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ABC Transporters: Potentially Selective Targets for the Treatment of Multi-drug Resistant Tumours Dr. Elias Georges. Potential Conflict of Interest. Research Contract / 2005 – 2007 YM Biosciences Biotechnology / 1997 – Aurelium BioPharma Inc.
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ABC Transporters: Potentially Selective Targets for the Treatmentof Multi-drug Resistant TumoursDr. Elias Georges
Potential Conflict of Interest • Research Contract / 2005 – 2007 • YM Biosciences • Biotechnology / 1997 – • Aurelium BioPharma Inc.
ABC Transporters: Potentially Selective Targets for the Treatment of Multi-drug Resistant TumoursQ-CROCMarch 27th, 2009 Elias Georges, Ph.D.
Estimated Cancer Cases… • Approximately 93,000 women in the U.S. were estimated to have resistant or refractory ovarian cancer in 2008…..
Mechanisms of Drug Resistance… Gottesman MM, 2002
Domain Organization of ABC Proteins… John A. Shelps et al. 2004
High Resolution Structure of ABCB1 Based Bacterial ABC Transporters Omote & Al Shawi, 2006; Shiling R. et. al. 2006
P-gp MRP1 MTX VBL LTC4 PTX VCR GSH BR-Glu Vp-16 DNR Bis E2-Glu Rhod Dox Mitox Epi Topo Praz Lys SN-38 BCRP CPT-11 Overlapping Substrate Specificity… Chemo-immunity
Different Approaches to Selectively Target ABC-Mediated MDR Cells Normal Function (drug efflux) ABC-mediate Apoptosis (Collateral Sensitivity) Reversing Agents (Blocker Drug) Regulate (Protein-interactions) Bypass (non-substrates)
O N S N S O H O H O MK571 C l O 1 2 5 1 2 5 I I C N O H N 3 O H 3 O C O I A C I I A A Q C H IAARh123 3 C l C H 3 + - N a O O C H 3 N N H H N O H 3 125 I Mapping Drug & Ligand Binding Domains N N N N N N H IAAGSH H IAALTC4 O O HA epitope; trypsin & V8 sites
Mapping Drug Binding Domains of ABCC1 Deeley & Cole, 2006 Karwatsky, J. & Georges, E 2004
Reversing Agent of ABCB1 MDR P-glycoprotein-mediated MDR: Reversal agent Pouliot JP, et al. 1998
Different Approaches to Selectively Target ABC-Mediated MDR Cells Normal Function (drug efflux) ABC-mediate Apoptosis (Collateral Sensitivity) Reversing Agents (Blocker Drug) • Regulation of ABC proteins functions by inhibition of protein Interactions • Selective targeting of MDR cells through Activation rather than inhibition of ABC proteins ATPase activities Regulate (Protein-interactions) Bypass (non-substrates)
Selective Targeting of ABC Proteins in MDR Cells Targeting ABCC1-expressing MDR Cells ABCC1 Efflux of GSH, GSH Conjugates and Cytotoxic Drugs Cole S.
Hyper-sensitivity of ABCC1 MDR CellsABCC1 Positive Tumour Cells are Hyper-sensitive to Specific Drugs Buthionine Sulfoximine (BSO): γ-GCS inhibitor Verapamil (Vrp): MRP1 modulator Apigenin (Api): MRP1 modulator H69 or HeLa: square H69AR or HeLaMRP1: Triangles H69PR: Diamonds Laberge et al. 2007
ABCC1 Activators Induce Apoptosis….GSH Depletion Leads to ROS Accumulation and Subsequent Apoptosis Verapamil Verapamil Apigenin Apigenin BSO BSO Apoptosis ROS GSH BSO: 50 µM; Vrp: 50 µM; Api: 25 µM Laberge et al. 2007
Collateral- or Hyper-sensitivity of MDR Cells June L. Biedler, Cancer Res. 1994
Hyper-sensitivity of MDR Cells to Verapamil Karwatsky et al. Biochemistry 2003
Verapamil Hyper-sensitivity Requires Active ABCB1 ATPase Karwatsky et al. Biochemistry 2003
Verapamil Hyper-sensitivity Correlates with Drug Effect on ABCB1 ATPase Laberge, et. al. 2009
Mechanism of Verapamil Hyper-sensitivity Higher ROS due to Increased ATPase Activity Higher basal ATPase in MDR cells Verapamil increases ROS & decreases cellular GSH levels in MDR cells Verapamil decreases cellular ATP in MDR cells Karwatsky et al. Biochemistry 2003
ABCB1 Expression Directly Modulates Hyper-sensitivity to Verapamil in MDR cells
Electron Transport Chain inhibitors and ROS Production H2O2 Cytoplasm CuZn-SOD H2O2 O·2 O2 e- Mitochondria Intermembrane space Antimycin A Rotenone GSSG GSH Mn-SOD e- H2O2 O·2 O2 H2O GPx Mitochondria Matrix
Hyper-sensitivity to ETC Inhibitors Requires ABCB1 Expression and ATPase Activity Laberge et. al., 2009
ETC Inhibitors Amplify Hyper-sensitivity of MDR Cells to Verapamil Laberge et. al., 2009
ABCB1 Hyper-sensitivity to Verapamil in CHRC5: Working Model Other compounds? Direct Link to ABCB1 High expression and Active ABCB1 Other P-gp ATPase stimulator: Pro, DOC, Tmx, Cort Missing Link Factor S….. Mitochondria’s ETC/ROS? Karwatsky et al. Biochemistry 2003
Post-docs, Graduate students and Collaborators Roni Daoud, Ph.D. Omar Alqawi, Ph.D. Joel Karwatski, Ph.D. Remi-Martin Laberge, Ph.D. Jean-Francois Pouliot, Ph.D. Francoise L’Heureaux MSc. Ying Wang, Ph.D. Marko Vezmar, MSc. Max Lincoln, MSc. Raghuram Ambadipudi, MSc. Gabriela Certad, MD Abraham Abraham, MSc. Mara Leimanis, Ph.D. Margaret Liu, MSc. Ashutosh Singh Sonia Eday, MSc. Dr. Philippe Gros Biochemistry, McGill Dr. Roger Prichard Parasitology, McGill Dr. Leann Tilley Dr. Leslie Deady La Trobe University, Australia Aurelium BioPharma Inc.
Different Approaches to Selectively Target ABC-Mediated MDR Cells Normal Function (drug efflux) ABC-mediate Apoptosis (Collateral Sensitivity) Reversing Agents (Blocker Drug) ABC Proteins are Enzymes… • Regulation of ABC proteins functions by inhibition of protein Interactions • Selective targeting of MDR cells through Activation rather than inhibition of ABC proteins ATPase activities Regulate (Protein-interactions) Bypass (non-substrates)
ABC Protein Interactions.. Possible Regulation of ABC Protein Functions • ABC Linker Domains • Low sequence homology between different ABC proteins • Highly charged domain • Encodes multiple PKA/PKC sites • Function unknown…. Georges, E. 2007
ABCB1 Linker Domain Interacts with Tubulin Possible Regulation Through Protein Interactions Signal ABCB1 Expression Georges, E. 2007
Differentially Expressed ProteinsProteomics Approach 116 97.4 66 45 31 21.5 14.4 Resistance Associated Proteins in Breast MW (kDa) Chemo-responsive breast tumours Chemo-resistant breast tumours Blue: increased Red: Unique pI 4 7 4 7 Georges, E. et. al. 2008
Differentially Expressed Genes A Genomic Approach Resistance Associated Genes in Breast • 838 downregulated genes with > 15x • 946 upregulated genes with > 15x Georges, E. et. al. 2008