Cheminformatic Approach for Deconvolution of Active Compounds in a Complex Mixture - phytoserms in Licorice

ABSTRACT After the validation of our in silico models by using the previous knowledge in this area the alerting phytochemicals from two Glycyrrhiza species (G. glabra and G. uralensis) were clustered. Exhaustive computational mining of licorice metabolome against selected endocrinal and metabolic targets led to the discovery of a unique class of compounds which belong to the dihydrostilbenoids (DHS) class appended with prenyl groups at various positions. To the best of our knowledge this interesting group of compounds has not been studied for their estrogenic activities or PXR activation. In addition some of the bis-prenylated DHS have been reported to be present only in G. uralensis. Another aspect of the current project was to predict the phase I primary metabolites of compounds found in both species of Glycyrrhiza and assess them with computational tools to predict their binding potential against both isoforms of hERs or drug metabolizing enzymes such as (CYP) inhibition models. Our investigations revealed estrogenic character for most of the predicted metabolites and have confirmed earlier reports of potential CYP3A4 and CYP1A2 inhibition. Compilation of such data is essential to gain a better understanding of the efficacy/safety of licorice extracts used in various botanical formularies. This approach with the involved cheminformatic tools has proven effective to yield rich information to support our understanding of traditional practices. It also can expand the role of botanical drugs for introducing new chemical entities (NCEs) and/or uncovering their liabilities at early stages. In this work we endeavored to comprehend the mechanism associated with the efficacy and safety of components reported in the licorice plant. We utilized smart screening techniques such as cheminformatics tools to reveal the high number of secondary metabolites produced by licorice which are capable of interfering with the human Estrogen Receptors (hERs) and/or PXR or other vital cytochrome P450 enzymes. The genus Glycyrrhiza encompasses several species exhibiting complex structural diversity of secondary metabolites and hence biological activities. The intricate nature of botanical remedies such as licorice rendered them obsolete for scientific research or medical industry. Understanding and finding the mechanisms of efficacy or safety for a plant-based therapy is very challenging yet it remains crucial and warranted. The licorice plant is known to have Selective Estrogen Receptor Modulatory effects (SERMs) with a spectrum of estrogenic and anti-estrogenic activities attributed to women’s health. On the contrary licorice extract was shown to induce pregnane xenobiotic receptor (PXR) which may manifest as a potential route for deleterious effects such as herb-drug interaction (HDI). While many studies attributed these divergent activities to a few classes of compounds such as liquiritigenin (a weak estrogenic SERM) or glycyrrhizin (weak PXR agonist) no attempt was made to characterize the complete set of compounds responsible for these divergent activities. A plethora of licorice components is undermined which might have the potential to be developed into novel phytoSERMS or to trigger undesirable adverse effects by altering drug metabolizing enzymes and thus pharmacokinetics. Thus we have ventured to synthesize a set of constitutional isomers of stilbenoids and DHS (archetypal of those found in licorice) with different prenylation patterns. Sixteen constitutional isomers of stilbenoids (M2-M10) and DHS (M12-M18) were successfully synthesized of which six of them (M8 M9 M14 M15 M17 and M18) were synthesized for the first time to be further tested and validated with cell-based methods for their estrogenic activities. We have unveiled a novel class of compounds which possess a strong PXR activation. These results which were in accord with the in silico prediction were observed for multiple synthesized prenylated stilbenoid and DHS by the luciferase reporter gene assay at µM concentrations. Moreover this activation was further validated by the six-fold increase in mRNA expression of Cytochrome P450 3A4 (CYP3A4) where three representative compounds (M7 M10 and M15) exceeded the activation fold of the positive control

Design of Nanoparticles in Cancer Therapy Based on Tumor Microenvironment Properties

Cancer is one of the leading causes of death worldwide, and battling cancer has always been a challenging subject in medical sciences. All over the world, scientists from different fields of study try to gain a deeper knowledge about the biology and roots of cancer and, consequently, provide better strategies to fight against it. During the past few decades, nanoparticles (NPs) have attracted much attention for the delivery of therapeutic and diagnostic agents with high efficiency and reduced side effects in cancer treatment. Targeted and stimuli-sensitive nanoparticles have been widely studied for cancer therapy in recent years, and many more studies are ongoing. This review aims to provide a broad view of different nanoparticle systems with characteristics that allow them to target diverse properties of the tumor microenvironment (TME) from nanoparticles that can be activated and release their cargo due to the specific characteristics of the TME (such as low pH, redox, and hypoxia) to nanoparticles that can target different cellular and molecular targets of the present cell and molecules in the TME

MICROBIAL PARTNERS IN HEALTH: BROADENING OUR UNDERSTANDING OF HOST-MICROBIOME RELATIONSHIPS

While microbes inhabit a wide array of environments, their ability to live within host tissue and become tolerated as part of a select microbial community is perhaps one of the most impressive feats of microbial resilience and survival. Host microbiome establishment and maintenance requires both host-microbe and microbe-microbe interactions. Among plant hosts, benefits from associated microbiomes are known to include improved growth, development and resistance to abiotic and biotic stresses. Mammalian microbiomes are known to improve host digestion, influence inflammation and even improve immune response to pathogens. While host-associated microbial communities across all domains of life are incredibly diverse, a growing number of studies are finding host-specific taxonomic trends, suggesting microbiome conservation and evolutionary selection. However, we have come to recognize that there is often functional redundancy between taxa. Therefore, investigative focus on microbiome composition potentially neglects pivotal and influential microbial players. Shifting focus to function over form creates the opportunity to tease apart the driving forces of unique microbiome constituents. This allows for identification of strains and genes of interest as well as microbial selections. To that end, here we describe the relationships between hosts and microbiomes as well as between microbes in two vastly different host systems (Figure 1.1). First, we suggest that plant root-associated Streptomyces isolates harboring genes encoding an enzyme and its co-factor are more tolerant of phenolic compounds generated by roots. Next, we address the capability of these Streptomyces isolates to employ their metabolic repertoires to influence the composition of the root microbiome. Finally, we define a previously under-described role for the gut microbiome in malaria immunology and suggest that gut microbial composition can modulate the severity of malarial disease. Together, these findings demonstrate the broad implications of microbiome composition across diverse hosts and environments, revealing unexplored opportunities for therapeutic interventions aimed at improving plant and human health

Cancer Nanomedicine

This special issue brings together cutting edge research and insightful commentary on the currentl state of the Cancer Nanomedicine field

Non psychotropic cannabinoids for the treatment of inflammatory diseases

Medicinal cannabis and purified cannabinoids have garnered worldwide attention since millions of patients can benefit from their medical properties. Over the past few years, the endocannabinoid system (ECS) has emerged as a crucial player for the regulation of physiological mechanisms related to inflammatory and immunomodulatory processes as well as food intake and energy metabolism. Hence, its pharmacological manipulation represents a novel strategy for the management of inflammatory diseases such as metabolic syndrome (MetS) and rheumatoid arthritis (RA) To investigate the real potential of medicinal marijuana, pre-clinical and clinical studies have been launched, with positive results for some neurological conditions like multiple sclerosis and some genetic juvenile forms of epilepsy. All these studies have used neutral cannabinoids, especially Δ9-THC and CBD, with little attention to the genuine phytocannabinoids of the plant, namely their acidic forms, such as the native precursor of Δ9-THC, the Δ9-THC acid A (Δ9-THCA-A), a non-psychoactive phytocannabinoid that remains largely unexplored. In addition, the modification of CBD scaffold was carried out in order to increase its application in different diseases. Δ9-THCA has two isomers, Δ9-THCA-A, in which the carboxylic acid group is in the 1 position, between the hydroxy group and the carbon chain, and Δ9-THCA -B, in which the carboxylic acid group is in the 3 position. Δ9-THCA-A is the most abundant isomer. In the present work, the CBD aminoquinone derivative VCE-004.8, a dual PPARγ and CB2 receptor agonist that also activates the HIF pathway, and Δ9-THCA-A, a partial and selective PPARγ modulator, showed lower adipogenic activity than the full PPARγ agonist rosiglitazone (RGZ) and did not affect osteoblastogenesis in hMSC. In an in vivo murine model of high fat diet (HFD)-induced obesity, both VCE-004.8 and Δ9-THCA-A treatments induced a significant reduction in body weight gain, total fat mass, adipocyte volume, plasma triglyceride levels, and liver steatosis in HFD mice. In addition, the treatments improved sensitivity to insulin and regulated the expression of other metabolic biomarkers in obese mice. Thus, VCE-004.8 and Δ9-THCA-A were qualified as novel therapeutic candidates for the management and improvement of the symptoms of obesityassociated metabolic syndrome and inflammation. In addition, subsequent in vitro and in silico analyses also showed that Δ9-THCA-A can act as an orthosteric CB1 agonist and also as a positive allosteric modulator in the presence of CP-55,940. Moreover, Δ9-THCA-A seemed to be an inverse agonist for CB2. In vivo experiments in a murine model of collagen-induced arthritis (CIA) demonstrated that Δ9-THCA-A prevented the inflammation and cartilage damage on knee joints. The anti-arthritic effect of Δ9-THCA-A was greatly prevented by either SR141716 or T0070907. Analysis of plasmatic biomarkers as well as determination of cytokines and anti-collagen antibodies confirmed that Δ9-THCA-A is mediating its activity mainly through PPARγ and CB1 pathways. Thus, our studies document that Δ9-THCA-A exerted anti-arthritis activity through CB1/PPARγ pathways highlighting its potential for the treatment of chronic inflammatory diseases such as RA.El cannabis medicinal y los cannabinoides purificados han captado la atención mundial, ya que millones de pacientes pueden beneficiarse de sus propiedades terapéuticas. En los últimos años, el sistema endocannabinoide (SEC) ha presentado un papel crucial en la regulación de mecanismos fisiológicos relacionados con procesos inflamatorios e inmunomoduladores, así como con la ingesta de alimentos y el metabolismo energético. Por lo tanto, su manipulación farmacológica representa una nueva estrategia para el tratamiento de enfermedades inflamatorias como son síndrome metabólico y artritis reumatoide (AR). Para investigar el verdadero potencial medicinal de la marihuana, se han realizado estudios preclínicos y clínicos con resultados positivos en determinadas condiciones neurológicas como la esclerosis múltiple y ciertas formas genéticas de epilepsia juvenil. Todos estos estudios han utilizado cannabinoides neutros, especialmente Δ9-THC y CBD, sin prestar atención a los auténticos fitocannabinoides de la planta, sus formas ácidas, como es el precursor nativo del Δ9-THC, el Δ9-THC ácido (Δ9-THCA-A), un fitocannabinoide no psicoactivo que permanece en gran medida inexplorado. Además, se ha realizado una modificación de la estructura química del CBD con el fin de aumentar su aplicación en el tratamiento de diferentes enfermedades. En el presente trabajo, el derivado aminoquinona del CBD, VCE-004.8, un agonista dual de los receptores PPARγ y CB2, que también activa la ruta de HIF, y Δ9-THCA-A, un modulador selectivo y parcial de PPARγ, mostraron una menor actividad adipogénica que rosiglitazona (RGZ), un agonista completo de PPARγ, y además, no presentaron efectos adversos asociados a los agonistas completos de PPARγ, como por ejemplo inhibición de osteoblastogénesis. En un modelo murino in vivo de obesidad inducida por una dieta alta en grasa (HFD), el tratamiento con VCE-004.8 y Δ9-THCA-A indujo una reducción significativa del peso corporal, masa grasa total, volumen de adipocitos, niveles plasmáticos de triglicéridos y esteatosis en ratones obesos. Además, también mejoraron la sensibilidad a la insulina y regularon la expresión de otros biomarcadores metabólicos. Por otro lado, el análisis posterior in vitro e in silico también demostró que Δ9-THCA-A puede actuar como un agonista ortostérico de CB1 y también como un modulador alostérico positivo en presencia de CP-55,940. Además, se muestra que Δ9-THCA-A es un agonista inverso de CB2. Experimentos in vivo en un modelo murino de artritis inducida por colágeno (CIA) demostraron que el tratamiento con Δ9-THCA-A previno la inflamación y el daño del cartílago en las articulaciones de la rodilla. El efecto antiartrítico de Δ9-THCA-A fue evitado en gran medida por SR141716 o T0070907. El análisis de biomarcadores plasmáticos, así como la determinación de citoquinas y anticuerpos anticolágeno confirmaron que Δ9-THCA-A actúa principalmente a través de las vías PPARγ y CB1. Nuestros hallazgos establecen que VCE-004.8 a través de PPARγ, CB2 y HIF, y Δ9- THCA-A a través de PPARγ, tienen un potencial terapéutico para el tratamiento de la obesidad y síndrome metabólico, sin presentar los efectos nocivos en adipogénesis y osteoblastogénesis asociados a los agonistas completos de PPARγ. Además, Δ9-THCA-A modula al receptor CB1 tanto a través del sitio de unión ortostérico como alostérico. Es más, Δ9-THCA-A también previene el desarrollo de artritis, principalmente a través de las vías CB1/PPARγ, destacando su potencial para el tratamiento de enfermedades inflamatorias crónicas como es la artritis reumatoide

Oral bioavailability of carbohydrate mimetics : an "in vitro" and "in vivo" evaluation

Carbohydrates play a crucial role in metabolism, cell recognition, cell differentiation, and adhesion processes. Therefore, these molecules represent a potent source for the development of new treatments against many different diseases with an unmet medical need. However, compounds of this class have major inherent drawbacks related to their chemical structure. Carbohydrates are complex and hydrophilic structures with a large polar surface area, which commonly results in poor pharmacokinetic (PK) properties including low oral bioavailability and short plasma half-life due to fast renal clearance. To overcome the poor PK properties of the natural ligands, specific structural modifications have to be implemented in the drug development process from the beginning. Improvements of newly synthesized compounds have to be constantly monitored with in vitro and in vivo PK measurements, allowing a direct feedback for further structural modifications. To this purpose, a PADMET platform (Physicochemical properties, Absorption, Distribution, Metabolism, Elimination, Toxicity) of in vitro assays, addressing different aspects that influence the PK properties of a molecule was developed and optimized. The intestinal absorption is a major hurdle to achieve sufficient oral bioavailability of carbohydrate mimetics and therefore,the focus of this work is set on the permeability of carbohydrate mimetic by passive permeation or active transport. In this thesis, three different targets for the development of potent lead structures starting from natural carbohydrates are discussed: • E-selectin is a lectin expressed on endothelial cells upon an inflammatory stimulus and is crucial for the recruitment of leukocytes to the side of inflammation. Therefore, E- selectin has been recognized as a potent target for the treatment of various diseases with an inflammatory component. The carbohydrate epitope recognized by E-selectin is the tetrasaccharide sialyl Lewisx (sLex). For the treatment of chronic inflammatory diseases, an oral administration is of interest. However, the development of an orally bioavailable E-selectin antagonist from sLex is challenging due to its chemical properties. To overcome the hurdle of insufficient intestinal absorption, an ester prodrug strategy and a bioisosteric replacement were followed and further evaluated by in vivo PK studies in mice. The ester prodrug approach resulted in insufficient oral bioavailability but improvement of the apparent plasma half-life, whereas the bioisosteric approach lead to the first orally bioavailable E-selectin antagonist. • Uropathogenic Eschericha coli (UPEC) are the main cause of urinary tract infections(UTI). UPEC are expressing the virulence factor FimH on the distal tip of type 1 fimbriae, which binds to mannosides on the luminal surface of the bladder to prevent bacteria from being washed out by urine flow. FimH is therefore a promising target for an antiadhesive treatment of UTIs in order to replace current antibiotic treatment strategies. In this work, known biphenyl-α-D-manno-pyranosides were further developed in terms of affinity and in vitro PK properties. • Sialic acids bound or in free circulation are regulated by sialyltransferases and neuraminidases (NEU). Selective inhibitors for the human neuraminidase NEU3 is of interest to study the physiological and pathophysiological role of neuraminidases and further evaluate the potential to develop therapeutics. Here, the development of specific NEU3 inhibitors, as well as the attempt to optimize their PK properties is reported

The development and applications of ceragenins and bone-binding antimicrobials to prevent osteomyelitis in orthopaedic patients

Bone infection remains a high-burden disease in orthopaedic and trauma patients with fractures and implantations. Osteomyelitis is difficult to cure in clinical settings, especially if antimicrobial resistance or biofilm is involved, which may prolong the treatments with antibiotics and require multiple surgeries, severely affecting the patients' quality of life and mobility. Osteomyelitis can lead to osteonecrosis, septicaemia, amputation, multi-organ dysfunction, and death in severe cases. Preclinical models are essential for efficacy testing to develop new prophylactic and therapeutic interventions. Previously bone infection models in rats involved fractures and implantations, making it complicated to perform. In this study, we have developed and optimised murine models with a tibial drilled hole (TDH) and needle insertion surgery (NIS) that are reliable, reproducible, and cost-effective for studying implant- related and biofilm bone infections and efficacy testing. Ceragenins (CSAs) are a novel class of broad-spectrum antimicrobials that mimic the activities of antimicrobial peptides. They are effective against bacterial, viral, fungal, and parasitic infections with low minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs). CSAs can also penetrate biofilm and kill antimicrobial-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus epidermidis (MRSE). In recent years, CSA-131 has been approved by the FDA for endotracheal tube coating to prevent infection in intubated and critical patients. In our study, we applied CSA-90 (which belongs to the same family as CSA-131) to implant coating and prevented osteomyelitis in a mouse model and demonstrated the osteogenic properties of CSA- 90, which promotes bone healing and reunion of the bone defects. CSA-90 has been classified as a potential drug to prevent and treat osteomyelitis. However, conventional methods of antibiotic delivery to the bone are inefficient. To increase the bone-binding property of CSA-90, we invented a new molecule by attaching alendronate (bisphosphonate) to CSA-90 and named it bone-binding antimicrobial-1 (BBA-1). In vitro, we determined the bone-binding properties of BBA-1 and confirmed its antimicrobial activities against S. aureus. Later, we conducted a preclinical trial to test the in vivo efficacy of BBA-1 and showed that BBA-1 could prevent osteomyelitis in mice and has low cytotoxicity. Multiple myeloma (MM) is an aggressive cancer of plasma cells. Although chemotherapy, corticosteroids, and radiation therapy manage multiple myeloma, MM has no cure. Most MM patients (>90%) suffer myeloma-skeletal disease, including local osteolytic lesions and osteomyelitis. Thus, we dedicate the clinical application of BBA-1 to MM patients. To pursue clinical trials, preclinical trials must be conducted. In our attempts, we proposed a feasible murine model that can induce bone infections in MM mice and elucidated how MM patients will benefit from BBA-1

Molecular insights and oligonucleotide based targeted gene therapy against cancer

The thesis helps to unravel the function of T lymphoma invasion and metastasis protein (TIAM1) and nucleolin, a nucleolar protein in retinoblastoma tumorigenesis. Aptamer based targeted imaging; drug and gene delivery to retinoblastoma and epithelial cancer cells was attained. The work work finally opened up avenues for cancer stem cell targeting using aptamers, imaging of cancer cells using novel bio-orthogonal agent and use of aptamer for blocking the miRNA-17-92 cluster maturation