Our Science – Ambudkar Website
Suresh V. Ambudkar, Ph.D.
Project Title: Biochemistry of Multidrug Transporters
Our long-term goal is to elucidate the role of ATP-binding cassette (ABC) drug transporters in the development of multidrug resistance (MDR) in cancers and facilitate new therapeutic strategies. For these studies we are employing innovative approaches including biophysical studies, directed mutagenesis, molecular modeling to elucidate molecular mechanisms of the ATP hydrolysis catalytic cycle and drug transport, the use of Fab of monoclonal antibodies and various mutant proteins arrested at various steps in the catalytic cycle to enable us to fix the transporter in a particular conformation for the resolution of the structure of Pgp by X-ray crystallography. We continue to use a mouse model system to validate the modulatory effect of natural products and other small molecule modulators identified in screens using cell-based and biochemical assays. In addition, we use microarrays, qRT-PCR, RNAi technology, TLDA microfluidic chips, and ChIP assays to profile expression of ABC transporters to study the role of these transporters in the early stages of the development of MDR. Such studies should provide insights into the mechanism of action and regulation of these transporters and aid in the development of strategies to increase the efficiency of chemotherapy in cancers.
Of the 48 ABC proteins in the human genome, at least 20 members of this superfamily have been linked to disorders exhibiting Mendelian inheritance. The various disease conditions associated with ABC transporters include cancer, cystic fibrosis, Tangier disease, Stargardt disease, age-related macular degeneration, Dubin-Johnson syndrome, immune deficiency, adrenoleukodystrophy, gout and defects in cholesterol and lipid transport. Our findings and various reagents and technologies developed during the course of this work will greatly facilitate the studies on these disease-linked ABC proteins.
(1) Elucidation of the catalytic cycle of ATP hydrolysis and transport pathway of Pgp and role of conserved motifs in the ATP-binding cassette
We are continuing our studies on the catalytic cycle and transport pathway of Pgp. Based on the thermodynamic and kinetic properties, we have identified the E-S and E-P stable reaction intermediates of the Pgp-mediated ATPase reaction. Using the Walker B E556Q/E1201Q double mutant and non-hydrolysable ATP analog ATP-γ-S, we can precisely attribute the high to low affinity switch in the transport substrate binding site to the formation of the E-S reaction intermediate. To monitor the conformational changes occurring during ATP hydrolysis and drug transport, we have begun to use an EPR spectroscopy and spin labeling approach. Based on a homology model of human Pgp constructed using Sav1866 and mouse mdr1a structure as a template, we have introduced either a single cys residue or two cys residues at various locations in the cys-less Pgp, including regions from extracellular loops, transmembrane domains, intracellular loops, and NBDs. We have generated 20 double and 15 single cys mutants so far. These mutants, after their expression in High-Five insect cells, have been purified and found to retain function to the same level as wild-type protein. We have optimized the conditions for labeling of these mutant proteins in detergent solution with the spin label MTSL for EPR spectroscopy analysis. In the next 2-3 years, our plan will be to use continuous wave and pulse double electron-electron resonance (DEER) ESR spectroscopy in collaboration with Dr. Jack Freed at an NIH-funded facility (Department of Chemistry and Chemical Biology, Cornell University, Ithaca) to monitor conformational changes in the presence and absence of drug-substrate and ATP. The DEER ESR spectroscopy studies with the double cys mutants will also allow us to validate the homology model of human Pgp in both open and closed conformations. To complement these studies, data mining, sequence alignment of ~38K ABC domains and site-directed mutagenesis approaches are being used to assess the role of critical residues in the conserved subdomains in the NBDs in ATP-binding and hydrolysis. We substituted the conserved H residue with Q, A, E, K and Y in the H-loop in each NBD individually or together and found that only the H587Q and H1232Q variants exhibit normal drug transport. The homology model of the Pgp NBDs based on the structure of the HlyB dimer suggests that the conserved H587 and H1232 interact with the γ-P of ATP and/or other residues in the ATP-binding pocket through H-bonding. In the next phase, we plan to focus on residues in the D- and Q-loop in ATP sites and in the intracellular loops to distinguish between residues that are critical for ATP hydrolysis and those that are involved in communication between the drug-substrate binding site and the NBDs.
(2) Development of potent natural product and other small molecule non-toxic modulators of ABC transporters: screening and validation in a mouse model system
We continue to identify natural products such as curcumin and plumbagin that interact with ABC drug transporters. In addition, we continue to validate the use of the natural product non-toxic modulator curcumin to reverse drug resistance in cancer patients. In collaboration with Dr. Bjorn Bauer (University of Minnesota), we use rat brain capillaries as an ex vivo model of blood-brain barrier to demonstrate that curcumin at nanomolar concentration blocks the function of both Abcg2 and Pgp. In addition, using Abcg2 knockout mice, we further confirmed that oral curcumin increased the C-max and relative bioavailability of sulfasalazine by selectively inhibiting ABCG2 function. To increase the bioavailability of curcumin, in future work we will test a combination of piperine (an active ingredient of black pepper) and curcumin in a xenograft mouse model, as our cell-based assays have shown that piperine potentiates the modulatory effect of curcumin. We have found that small molecule tyrosine kinase inhibitors including imatinib, nilotinib, sunitinib, erlotinib and lapatinib modulate the function of Pgp and ABCG2 by interacting at the drug-substrate sites, even though these inhibitors compete for the ATP-binding site on the tyrosine kinase. We plan to continue to study the small molecule tyrosine kinase inhibitors for their potential use as inhibitors of ABC drug transporters. To aid in future studies, we have synthesized a fluorescent bodipy derivative of nilotinib that can be used to monitor nilotinib accumulation in vivo. In addition, we have synthesized 26 derivatives of nilotinib in collaboration with Dr. Craig Thomas at NCGC, NHGRI to identify derivatives of nilotinib that inhibit only the BCR-ABL kinase or those that interact only with ABC drug transporters.
(3) Resolution of three-dimensional structure of human Pgp
Resolution of the three-dimensional structure of Pgp is an ongoing project in collaboration with Dr. Di Xia in LCB. Currently, we have been able to obtain 10-12 mg of pure Pgp/ml using a baculovirus-insect cell expression system. In addition to wild-type protein, several mutants including the E556Q/E1201Q double mutant, which is trapped in an E-S pre-hydrolysis transition-like state in the presence of ATP, have also been purified. To further optimize fixed conformation of the transporter to generate crystals, we plan to use another approach wherein the Fab of the conformation-sensitive monoclonal antibody UIC2 is incubated along with purified Pgp during crystallization. The use of UIC2 Fab will allow stabilization of extracellular loops and TM domains, while the use of the E-Q double mutant will fix the conformation of NBDs in the presence of ATP. We will focus on screening several conditions for Pgp-UIC2 Fab crystal growth in the presence and absence of nucleotides or non-hydrolysable nucleotide analogs and high-affinity transport substrates or modulators such as bodipy-verapamil and tariquidar to obtain crystals that will diffract at high resolution.
(4) Determination of molecular mechanisms of drug resistance in single- and multi-step selection with anticancer agents in cancer cells
To understand the mechanism of development of MDR under clinical conditions, we established single-step doxorubicin-selected MCF-7 sublines using very low concentrations (14 or 21 nM). We have found that ABCG2 is the major transporter responsible for the development of resistance, which was confirmed using ABCG2-siRNA. ChIP assays confirmed that epigenetic changes, including histone hyperacetylation, cause upregulation of ABCG2 expression. In future work, we plan to subject these subclones to multi-step selection with doxorubicin to determine at what stage the ABCG2 expression is down regulated and expression of ABCB1 is increased. In related work with the breast cancer cell line MCF-7/ADR, which is multi-step selected and maintained in a high concentration of doxorubicin, we found that there is significant enrichment (30-50%) of the CD44+/CD24- population. This population exhibits enhanced capacity for self-renewal, migration and proliferation in three-D cultures in vitro and formation of tumors in vivo, suggesting that cells with characteristics of breast cancer stem cells (CSCs) are enriched following prolonged drug treatment. In future work we will test whether culturing MCF-7/ADR cells in drug-free medium for 4-6 weeks will partially or fully revert the CSC phenotype. In addition, we plan to determine whether enrichment of CSCs in response to drug treatment is associated with an Epithelial-Mesenchymal Transition (EMT)-like phenotype. Such studies should help us to establish a link between MDR, metastasis, EMT and CSC phenotypes.
1. Michael M. Gottesman, M.D.
Laboratory of Cell Biology, CCR, NCI, NIH
2. Di Xia, Ph.D.
Laboratory of Cell Biology, CCR, NCI, NIH
3. Susan E. Bates, M.D.
Medical Oncology Branch, CCR, NCI, NIH
4. Lyuba Varticovski, M.D.
Laboratory of Receptor Biology and Gene Expression, CCR, NCI, NIH
5. Stuart H. Yuspa, M.D.
Laboratory of Cancer Biology and Genetics, NCI, NIH
6. Craig J. Thomas, Ph.D.
NIH Chemical Genomics Center, NHGRI, NIH
7. Bjorn Bauer, Ph.D.
Department of Pharmaceutical Sciences, College of Pharmacy, University of Minnesota, Duluth, MN 55812
8. Michael Mayer, Ph.D.
Department of Biomedical Engineering and Department of Chemical Engineering, University of Michigan, Ann Arbor, MI
9. Jack Freed, Ph.D.
Department of Chemistry and Chemical Biology, Baker Laboratory,
Cornell University, Ithaca, NY
10. Rajendra Prasad, Ph.D.
School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India
11. Li-wu Fu, M.D., Ph.D.
State Key Laboratory of Oncology in South China, Cancer Center, Sun Yat-Sen
University, Guangzhou 510060, China
This page was last updated on 2/19/2013.