Methyltransferases prefer monomer over core-trimmed nucleosomes as in vitro substrates.

Epigenetics is an area of increasing interest for drug discovery, driving the need for assays that use nucleosome substrates. Our studies showed that SUV39H1, a histone lysine methyltransferase, and Dnmt3b/Dnmt3L, a DNA methyltransferase, both exhibited approximately five times more activity on monomer nucleosomes than on DNA-core-trimmed nucleosomes in a scintillation proximity assay (SPA). The methyltransferases recognize and have a preference for nucleosomes with longer DNA strands. Our findings suggest that the use of monomer nucleosomes as substrates using SPA technology could lead to more robust screening assays and potentially more specific small molecule inhibitors of epigenetic enzymes.

Baculovirus production of fully-active phosphoinositide 3-kinase alpha as a p85alpha-p110alpha fusion for X-ray crystallographic analysis with ATP competitive enzyme inhibitors.

Phosphoinositide 3-kinases have been targeted for therapeutic research because they are key components of a cell signaling cascade controlling proliferation, growth, and survival. Direct activation of the PI3Kalpha pathway contributes to the development and progression of solid tumors in breast, endometrial, colon, ovarian, and gastric cancers. In the context of a drug discovery effort, the availability of a robust crystallographic system is a means to understand the subtle differences between ATP competitive inhibitor interactions with the active site and their selectivity against other PI3Kinase enzymes. To generate a suitable recombinant design for this purpose, a p85alpha-p110alpha fusion system was developed which enabled the expression and purification of a stoichiometrically homogeneous, constitutively active enzyme for structure determination with potent ATP competitive inhibitors (Raha et al., in preparation) [56]. This approach has yielded preparations with activity and inhibition characteristics comparable to those of the full-length PI3Kalpha from which X-ray diffracting crystals were grown with inhibitors bound in the active site.

Modular Protein Ligation: A New Paradigm as a Reagent Platform for Pre-Clinical Drug Discovery.

Significant resource is spent by drug discovery project teams to generate numerous, yet unique target constructs for the multiple platforms used to drive drug discovery programs including: functional assays, biophysical studies, structural biology, and biochemical high throughput screening campaigns. To improve this process, we developed Modular Protein Ligation (MPL), a combinatorial reagent platform utilizing Expressed Protein Ligation to site-specifically label proteins at the C-terminus with a variety of cysteine-lysine dipeptide conjugates. Historically, such proteins have been chemically labeled non-specifically through surface amino acids. To demonstrate the feasibility of this approach, we first applied MPL to proteins of varying size in different target classes using different recombinant protein expression systems, which were then evaluated in several different downstream assays. A key advantage to the implementation of this paradigm is that one construct can generate multiple final products, significantly streamlining the reagent generation for multiple early drug discovery project teams.

Intravascular location of breast cancer cells after spontaneous metastasis to the lung.

In this study, we examined the hypothesis that early pulmonary metastases form within the vasculature. We introduced primary tumors in immunocompromised mice by subcutaneous injection of murine breast carcinoma cells (4T1) expressing green fluorescent protein. Isolated ventilated and perfused lungs from these mice were examined at various times after tumor formation by fluorescent microscopy. The vasculature was visualized by counterstaining with 1,1-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (DiI)-acetylated low-density lipoprotein. These experiments showed that metastatic cells derived by spontaneous metastases were intravascular, and that early colony formation was intravascular. The location of the tumor cells was confirmed by deconvolution analysis. This work extends our previous study(1) that sarcoma cells injected intravenously form intravascular colonies to spontaneous metastasis and to a carcinoma model system. Many of the tumor cells seen were single implying that tumor cells may travel as single cells. These results support a model for pulmonary metastasis in mice in which 1) tumor cells can attach to lung endothelium soon after arrival; 2) surviving tumor cells proliferate intravascularly in this model; and 3) extravasation of the tumor occurs when intravascular micrometastatic foci outgrow the vessels they are in.