Discovery of a PDZ Domain Inhibitor Targeting the Syndecan/Syntenin Protein-Protein Interaction: A Semi-Automated "Hit Identification-to-Optimization" Approach.

The rapid identification of early hits by fragment-based approaches and subsequent hit-to-lead optimization represents a challenge for drug discovery. To address this challenge, we created a strategy called "DOTS" that combines molecular dynamic simulations, computer-based library design (chemoDOTS) with encoded medicinal chemistry reactions, constrained docking, and automated compound evaluation. To validate its utility, we applied our DOTS strategy to the challenging target syntenin, a PDZ domain containing protein and oncology target. Herein, we describe the creation of a "best-in-class" sub-micromolar small molecule inhibitor for the second PDZ domain of syntenin validated in cancer cell assays. Key to the success of our DOTS approach was the integration of protein conformational sampling during hit identification stage and the synthetic feasibility ranking of the designed compounds throughout the optimization process. This approach can be broadly applied to other protein targets with known 3D structures to rapidly identify and optimize compounds as chemical probes and therapeutic candidates.

Nuclear ingression of cytoplasmic bodies accompanies a boost in autophagy.

Membrane contact sites are functional nodes at which organelles reorganize metabolic pathways and adapt to changing cues. In Saccharomyces cerevisiae, the nuclear envelope subdomain surrounding the nucleolus, very plastic and prone to expansion, can establish contacts with the vacuole and be remodeled in response to various metabolic stresses. While using genotoxins with unrelated purposes, we serendipitously discovered a fully new remodeling event at this nuclear subdomain: the nuclear envelope partitions into its regular contact with the vacuole and a dramatic internalization within the nucleus. This leads to the nuclear engulfment of a globular, cytoplasmic portion. In spite of how we discovered it, the phenomenon is likely DNA damage-independent. We define lipids supporting negative curvature, such as phosphatidic acid and sterols, as bona fide drivers of this event. Mechanistically, we suggest that the engulfment of the cytoplasm triggers a suction phenomenon that enhances the docking of proton pump-containing vesicles with the vacuolar membrane, which we show matches a boost in autophagy. Thus, our findings unveil an unprecedented remodeling of the nucleolus-surrounding membranes with impact on metabolic adaptation.

Identification of a Crosstalk among TGR5, GLIS2, and TP53 Signaling Pathways in the Control of Undifferentiated Germ Cell Homeostasis and Chemoresistance.

Spermatogonial stem cells regenerate and maintain spermatogenesis throughout life, making testis a good model for studying stem cell biology. The effects of chemotherapy on fertility have been well-documented previously. This study investigates how busulfan, an alkylating agent that is often used for chemotherapeutic purposes, affects male fertility. Specifically, the role of the TGR5 pathway is investigated on spermatogonia homeostasis using in vivo, in vitro, and pharmacological methods. In vivo studies are performed using wild-type and Tgr5-deficient mouse models. The results clearly show that Tgr5 deficiency can facilitate restoration of the spermatogonia homeostasis and allow faster resurgence of germ cell lineage after exposure to busulfan. TGR5 modulates the expression of key genes of undifferentiated spermatogonia such as Gfra1 and Fgfr2. At the molecular level, the present data highlight molecular mechanisms underlying the interactions among the TGR5, GLIS2, and TP53 pathways in spermatogonia associated with germ cell apoptosis following busulfan exposure. This study makes a significant contribution to the literature because it shows that TGR5 plays key role on undifferentiated germ cell homeostasis and that modulating the TGR5 signaling pathway could be used as a potential therapeutic tool for fertility disorders.

[Bispecific antibodies in onco-hematology: Applications and perspectives]. / Anticorps bispecifiques en oncohématologie : applications et perspectives.

Bispecific antibodies are novel approaches of immunotherapy engaging immune cells to destroy tumor cells. Their structure is variable and underlies their pharmacocinetic properties. These coumpounds are now being evaluated across multiple hematological malignancies. The anti-CD3/CD19 antibody blinatumomab is the first in class and have been approved for the treatment of patients with Ph-negative B-cell acute lymphoblastic leukemia. Other emerging applications are lymphoma, multiple myeloma and acute myeloid leukemia. The safety profile of bispecific antibodies is acceptable while limited by neurotoxicity and cytokine-release syndrome. The present review aims to depict the landscape of emerging bispecific antibodies currently in development for hematological malignancies.

Analysis of the Reversible Impact of the Chemodrug Busulfan on Mouse Testes.

Spermatogenesis is a process within the testis that leads to the production of spermatozoa. It is based on a population of spermatogonial stem cells, which have the capacity to self-renew and to differentiate throughout life to ensure the functions of reproduction are maintained. Male fertility disorders are responsible for half of the cases of infertility in couples worldwide. It is well known that cancer treatments are associated with reversible or irreversible fertility disorders. Busulfan (Bu) is an alkylating agent that significantly inhibits spermatogenesis. The present study relied on a combination of in vivo and in vitro approaches as well as RNAseq analysis to characterize the effects of Bu, in which mouse testes were used as a model. An in silico analysis revealed that many of the Bu-modulated genes are potentially regulated by the SIN3 Transcription Regulator Family Member A (SIN3A) and E2F Transcription Factor (E2F) families of transcription factors. The results demonstrate that the deregulated genes function in processes related to the cell cycle, DNA repair, and cell death mechanisms, including the Tumor Protein 53 (TP53) pathway. This reinforces the role of the TP53 signaling pathway as a major player in Bu effects. In addition, Bu altered the patterns of mRNA accumulation for various genes in undifferentiated spermatogonia. This work provides significant insight into the kinetics and impacts of busulfan, which could pave the way for developing strategies to minimize the impact of chemodrugs and, thus, could lead to germ cell lineage regeneration following anticancer treatments.

Fragment-based drug design targeting syntenin PDZ2 domain involved in exosomal release and tumour spread.

Syntenin stimulates exosome production and its expression is upregulated in many cancers and implicated in the spread of metastatic tumor. These effects are supported by syntenin PDZ domains interacting with syndecans. We therefore aimed to develop, through a fragment-based drug design approach, novel inhibitors targeting syntenin-syndecan interactions. We describe here the optimization of a fragment, 'hit' C58, identified by in vitro screening of a PDZ-focused fragment library, which binds specifically to the syntenin-PDZ2 domain at the same binding site as the syndecan-2 peptide. X-ray crystallographic structures and computational docking were used to guide our optimization process and lead to compounds 45 and 57 (IC50 = 33 µM and 47 µM; respectively), two representatives of syntenin-syndecan interactions inhibitors, that selectively affect the syntenin-exosome release. These findings demonstrate that it is possible to identify small molecules inhibiting syntenin-syndecan interaction and exosome release that may be useful for cancer therapy.

FXRα modulates leydig cell endocrine function in mouse.

The hypothalamic-pituitary axis exert a major control over endocrine and exocrine testicular functions. The hypothalamic-pituitary axis corresponds to a cascade with the Gonadotropin Releasing Hormone secreted by the hypothalamus, which stimulates the synthesis and the release of Luteinizing Hormone (LH) and Follicle Stimulating Hormone by the gonadotropic cells of the anterior pituitary. The LH signaling pathway controls the steroidogenic activity of the Leydig cells via the activation of the luteinizing hormone/choriogonadotropin receptor. In order to avoid a runaway system, sex steroids exert a negative feedback within hypothalamus and pituitary. Testicular steroidogenesis is locally controlled within Leydig cells. The present work reviews some local regulations of steroidogenesis within the Leydig cells focusing mainly on the roles of the Farnesoid-X-Receptor-alpha and its interactions with several orphan members of the nuclear receptor superfamily. Further studies are required to reinforce our knowledge of the regulation of testicular endocrine function, which is necessary to ensure a better understanding of fertility disorders and then proposed an adequate treatment of the diseases.

Fxralpha gene is a target gene of hCG signaling pathway and represses hCG induced steroidogenesis.

The bile acid receptor Farnesoid-X-Receptor alpha (FXRα), a member of the nuclear receptor superfamily, is well known for its roles in the enterohepatic tract. In addition, FXRα regulates testicular physiology through the control of both endocrine and exocrine functions. The endocrine function of the Leydig cells is mainly controlled by the hypothalamo-pituitary axis viaLH/chorionic gonadotropin (CG). If FXRα was demonstrated to control the expression of the Lhcgr gene, encoding the LH receptor; the impact of the LH/CG signaling on the Fxrα expression has not been defined so far. Here, we demonstrate that hCG increases the Fxrα gene expression through the protein kinase-A signaling pathway. Fxrα is then involved in a negative feedback of steroid synthesis. These data improve our knowledge of the local control of the testicular steroidogenesis with the identification of the link between the hypothalamo-pituitary axis and the FXRα signaling pathway.

Farnesoid X receptor alpha (FXRα) is a critical actor of the development and pathologies of the male reproductive system.

The farnesoid-X-receptorα (FXRα; NR1H4) is one of the main bile acid (BA) receptors. During the last decades, through the use of pharmalogical approaches and transgenic mouse models, it has been demonstrated that the nuclear receptor FXRα controls numerous physiological functions such as glucose or energy metabolisms. It is also involved in the etiology or the development of several pathologies. Here, we will review the unexpected roles of FXRα on the male reproductive tract. FXRα has been demonstrated to play functions in the regulation of testicular and prostate homeostasis. Even though additional studies are needed to confirm these findings in humans, the reviewed reports open new field of research to better define the effects of bile acid-FXRα signaling pathways on fertility disorders and cancers.

Encapsulation of ε-viniferin in onion-type multi-lamellar liposomes increases its solubility and its photo-stability and decreases its cytotoxicity on Caco-2 intestinal cells.

ε-Viniferin, a resveratrol dimer, is a naturally occurring stilbene that has been studied so far for its potential beneficial effects on human health. Its low water solubility, its photo-sensitivity and its low bioavailability make its applications in the food industry complicated. To overcome these limitations, ε-viniferin was encapsulated in phospholipid-based multi-lamellar liposomes (MLLs) called spherulites or onions. In the best case, an encapsulation efficiency of 58 ± 3% and a bioactive loading of 4.2 ± 0.5% were reached. Encapsulation of ε-viniferin drastically increased its water solubility by more than 5 orders to reach 17.4 g L-1 and provided protection against its UV-induced isomerization. While ε-viniferin was shown to be significantly toxic to Caco-2 intestinal-like cells for concentrations higher than 25 µM, once encapsulated in MLLs, those cells did not experience any mortality even for the highest tested stilbene concentration (100 µM) as revealed by red neutral assay.

Nuclear Receptor Metabolism of Bile Acids and Xenobiotics: A Coordinated Detoxification System with Impact on Health and Diseases.

Structural and functional studies have provided numerous insights over the past years on how members of the nuclear hormone receptor superfamily tightly regulate the expression of drug-metabolizing enzymes and transporters. Besides the role of the farnesoid X receptor (FXR) in the transcriptional control of bile acid transport and metabolism, this review provides an overview on how this metabolic sensor prevents the accumulation of toxic byproducts derived from endogenous metabolites, as well as of exogenous chemicals, in coordination with the pregnane X receptor (PXR) and the constitutive androstane receptor (CAR). Decrypting this network should provide cues to better understand how these metabolic nuclear receptors participate in physiologic and pathologic processes with potential validation as therapeutic targets in human disabilities and cancers.

Cholesterol: A Gatekeeper of Male Fertility?

Cholesterol is essential for mammalian cell functions and integrity. It is an important structural component maintaining the permeability and fluidity of the cell membrane. The balance between synthesis and catabolism of cholesterol should be tightly regulated to ensure normal cellular processes. Male reproductive function has been demonstrated to be dependent on cholesterol homeostasis. Here we review data highlighting the impacts of cholesterol homeostasis on male fertility and the molecular mechanisms implicated through the signaling pathways of some nuclear receptors.

Potential risk of medication discrepancies and reconciliation errors at admission and discharge from an inpatient medical service.

BACKGROUND: Medication discrepancies, defined as unexplained variations among drug regimens at care transitions, are common. Some are unintended and cause reconciliation errors that are potentially detrimental for patients. OBJECTIVE: To determine the prevalence of medication discrepancies and reconciliation errors at admission and discharge in hospitalized patients and explore risk factors for reconciliation errors and their potential clinical impact. METHODS: An observational prospective study was conducted at a general teaching hospital. Patients who were admitted to the internal medicine service and were receiving chronic preadmission treatment were included in the study. Preadmission treatment was compared with the treatment prescribed on admission (first 48 hours) and at hospital discharge, and discrepancies and reconciliation errors were identified. The primary endpoint was the presence of reconciliation errors at admission and/or discharge. Potential risk factors (patient-, medication-, and system-related) for reconciliation errors were analyzed using a multivariate logistic regression model. RESULTS: Of the 120 patients enrolled in the study between April and August 2009, 109 (90.8%) showed 513 discrepancies. The prevalence of patients with reconciliation errors was 20.8% (95% CI 13.6 to 28.1). Intended medication discrepancies were more frequent at admission (96.6%) than at discharge (75.5%), while reconciliation errors were more frequent at discharge (24.5%) than at admission (3.4%). The prevalence ratio (admission vs discharge) was 2.4 (95% CI 1.9 to 3.0) for discrepancies and 0.65 (95% CI 0.32 to 1.32) for reconciliation errors. The logistic regression analysis revealed an association between the number of discrepancies at admission (OR 1.21; 95% CI 1.01 to 1.44) and age (OR 1.05; 95% CI 0.99 to 1.10) and an increased risk of reconciliation errors. CONCLUSIONS: Medication reconciliation strategies should focus primarily on avoiding errors at discharge. Since medication discrepancies at admission may predispose patients to reconciliation errors, early detection of such discrepancies would logically reduce the risk of reconciliation errors. Medication reconciliation programs must implement a process for gathering accurate preadmission drug histories and must submit this information to a critical assessment of patients' needs.