of the first conference were published in the Anticancer Drugs journal as a supplement. The second meeting, namely The Second International Conference on Natural Products for Cancer Prevention and Therapy, was held at Erciyes University, Kayseri, Turkey, between 8 and 11 November 2017. The abstracts of all of the presentations from the second meeting were published in the Special Issue of Proceedings by the MDPI publishing group. Furthermore, the conference report and the selected full-length papers based on the presentations at the meeting, as well as other papers based on natural products for cancer prevention and therapy, were published as a Special Issue of the Nutrients journal from MDPI. This Special Issue has contributions from various participants of the aforementioned conference, as well as other cancer and natural product researchers. These contributions include original research papers, authoritative and up-to-date reviews, and commentaries on the following topics and areas: •Natural products for the prevention and therapy of oncologic diseases •Mechanism of natural agents for anticancer and cancer preventive effects •In vitro, in vivo, and clinical studies related to natural agents and cancer • Combinatorial effects of phytochemicals and cancer chemotherapeutic drugs •Challenges and innovative approaches for anticancer drug development based on natural products •Emerging studies on anticancer phytochemicals

Optimization of ERK signalling by transphosphorylation across different scaffold protein species: implication in cancer therapeutics

RESUMEN: La ruta de señalización Ras/ERK está claramente relacionada con el desarrollo y progresión de varias enfermedades. Alrededor del 40% de cánceres humanos portan mutaciones en alguno de los constituyentes de esta cascada. Uno de los mecanismos de regulación son las proteínas scaffold, que modulan la intensidad, amplitud y duración de las señales de ERK. Por esta razón algunos scaffolds tienen potencial como diana antitumoral. A tenor de nuevas evidencias que desvelan la interacción entre diferentes especies de proteínas scaffold, hemos propuesto la hipótesis de que las proteínas scaffold interaccionan entre sí para regular el flujo de las señales de ERK de manera coordinada. Esto podría ser una posible explicación de la ineficacia de un novedoso inhibidor de KSR. De hecho, hemos demostrado que KSR1 e IQGAP1 deficientes para la unión de MEK son capaces de incorporar ERK fosforilado. En este respecto, descubrimos que KSR1 e IQGAP1 pueden interaccionar directamente entre sí, permitiendo la “transfosforilación” de ERK unido a la proteína mutante deficiente para unir MEK. Así, asociaciones entre diferentes proteínas scaffold añadirían un grado más a la regulación de una vía de señalización, ya altamente regulada, lo que puede ofrecernos nuevos medios para manipular las señales de ERK, incluso con fines terapéuticos.ABSTRACT: The Ras/ERK pathway is noticeably linked to the development and progression of human malignancies. About 40% of human cancers harbor activating mutations in constituents of this pathway. One of the regulatory mechanisms is carried out by scaffold proteins, that modulate the intensity, amplitude and duration of ERK signals. For this reason, some scaffolds harbor a potential as antitumoral targets. In light of recent evidence unveiling direct interactions among different scaffold proteins species, we have considered the hypothesis that scaffold proteins must somehow interact with each other in order to regulate the flow of signals through the Ras/ERK pathway in a coordinated manner. This could be a possible explanation for the inefficacy of a novel small molecule KSR inhibitor. Indeed, we have shown that KSR1 and IQGAP1 mutants defective for binding MEK, can still associate to phosphorylated ERK. In this respect, we have found that KSR1 and IQGAP1 can directly interact among themselves, and in so doing they can “transphosphorylate” each other, enabling ERK phosphorylation in MEK-binding deficient mutants. Thus, associations among different scaffold proteins will add one further degree of regulation for an already tightly regulated cascade and could provide a novel means for manipulating ERK signals even with therapeutic purposes.La presente tesis doctoral titulada “Optimization of ERK signalling by transphosphorylation across different scaffold protein species: implication in cancer therapeutics” ha sido realizada en el Instituto de Biomedicna y Biotecnologia de Cantabria (IBBTEC) en el laboratorio de Regulacion espacial de las senales RAS/ERK en cancer gracias a la ayuda del Programa de personal investigador en formacion predoctoral en el area de biomedicina, biotecnologia y ciencias de la salud de la Universidad de Cantabria ( BOC 30.09.2015) y a los proyectos financiados por: CIBEROCIC ISCII, AECC, MINISTERIO DE ECONOMÍA Y COMPETITIVIDAD Durante el presente trabajo Ana Martin Vega ha realizado una estancia predoctoral de 3 meses en el laboratorio del Dr. David Engelberg en el instituto Alexander Silberman of Life Science en la Universidad Hebrea de Jerusalen, Israel, gracias a la financiacion para estancias predoctorales de la Universidad de Cantabri