Binh Thanh Vu

Discovery of RG7112: A Small-Molecule MDM2 Inhibitor in Clinical Development.

The p53 tumor suppressor is a potent transcription factor that plays a key role in the regulation of cellular responses to stress. It is controlled by its negative regulator MDM2, which binds directly to p53 and inhibits its transcriptional activity. MDM2 also targets p53 for degradation by the proteasome. Many tumors produce high levels of MDM2, thereby impairing p53 function. Restoration of p53 activity by inhibiting the p53-MDM2 interaction may represent a novel approach to cancer treatment. RG7112 (2g) is the first clinical small-molecule MDM2 inhibitor designed to occupy the p53-binding pocket of MDM2. In cancer cells expressing wild-type p53, RG7112 stabilizes p53 and activates the p53 pathway, leading to cell cycle arrest, apoptosis, and inhibition or regression of human tumor xenografts.

Metal impurities cause false positives in high-throughput screening campaigns.

Organic impurities in compound libraries are known to often cause false-positive signals in screening campaigns for new leads, but organic impurities do not fully account for all false-positive results. We discovered inorganic impurities in our screening library that can also cause positive signals for a variety of targets and/or readout systems, including biochemical and biosensor assays. We investigated in depth the example of zinc for a specific project and in retrospect in various HTS screens at Roche and propose a straightforward counter screen using the chelator TPEN to rule out inhibition caused by zinc.

Deconstruction of a Nutlin: Dissecting the Binding Determinants of a Potent Protein–Protein Interaction Inhibitor

Protein-protein interaction (PPI) systems represent a rich potential source of targets for drug discovery, but historically have proven to be difficult, particularly in the lead identification stage. Application of the fragment-based approach may help toward success with this target class. To provide an example toward understanding the potential issues associated with such an application, we have deconstructed one of the best established protein-protein inhibitors, the Nutlin series that inhibits the interaction between MDM2 and p53, into fragments, and surveyed the resulting binding properties using heteronuclear single quantum coherence nuclear magnetic resonance (HSQC NMR), surface plasmon resonance (SPR), and X-ray crystallography. We report the relative contributions toward binding affinity for each of the key substituents of the Nutlin molecule and show that this series could hypothetically have been discovered via a fragment approach. We find that the smallest fragment of Nutlin that retains binding accesses two subpockets of MDM2 and has a molecular weight at the high end of the range that normally defines fragments.

Substituent effects on intramolecular dipolar cycloadditions: The gem-dicarboalkoxy effect

Abstract A gem -dicarboalkoxy effect (rel. rate >20) has been measured and good diastereoselectivity (∼9:1) has been seen for the intramolecular dipolar cycloaddition of 3-substituted 5-hexenyl nitrile oxides.

Targeting breast cancer stem cells by dendritic cell vaccination in humanized mice with breast tumor: preliminary results

Background Breast cancer (BC) is one of the leading cancers in women. Recent progress has enabled BC to be cured with high efficiency. However, late detection or metastatic disease often renders the disease untreatable. Additionally, relapse is the main cause of death in BC patients. Breast cancer stem cells (BCSCs) are considered to cause the development of BC and are thought to be responsible for metastasis and relapse. This study aimed to target BCSCs using dendritic cells (DCs) to treat tumor-bearing humanized mice models. Materials and methods NOD/SCID mice were used to produce the humanized mice by transplantation of human hematopoietic stem cells. Human BCSCs were injected into the mammary fat pad to produce BC humanized mice. Both hematopoietic stem cells and DCs were isolated from the human umbilical cord blood, and immature DCs were produced from cultured mononuclear cells. DCs were matured by BCSC-derived antigen incubation for 48 hours. Mature DCs were vaccinated to BC humanized mice with a dose of 106 cells/mice, and the survival percentage was monitored in both treated and untreated groups. Results The results showed that DC vaccination could target BCSCs and reduce the tumor size and prolong survival. Conclusion These results suggested that targeting BCSCs with DCs is a promising therapy for BC.

Isolation of Breast Cancer Stem Cells by Single-Cell Sorting

Breast cancer is the most common cancer in women, with more than 1,000,000 new cases and more than 410,000 deaths each year [38]; [39]. At present, breast cancer is mainly treated by surgical therapy as well as cytotoxic, hormonal and immunotherapeutic agents. These methods achieve response rates ranging from 60 to 80% for primary breast cancers and about 50% of metastases [22]; [24]. However, up to 20 to 70% of patients relapse within 5 years [10].

Chick chorioallantoic membrane assay as an in vivo model to study the effect of nanoparticle-based anticancer drugs in ovarian cancer

New therapy development is critically needed for ovarian cancer. We used the chicken egg CAM assay to evaluate efficacy of anticancer drug delivery using recently developed biodegradable PMO (periodic mesoporous organosilica) nanoparticles. Human ovarian cancer cells were transplanted onto the CAM membrane of fertilized eggs, resulting in rapid tumor formation. The tumor closely resembles cancer patient tumor and contains extracellular matrix as well as stromal cells and extensive vasculature. PMO nanoparticles loaded with doxorubicin were injected intravenously into the chicken egg resulting in elimination of the tumor. No significant damage to various organs in the chicken embryo occurred. In contrast, injection of free doxorubicin caused widespread organ damage, even when less amount was administered. The lack of toxic effect of nanoparticle loaded doxorubicin was associated with specific delivery of doxorubicin to the tumor. Furthermore, we observed excellent tumor accumulation of the nanoparticles. Lastly, a tumor could be established in the egg using tumor samples from ovarian cancer patients and that our nanoparticles were effective in eliminating the tumor. These results point to the remarkable efficacy of our nanoparticle based drug delivery system and suggests the value of the chicken egg tumor model for testing novel therapies for ovarian cancer.

Impact of Pore–Walls Ligand Assembly on the Biodegradation of Mesoporous Organosilica Nanoparticles for Controlled Drug Delivery

Porous materials with molecular-scale ordering have attracted major attention mainly because of the possibility to engineer their pores for selective applications. Periodic mesoporous organosilica is a class of hybrid materials where self-assembly of the organic linkers provides a crystal-like pore wall. However, unlike metal coordination, specific geometries cannot be predicted because of the competitive and dynamic nature of noncovalent interactions. Herein, we study the influence of competing noncovalent interactions in the pore walls on the biodegradation of organosilica frameworks for drug delivery application. These results support the importance of studying self-assembly patterns in hybrid frameworks to better engineer the next generation of dynamic or “soft” porous materials.

Culture and Differentiation of Cytokine-Induced Killer Cells from Umbilical Cord Blood-Derived Mononuclear Cells

Cytokine-induced killer cells (CIK) are cytotoxic T cells, which have both NK and T cell properties. These cells are characterized by potent, non-MHC-restricted cytotoxicity and reduced alloreactivity, which make them appealing for use in adoptive immunotherapy of cancer and virus infections. In this study, CIK cells were generated by stimulating umbilical cord blood-derived mononuclear cells (UCB-MNCs) with interferon-gamma (IFN-γ) on day 0. Anti-CD3 antibody and interleukin-2 (IL-2) were added after 24 h at four different experimental concentration combinations in order to identify the optimal cytokine amounts for CIK cell proliferation. Cells were collected at four time points over a 21-day period (day 0, 7, 14, 21) for analysis of cell marker presentation using flow cytometry, as well as transcription-level cytokine production using RT-PCR. The results showed that in the 21-day culture, killer (LAK) cells, and cytokine-induced killer (CIK) cells. Among them, CIK cells appear to be the most promising cytotoxic effector cell type. CIK cells are a heterogeneous subset of T lymphocytes with NK functional properties. The qualifier “cytokine- induced killer” indicates that they are generated via administration of cytokines during in vitro culture [1, 2]. Cells which have the most potential effector function in CIK culture co-express CD3 and CD56 surface molecules; possess a potent, MHC-unrestricted tumor-killing ability; and significantly reduced alloreactivity [3]. CIK cells recognize tumor cells through the binding of CIK NKG2D receptors with tumor cell ligands such as MHC class I polypeptide- related sequences A and B (MICA and MICB); they then the average final expansion levels of 56 + CIK cell CD were in the range of hundredfold, accounted for 26% in the bulk culture. Most important, these cells strongly expressed granzyme B (80.87%), a potent factor involved in cell- mediated cytotoxicity. These CIK cells also transcriptionally overexpressed the three cytokine genes that produce IFN-γ, tumor necrosis factor-alpha (TNF-α), and IL-2; these are key for immune cell mobilization against tumors as well as foreign pathogens. Our research establishes an effective cytokine con- centration and time protocol for use in generation of CIK cells from UCB-MNCs, potentiating greater applications of CIK cell-adoptive immunotherapy in both research and clinical settings. Thus, the 3rd and 4th experimental conditions both stimulated CIK cell differentiation with 50 ng/ml of anti-CD3 antibody, but with IL-2 concentrations of 500 and 1000 U/ml, respectively.

Optimization of Culture Medium for the Isolation and Propagation of Human Breast Cancer Cells from Primary Tumor Biopsies

Breast cancer cells from patients hold an important role in antigen production for immunotherapy, drug testing, and cancer stem cell studies. To date, although many studies have been conducted to develop protocols for the isolation and culture of breast cancer cells from tumor biopsies, the efficiencies of these protocols remain low. This study aimed to identify a suitable medium for the isolation and propagation of primary breast cancer cells from breast tumor biopsies. Breast tumor biopsies were obtained from hospitals after all patients had given their written in-formed consent and were cultured according to the expanding tumor method in 3 different media: DMEM/F12 (Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12) supplemented with 10% FBS (Fetal bovine serum) and 1% antibiotic-antimycotic (Medium D); Medium 171 supplemented with 1X MEGS (Mammary Epithelial Growth Supplement) and 1% antibiotic-antimycotic (Medium M); or a 1:1 mixture of Medium D and Medium M (Medium DB). The cell culture efficiency was evaluated by several criteria, including the time of cell appearance, cell morphology, capability of proliferation, cell surface marker expression, ALDH (Aldehyde dehydrogenases) activity, karyotype, and tumor formation capacity in immune-deficient mice. Notably, primary cancer cells cultured in Medium DB showed a high expression of breast cancer stem cell surface markers (including CD44+CD24− and CD49f+), low expression of stromal cell surface markers (CD90), high ALDH activity, an abnormal karyotype, and high tumor formation capacity in immune- deficient mice. These findings suggested that Medium DB was suitable to support the survival and proliferation of primary breast cancer cells as well as to enrich breast cancer stem cells.