Multiple myeloma is the second most common hematologic malignancy in the United States. While the disease remains incurable, there are widespread translational efforts to improve chemotherapeutic options in this disease. Molecularly targeted therapies, particularly those directed at the proteasome, but more recently kinase targets as well, are showing clinical efficacy. Therapeutically inhibiting specific enzymes creates a critical need for assays capable of measuring the activities of these proteins in myeloma disease models and in patient samples. The ability to measure relevant enzyme activity in primary tumor samples at baseline and/or after treatment would provide the ability to tailor patient therapy based on aberrant signal transduction, validate mechanisms of resistance in patients, and would offer an invaluable pharmacodynamic tool to assess whether resistance is associated with inadequate target inhibition. An interdisciplinary collaboration is proposed to create the analytical and chemical tools needed to directly measure the enzymatic activities of protein kinases and the proteasome in cells taken directly from patients with multiple myeloma. The investigators will develop unique fluorescent reagents to report the activity of the protein targets of molecular-based therapies currently in use or in clinical trials. Kinase substrates will be modified to create long- lived compounds by attachment of stably folded beta-hairpin structures or """"""""protectides"""""""". These beta-hairpins will also be used to create substrates with secondary structure for metabolism by the proteasome. Membrane-permeant forms of these protectide-substrates will be capable of accurately reporting the enzymatic activity within a living cell. Microelectrophoretic separations combined with low-level fluorescence detection will enable the quantitative analysis of these molecules from single mammalian cells. This capability will resolve three major issues currently faced in the biochemical analysis of clinical myeloma samples: lack of direct measurement of the enzymatic activity of target proteins;sample size requirements that are impractically large for clinical implementation;and sample heterogeneity that can mask pertinent aspects related to patient response. Protein kinase B, protein kinase C, and the chymotrypsin-like activity of the proteasome were chosen for this work by virtue of their pertinence to both research and clinical practice in multiple myeloma.

Public Health Relevance

The goal of the proposed work is to develop an assay to study active signal transduction pathways in the tumor cells of patients. This would enhance diagnostics and prognostics as well aid therapeutic planning.
The final aim of the work focuses on multiple myeloma, a disease with a very poor survival rate and the second most common hematologic malignancy in the United States where advances in therapy are being aggressively sought.

Agency
National Institute of Health (NIH)
Institute
National Institute of Biomedical Imaging and Bioengineering (NIBIB)
Type
Research Project (R01)
Project #
5R01EB011763-03
Application #
8217264
Study Section
Enabling Bioanalytical and Biophysical Technologies Study Section (EBT)
Program Officer
Zullo, Steven J
Project Start
2010-04-01
Project End
2014-01-31
Budget Start
2012-02-01
Budget End
2013-01-31
Support Year
3
Fiscal Year
2012
Total Cost
$518,482
Indirect Cost
$168,156
Name
University of North Carolina Chapel Hill
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
608195277
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Fayer, Effrat L; Gilliland, William M; Ramsey, J Michael et al. (2017) N-Gemini peptides: cytosolic protease resistance via N-terminal dimerization of unstructured peptides. Chem Commun (Camb) 54:204-207
Melvin, Adam T; Dumberger, Lukas D; Woss, Gregery S et al. (2016) Identification of a p53-based portable degron based on the MDM2-p53 binding region. Analyst 141:570-8
Mainz, Emilie R; Dobes, Nicholas C; Allbritton, Nancy L (2015) Pronase E-Based Generation of Fluorescent Peptide Fragments: Tracking Intracellular Peptide Fate in Single Cells. Anal Chem 87:7987-95
Kovarik, Michelle L; Dickinson, Alexandra J; Roy, Pourab et al. (2014) Response of single leukemic cells to peptidase inhibitor therapy across time and dose using a microfluidic device. Integr Biol (Camb) 6:164-74
Dickinson, Alexandra J; Hunsucker, Sally A; Armistead, Paul M et al. (2014) Single-cell sphingosine kinase activity measurements in primary leukemia. Anal Bioanal Chem 406:7027-36
Park, Jessica H; Waters, Marcey L (2013) Positional effects of click cyclization on ?-hairpin structure, stability, and function. Org Biomol Chem 11:69-77
Kovarik, Michelle L; Shah, Pavak K; Armistead, Paul M et al. (2013) Microfluidic chemical cytometry of peptide degradation in single drug-treated acute myeloid leukemia cells. Anal Chem 85:4991-7
Melvin, Adam T; Woss, Gregery S; Park, Jessica H et al. (2013) Measuring activity in the ubiquitin-proteasome system: from large scale discoveries to single cells analysis. Cell Biochem Biophys 67:75-89
Melvin, Adam T; Woss, Gregery S; Park, Jessica H et al. (2013) A comparative analysis of the ubiquitination kinetics of multiple degrons to identify an ideal targeting sequence for a proteasome reporter. PLoS One 8:e78082
Yang, Shan; Proctor, Angela; Cline, Lauren L et al. (2013) ?-Turn sequences promote stability of peptide substrates for kinases within the cytosolic environment. Analyst 138:4305-11

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