Ruben Papoian

Drug-based modulation of endogenous stem cells promotes functional remyelination in vivo

Multiple sclerosis involves an aberrant autoimmune response and progressive failure of remyelination in the central nervous system. Prevention of neural degeneration and subsequent disability requires remyelination through the generation of new oligodendrocytes, but current treatments exclusively target the immune system. Oligodendrocyte progenitor cells are stem cells in the central nervous system and the principal source of myelinating oligodendrocytes. These cells are abundant in demyelinated regions of patients with multiple sclerosis, yet fail to differentiate, thereby representing a cellular target for pharmacological intervention. To discover therapeutic compounds for enhancing myelination from endogenous oligodendrocyte progenitor cells, we screened a library of bioactive small molecules on mouse pluripotent epiblast stem-cell-derived oligodendrocyte progenitor cells. Here we show seven drugs function at nanomolar doses selectively to enhance the generation of mature oligodendrocytes from progenitor cells in vitro. Two drugs, miconazole and clobetasol, are effective in promoting precocious myelination in organotypic cerebellar slice cultures, and in vivo in early postnatal mouse pups. Systemic delivery of each of the two drugs significantly increases the number of new oligodendrocytes and enhances remyelination in a lysolecithin-induced mouse model of focal demyelination. Administering each of the two drugs at the peak of disease in an experimental autoimmune encephalomyelitis mouse model of chronic progressive multiple sclerosis results in striking reversal of disease severity. Immune response assays show that miconazole functions directly as a remyelinating drug with no effect on the immune system, whereas clobetasol is a potent immunosuppressant as well as a remyelinating agent. Mechanistic studies show that miconazole and clobetasol function in oligodendrocyte progenitor cells through mitogen-activated protein kinase and glucocorticoid receptor signalling, respectively. Furthermore, both drugs enhance the generation of human oligodendrocytes from human oligodendrocyte progenitor cells in vitro. Collectively, our results provide a rationale for testing miconazole and clobetasol, or structurally modified derivatives, to enhance remyelination in patients.

Osteopontin is upregulated during in vivo demyelination and remyelination and enhances myelin formation in vitro.

We have used in vitro oligodendrocyte differentiation and the in vivo remyelination model, the cuprizone model, to identify genes regulating oligodendrocyte function and remyelination. One of the genes we identified, osteopontin (opn), is a secreted glycoprotein with cytokine-like, chemotactic, and anti-apoptotic properties that contains an Arg-Gly-Asp (RGD) cell adhesion motif-mediating interactions with several integrins. Both microglia and astrocytes in demyelinating brain regions of cuprizone-fed mice expressed OPN protein. Recombinant OPN protein produced in a baculovirus expression system induced proliferation of both the rat CG-4 and the mouse Oli-neu oligodendrocyte precursor (OLP)-like cell lines in a dose-dependent manner. In addition, recombinant OPN treatment stimulated both myelin basic protein (MBP) synthesis and myelin sheath formation in mixed cortical cultures from embryonic mouse brain, an in vitro primary culture model of myelination. Interestingly, myelinating mixed cultures prepared from OPN(-/-) mice contained significantly less MBP compared to wild-type cultures after 17 days in culture. We propose that in the central nervous system, OPN may act as a novel regulator of myelination and remyelination.

Microarray gene expression profiling of chronic active and inactive lesions in multiple sclerosis

Multiple sclerosis, a primary autoimmune disease of the central nervous system has been characterized by the presence of the demyelinating lesions (plaques) in the CNS. To further understand the gene transcription status of the two most common lesions, chronic active and chronic inactive, we have performed a cDNA microarray analysis of these two lesion type. Comparative analysis of differential gene expression of chronic active and inactive lesions have confirmed the existence of a significant difference in the transcriptional profiles of these two lesion types in both marginal and central areas. Different sets of genes were highlighted, including genes of inflammatory characteristics, apoptosis related and stress-induced, indicating their potential role in MS pathogenesis.

Unexploited therapies in breast and prostate cancer: blockade of the prolactin receptor.

Breast and prostate cancers are hormone-sensitive malignancies that afflict millions of women and men. Although prolactin (PRL) is known as a survival factor that supports tumor growth and confers chemoresistance in both cancers, its precise role in these tumors has not been studied extensively. Growth hormone and placental lactogen also bind PRL receptor (PRLR) and mimic some of the actions of PRL. Blockade of the PRLR represents a novel treatment for patients with advanced breast or prostate cancer with limited therapeutic options. This review discusses different approaches for generating PRLR antagonists. Emphasis is placed on technological advances which enable high-throughput screening for small molecule inhibitors of PRLR signaling that could serve as oral medications.

MIG--differential gene expression in mouse brain endothelial cells.

Different diseases of the CNS are associated with blood–brain barrier (BBB) damage and mononuclear cell infiltration. In order to study genes that may play a role in endothelial cell regulation in inflammatory CNS diseases, we performed differential gene expression (DGE) analysis using a mouse brain endothelial cell line. We found that interferon-γ (IFNγ)-induced monokine (MIG), a chemokine that plays a role in T lymphocyte and monocyte chemoattraction, is highly expressed in the presence of inflammatory cytokines.We show that MIG, produced by brain endothelial cells in vitro, is biologically active in attracting T lymphocytes and that it is possible to interfere with this mechanism of action using anti-MIG antibodies. We suggest that blocking MIG may be beneficial in CNS inflammation. We detected constitutive expression of the MIG receptor, CXCR3, on the surface of the endothelial cells and therefore hypothesize that it plays a role in maintaining the cytokine gradient at the region of CNS inflammation.

Hits of a High-Throughput Screen Identify the Hydrophobic Pocket of Autotaxin/Lysophospholipase D As an Inhibitory Surface

Autotaxin (ATX), a lysophospholipase D, plays an important role in cancer invasion, metastasis, tumor progression, tumorigenesis, neuropathic pain, fibrotic diseases, cholestatic pruritus, lymphocyte homing, and thrombotic diseases by producing the lipid mediator lysophosphatidic acid (LPA). A high-throughput screen of ATX inhibition using the lysophosphatidylcholine-like substrate fluorogenic substrate 3 (FS-3) and ∼10,000 compounds from the University of Cincinnati Drug Discovery Center identified several small-molecule inhibitors with IC50 vales ranging from nanomolar to low micromolar. The pharmacology of the three most potent compounds: 918013 (1; 2,4-dichloro-N-(3-fluorophenyl)-5-(4-morpholinylsulfonyl) benzamide), 931126 (2; 4-oxo-4-{2-[(5-phenoxy-1H-indol-2-yl)carbonyl]hydrazino}-N-(4-phenylbutan-2-yl)butanamide), and 966791 (3; N-(2,6-dimethylphenyl)-2-[N-(2-furylmethyl)(4-(1,2,3,4-tetraazolyl)phenyl)carbonylamino]-2-(4-hydroxy-3-methoxyphenyl) acetamide), were further characterized in enzyme, cellular, and whole animal models. Compounds 1 and 2 were competitive inhibitors of ATX-mediated hydrolysis of the lysophospholipase substrate FS-3. In contrast, compound 3 was a competitive inhibitor of both FS-3 and the phosphodiesterase substrate p-nitrophenyl thymidine 5′-monophosphate. Computational docking and mutagenesis suggested that compounds 1 and 2 target the hydrophobic pocket, thereby blocking access to the active site of ATX. The potencies of compounds 1–3 were comparable to each other in each of the assays. All of these compounds significantly reduced invasion of A2058 human melanoma cells in vitro and the colonization of lung metastases by B16-F10 murine melanoma cells in C57BL/6 mice. The compounds had no agonist or antagonist effects on select LPA or sphingosine 1-phosphate receptors, nor did they inhibit nucleotide pyrophosphatase/phosphodiesterase (NPP) enzymes NPP6 and NPP7. These results identify the molecular surface of the hydrophobic pocket of ATX as a target-binding site for inhibitors of enzymatic activity.

Inhibition of the Growth Factor MDK/Midkine by a Novel Small Molecule Compound to Treat Non-Small Cell Lung Cancer

Midkine (MDK) is a heparin-binding growth factor that is highly expressed in many malignant tumors, including lung cancers. MDK activates the PI3K pathway and induces anti-apoptotic activity, in turn enhancing the survival of tumors. Therefore, the inhibition of MDK is considered a potential strategy for cancer therapy. In the present study, we demonstrate a novel small molecule compound (iMDK) that targets MDK. iMDK inhibited the cell growth of MDK-positive H441 lung adenocarcinoma cells that harbor an oncogenic KRAS mutation and H520 squamous cell lung cancer cells, both of which are types of untreatable lung cancer. However, iMDK did not reduce the cell viability of MDK-negative A549 lung adenocarcinoma cells or normal human lung fibroblast (NHLF) cells indicating its specificity. iMDK suppressed the endogenous expression of MDK but not that of other growth factors such as PTN or VEGF. iMDK suppressed the growth of H441 cells by inhibiting the PI3K pathway and inducing apoptosis. Systemic administration of iMDK significantly inhibited tumor growth in a xenograft mouse model in vivo. Inhibition of MDK with iMDK provides a potential therapeutic approach for the treatment of lung cancers that are driven by MDK.

Targeting the hydrophobic pocket of autotaxin with virtual screening of inhibitors identifies a common aromatic sulfonamide structural motif.

Modulation of autotaxin (ATX), the lysophospholipase D enzyme that produces lysophosphatidic acid, with small‐molecule inhibitors is a promising strategy for blocking the ATX–lysophosphatidic acid signaling axis. Although discovery campaigns have been successful in identifying ATX inhibitors, many of the reported inhibitors target the catalytic cleft of ATX. A recent study provided evidence for an additional inhibitory surface in the hydrophobic binding pocket of ATX, confirming prior studies that relied on enzyme kinetics and differential inhibition of substrates varying in size. Multiple hits from previous high‐throughput screening for ATX inhibitors were obtained with aromatic sulfonamide derivatives interacting with the hydrophobic pocket. Here, we describe the development of a ligand‐based strategy and its application in virtual screening, which yielded novel high‐potency inhibitors that target the hydrophobic pocket of ATX. Characterization of the structure–activity relationship of these new inhibitors forms the foundation of a new pharmacophore model of the hydrophobic pocket of ATX.

Expansion of First-in-Class Drug Candidates That Sequester Toxic All-Trans-Retinal and Prevent Light-Induced Retinal Degeneration

All-trans-retinal, a retinoid metabolite naturally produced upon photoreceptor light activation, is cytotoxic when present at elevated levels in the retina. To lower its toxicity, two experimentally validated methods have been developed involving inhibition of the retinoid cycle and sequestration of excess of all-trans-retinal by drugs containing a primary amine group. We identified the first-in-class drug candidates that transiently sequester this metabolite or slow down its production by inhibiting regeneration of the visual chromophore, 11-cis-retinal. Two enzymes are critical for retinoid recycling in the eye. Lecithin:retinol acyltransferase (LRAT) is the enzyme that traps vitamin A (all-trans-retinol) from the circulation and photoreceptor cells to produce the esterified substrate for retinoid isomerase (RPE65), which converts all-trans-retinyl ester into 11-cis-retinol. Here we investigated retinylamine and its derivatives to assess their inhibitor/substrate specificities for RPE65 and LRAT, mechanisms of action, potency, retention in the eye, and protection against acute light-induced retinal degeneration in mice. We correlated levels of visual cycle inhibition with retinal protective effects and outlined chemical boundaries for LRAT substrates and RPE65 inhibitors to obtain critical insights into therapeutic properties needed for retinal preservation.

Combined Rational Design and a High-Throughput Screening Platform for Identifying Chemical Inhibitors of a Ras Activating Enzyme

Background: GEFs can serve as targets in Ras family GTPase activity signaling. Results: A virtual screening coupled with the experimental screening platform is established targeting a Ras GEF enzyme. Conclusion: The multiple tier screening platform is useful for lead discovery targeting GEFs of Ras-like small GTPases. Significance: This study provides a unique screening approach for inhibitors of Ras and Ras-like GTPase activities. The Ras family small GTPases regulate multiple cellular processes, including cell growth, survival, movement, and gene expression, and are intimately involved in cancer pathogenesis. Activation of these small GTPases is catalyzed by a special class of enzymes, termed guanine nucleotide exchange factors (GEFs). Herein, we developed a small molecule screening platform for identifying lead hits targeting a Ras GEF enzyme, SOS1. We employed an ensemble structure-based virtual screening approach in combination with a multiple tier high throughput experimental screen utilizing two complementary fluorescent guanine nucleotide exchange assays to identify small molecule inhibitors of GEF catalytic activity toward Ras. From a library of 350,000 compounds, we selected a set of 418 candidate compounds predicted to disrupt the GEF-Ras interaction, of which dual wavelength GDP dissociation and GTP-loading experimental screening identified two chemically distinct small molecule inhibitors. Subsequent biochemical validations indicate that they are capable of dose-dependently inhibiting GEF catalytic activity, binding to SOS1 with micromolar affinity, and disrupting GEF-Ras interaction. Mutagenesis studies in conjunction with structure-activity relationship studies mapped both compounds to different sites in the catalytic pocket, and both inhibited Ras signaling in cells. The unique screening platform established here for targeting Ras GEF enzymes could be broadly useful for identifying lead inhibitors for a variety of small GTPase-activating GEF reactions.