The Novel Bromodomain and Extraterminal Domain Inhibitor INCB054329 Induces Vulnerabilities in Myeloma Cells That Inform Rational Combination Strategies.

Purpose: Bromodomain and extraterminal domain (BET) proteins regulate the expression of many cancer-associated genes and pathways; BET inhibitors have demonstrated activity in diverse models of hematologic and solid tumors. We report the preclinical characterization of INCB054329, a structurally distinct BET inhibitor that has been investigated in phase I clinical trials.

Experimental Design: We used multiple myeloma models to investigate vulnerabilities created by INCB054329 treatment that could inform rational combinations.

Binding Assays for Bromodomain Proteins: Their Utility in Drug Discovery in Oncology and Inflammatory Disease.

Bromodomains are protein domains that recognize acetylated lysine residues and are important for recruiting a large number of protein and multiprotein complexes to sites of lysine acetylation. They play an important role in chromatin biology and are popular targets for drug discovery. Compound screening in this area requires the use of biochemical assays to assess the binding potency of potential drug candidates.

Multi-component immunoaffinity subtraction and reversed-phase chromatography of human serum.

Serum analysis represents an extreme challenge because of the dynamic range of the proteins of interest, and the high structural complexity of the constituent proteins. High-abundant proteins such as albumin, IgG, transferrin, haptoglobin, IgA and alpha1-anti-trypsin represent up to 85% of the total protein mass in serum (Fig. 1).

Combination of affinity depletion of abundant proteins and reversed-phase fractionation in proteomic analysis of human plasma/serum.

The tremendous complexity of the serum and plasma proteome presents extreme analytical challenges in comprehensive analysis due to the wide dynamic range of protein concentrations. Therefore, robust sample preparation methods remain one of the important steps in the proteome characterization workflow. We present the results on a new column for the specific depletion of 14 high-abundant proteins from human serum and plasma and the subsequent reversed-phase fractionation of the flow-through proteins.

High recovery HPLC separation of lipid rafts for membrane proteome analysis.

Proteomic analysis of complex samples can be facilitated by protein fractionation prior to enzymatic or chemical fragmentation combined with MS-based identification of peptides. Although aqueous soluble protein fractionation by liquid chromatography is relatively straightforward, membrane protein separations have a variety of technical challenges. Reversed-phase high performance liquid chromatography (RP-HPLC) separations of membrane proteins often exhibit poor recovery and bandwidths, and generally require extensive pretreatment to remove lipids and other membrane components.

Reversed-phase high-performance liquid chromatographic prefractionation of immunodepleted human serum proteins to enhance mass spectrometry identification of lower-abundant proteins.

Serum analysis represents an extreme challenge due to the dynamic range of the proteins of interest, and the high structural complexity of the constituent proteins. In serum, the quantities of proteins and peptides of interest range from those considered "high abundance", present at 2-70% by mass of total protein, to those considered "low abundance", present at 10(-12) M or less. This range of analytical target molecules is outside the realm of available technologies for proteomic analysis.

Differences among techniques for high-abundant protein depletion.

The need to identify protein or peptide biomarkers via readily available biological samples like serum, plasma, or cerebrospinal fluid is often hindered by a few particular proteins present at relatively high concentrations. The ability to remove these proteins specifically, reproducibly, and with high selectivity is increasingly important in proteomic studies, and success in this procedure is leading to an ever-increasing list of lower abundant proteins being identified in these biological fluids. The current work addresses some of the potential problems in depleting proteins in typical biomarker studies, including nonspecific binding during depletion procedures and whether low molecular weight (LMW) species bind to the column in a so-called "sponge" effect caused by the ability of albumin or other high-abundant proteins to bind peptides or protein fragments.

Mining the human cerebrospinal fluid proteome by immunodepletion and shotgun mass spectrometry.

We have analyzed the proteome of human cerebrospinal fluid with the help of shotgun mass spectrometry. In order to identify low-abundant proteins in these fluids, we have found it necessary to remove the abundant protein components from the mixture. Immunodepletion of the abundant proteins has allowed us to identify more than 100 proteins in cerebrospinal fluids from a patient suffering from normal pressure hydrocephalus.

Large-scale purification of active platelet integrin glycoprotein IIb-IIIa.

Glycoprotein IIb-IIIa is an abundant platelet receptor of the integrin family that plays a primary role in platelet aggregation. It exists on the platelet surface predominantly in a resting or inactive conformation that is converted to an active binding competent conformation upon platelet activation. There is much interest in studying the difference between active and inactive GP IIb-IIIa, developing therapeutic agents targeted towards GP IIb-IIIa and developing diagnostic assays for antibodies that recognize epitopes on GP IIb-IIIa.

Evidence that thrombocytopenia observed in humans treated with orally bioavailable glycoprotein IIb/IIIa antagonists is immune mediated.

Glycoprotein (GP) IIb/IIIa antagonists are effective therapeutic agents, but elicit thrombocytopenia with a frequency that approaches 2%. Here, we provide evidence that thrombocytopenia in humans treated with the GP IIb/IIIa antagonist roxifiban is immune mediated. Two patients underwent conversion to a highly positive drug-dependent antibody (DDAB) status temporally associated with thrombocytopenia.

Absence of post-translational aspartyl beta-hydroxylation of epidermal growth factor domains in mice leads to developmental defects and an increased incidence of intestinal neoplasia.

The BAH genomic locus encodes three distinct proteins: junctin, humbug, and BAH. All three proteins share common exons, but differ significantly based upon the use of alternative terminal exons. The biological roles of BAH and humbug and their functional relationship to junctin remain unclear.