Nancy Cheng

Human hepatic stem cells from fetal and postnatal donors

Human hepatic stem cells (hHpSCs), which are pluripotent precursors of hepatoblasts and thence of hepatocytic and biliary epithelia, are located in ductal plates in fetal livers and in Canals of Hering in adult livers. They can be isolated by immunoselection for epithelial cell adhesion molecule–positive (EpCAM+) cells, and they constitute ∼0.5–2.5% of liver parenchyma of all donor ages. The self-renewal capacity of hHpSCs is indicated by phenotypic stability after expansion for >150 population doublings in a serum-free, defined medium and with a doubling time of ∼36 h. Survival and proliferation of hHpSCs require paracrine signaling by hepatic stellate cells and/or angioblasts that coisolate with them. The hHpSCs are ∼9 μm in diameter, express cytokeratins 8, 18, and 19, CD133/1, telomerase, CD44H, claudin 3, and albumin (weakly). They are negative for α-fetoprotein (AFP), intercellular adhesion molecule (ICAM) 1, and for markers of adult liver cells (cytochrome P450s), hemopoietic cells (CD45), and mesenchymal cells (vascular endothelial growth factor receptor and desmin). If transferred to STO feeders, hHpSCs give rise to hepatoblasts, which are recognizable by cordlike colony morphology and up-regulation of AFP, P4503A7, and ICAM1. Transplantation of freshly isolated EpCAM+ cells or of hHpSCs expanded in culture into NOD/SCID mice results in mature liver tissue expressing human-specific proteins. The hHpSCs are candidates for liver cell therapies.

Mature human hepatocytes from ex vivo differentiation of alginate-encapsulated hepatoblasts.

Alginate gel was used to provide encapsulation to support the growth and eventually the differentiation of hepatic progenitors cells derived from human fetal livers. The encapsulated cells aggregated into spheroids within a few days in culture and continued to grow for at least 4 weeks in a serum-free medium. The hepatic progenitor cells in the spheroids undergo differentiation, as indicated by the appearance of functions of mature hepatocytes such as the detoxification of ammonia, albumin secretion, expression of the adult cytochrome P450 isozyme CYP3A4 and enzymatic activity typical of CYP2C9. Along with the expression of mature hepatic markers, these progenitor cells lost features typical of immature liver cells such as epithelial cell adhesion molecule. In addition to the acquisition of mature biochemical functions, the spheroids also developed a bile ducts, suggesting that they had differentiated into tissues resembling those in an intact liver.

A cellular chemical probe targeting the chromodomains of Polycomb repressive complex 1

We report the design and characterization of UNC3866, a potent antagonist of the methyl-lysine (Kme) reading function of the Polycomb CBX and CDY families of chromodomains. Polycomb CBX proteins regulate gene expression by targeting Polycomb Repressive Complex 1 to sites of H3K27me3 via their chromodomains. UNC3866 binds the chromodomains of CBX4 and CBX7 most potently with a Kd of ∼100 nM for each, and is 6- to 18-fold selective versus seven other CBX and CDY chromodomains while being highly selective versus >250 other protein targets. X-ray crystallography revealed that UNC3866 closely mimics the interactions of the methylated H3 tail with these chromodomains. UNC4195, a biotinylated derivative of UNC3866, was used to demonstrate that UNC3866 engages intact PRC1 and that EED incorporation into PRC1 is isoform-dependent in PC3 prostate cancer cells. Finally, UNC3866 inhibits PC3 cell proliferation, a known CBX7 phenotype, while UNC4219, a methylated negative control compound, has negligible effects.

UNC1062, a new and potent Mer inhibitor.

Abnormal activation of Mer kinase has been implicated in the oncogenesis of many human cancers including acute lymphoblastic and myeloid leukemia, non-small cell lung cancer, and glioblastoma. We have discovered a new family of small molecule Mer inhibitors, pyrazolopyrimidine sulfonamides, that potently inhibit the kinase activity of Mer. Importantly, these compounds do not demonstrate significant hERG activity in the PatchXpress assay. Through structure-activity relationship studies, 35 (UNC1062) was identified as a potent (IC50 = 1.1 nM) and selective Mer inhibitor. When applied to live tumor cells, UNC1062 inhibited Mer phosphorylation and colony formation in soft agar. Given the potential of Mer as a therapeutic target, UNC1062 is a promising candidate for further drug development.

Comparison of radioimmunoassay and ELISA methods for detection of antibodies to chromatin components.

A solid phase radioimmunoassay has been compared with an enzyme-linked immunosorbent assay (ELISA) for efficacy in measuring anti-chromatin antibodies. The low backgrounds achieved with the radioimmunoassay method produced a high signal-to-noise ratio and enabled detection of the human test antiserum at a dilution of 1:102,400. By contrast, the ELISA could detect the same antiserum only at a dilution of 1:3200 and above. The radioimmunoassay was consistently more sensitive than the ELISA for detection of anti-chromatin antibodies in a number of human and mouse sera and ascites fluid containing a monoclonal antibody. Factors affecting sensitivity in both assays are discussed.

Development of a Novel Screening Strategy Designed to Discover a New Class of HIV Drugs

Current antiretroviral treatments target multiple pathways important for human immunodeficiency virus (HIV) multiplication, including viral entry, synthesis and integration of the DNA provirus, and the processing of viral polyprotein precursors. However, HIV is becoming increasingly resistant to these “combination therapies.” Recent findings show that inhibition of HIV Gag protein cleavage into its two structural proteins, matrix (MA) and capsid (CA), has a devastating effect on viral production, revealing a potential new target class for HIV treatment. Unlike the widely used HIV protease inhibitors, this new class of inhibitor would target the substrate, not the protease enzyme itself. This approach offers a distinct advantage in that inhibitors of MA/CA would only need to affect a subset of the Gag molecules to disable viral replication. To discover MA/CA-specific inhibitors, we constructed a modified MA/CA fusion peptide (MA/CAΔ) that contains the HIV protease (PR) cleavage site as well as a tetracysteine motif for fluorescent labeling. The HIV PR cleavage of MA/CAΔ can then be monitored via fluorescence polarization (FP). We have adapted this FP assay for high-throughput screening and validated it according to industry standards using a 384-well plate format. We have currently tested 24,000 compounds in this assay and here detail the screening methodology and the results of this screening campaign.

Filtration improves the performance of a high-throughput screen for anti-mycobacterial compounds.

The tendency for mycobacteria to aggregate poses a challenge for their use in microplate based assays. Good dispersions have been difficult to achieve in high-throughput screening (HTS) assays used in the search for novel antibacterial drugs to treat tuberculosis and other related diseases. Here we describe a method using filtration to overcome the problem of variability resulting from aggregation of mycobacteria. This method consistently yielded higher reproducibility and lower variability than conventional methods, such as settling under gravity and vortexing.

A Thermodynamic Analysis of the Binding of Nucleic Acid to HIV-1 Reverse Transcriptase

Human immunodeficiency virus type 1 (HIV-1) encodes a Mg+2-dependent reverse transcriptase (E.C.2.7.7.49) that synthesizes a double-stranded DNA copy of genomic RNA. The enzyme purified from virions has been shown to consist of two polypeptides of molecular weights 66,000 and 51,000 (Hoffman et al., 1985; Di Marzo Veronese et al.,1986). The 66 kD subunit has both polymerase and RNase H activities. Like other retroviral reverse transcriptases, the associated RNase H activity is located on the carboxy terminal portion of the polypeptide (Johnson et al., 1986; Hansen et al., 1987; Tisdale et al., 1988). The 51 kD subunit is derived from the 66 kD polypeptide by cleavage at a protease sensitive site on the linker between the polymerase and RNase H domains (Lowe et al.,1988). Catalytically active HIV-1 reverse transcriptase has been cloned and expressed in E. coli (Larder et al., 1987). The recombinant, heterodimeric enzyme is kinetically indistinguishable from the native enzyme purified from virus.

Regulation of Enzymes by Ligand Induced Change in Polymerization

Most enzymes form homopolymers, some of which are stable while others readily change their aggregation state by dissociation or association of subunits. This constitutes a mechanism for regulation if the different molecular weight species vary in their specific activity, and if the equilibrium between these species is readily perturbed by physiological effectors. Several enzymes in pyrimidine metabolism appear to be regulated by this mechanism: uridine kinase (EC 2.7.1.48); β-alanine synthase (EC 3.5.1.6), the last enzyme in the catabolism of uracil; and the multifunctional protein UMP synthase which has 2 enzyme activities, orotate phosphoribosyltransferase (EC 2.4.2.10) and OMP decarboxylase (EC 4.1.1.23).

REGULATION OF ENZYMES BY LIGAND INDUCED CHANGE IN POLYMERIZATION: 211

Many enzymes readily change their aggregation state, by dissociation or association of subunits. This constitutes a mechanism for regulation, if the different molecular weight species vary in their specific activity, and if the equilibrium between these species is readily perturbed by physiological effectors. Our studies show that several enzymes involved in the synthesis or degradation of pyrimidines exhibit such regulation: The multifunctional protein UMP synthase (EC 2.4.2.10 + EC 4.1.1.23), and uridine kinase (EC 2.7.1.48) from Ehrlich ascites cells, and N-carbamoyl-β-alanine amidohydrolase (EC 3.5.1.6) from rat liver. Generally, substrates are positive effectors, while immediate or more distant end products are negative effectors.UMP synthase shows only positive regulation: The 50,000 dalton monomer is inactive; effectors result in association to the active dimer. Uridine kinase shows negative regulation: the native enzyme exists as polymers of 4 or more subunits, and CTP or UTP produce dissociation to the 31,000 dalton subunit and loss of activity. NCβA amidohydrolase shows bimodal regulation: the native enzyme has an Mr of 235,000; the substrate produces association to 470,000 daltons; the product, β-alanine, causes dissociation to 120,000 daltons.Ligand induced changes in polymerization may be a general mechanism for regulating enzyme activity.