NCI Funded Research Portfolio - Z01 BC 010030 Abstract

Z01 BC 010030 (Z01)
Title	Biochemical Analysis of Multidrug Resistance-linked Transport Proteins
Institution	NCI, Bethesda, MD
Principal Investigator	Ambudkar, Suresh	NCI Program Director	N/A
Cancer Activity	N/A	Division	CCR
Funded Amount	$863,565	Project Dates	10/01/2001 - N/A
Fiscal Year	2008	Project Type	Intramural
Research Topics (SICs) w/ Percent Relevance		Cancer Types (Disease Sites) w/ Percent Relevance
Antimicrobial Resistance (20.0%) Breast Cancer - Treatment (25.0%)		Breast Cancer (25.0%) Colon/Rectum (20.0%) Genital System - Male Other (15.0%) Liver Cancer (20.0%) Ovarian Cancer (5.0%) Prostate (15.0%)
Common Scientific Outline
Complementary and Alternative Treatment Approaches Systemic Therapies - Discovery and Development
Abstract	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 ES and EP stable reaction intermediates of the Pgp-mediated ATPase reaction. Recently, we observed that the ES conformation of Pgp (previously demonstrated in the E556Q/E1201Q mutant) can be obtained with the wild-type protein by use of the nonhydrolyzable ATP analog ATP-g-S. ATP-g-S, similar to ATP in E556Q/E1201Q mutant, is occluded into the wild-type Pgp NBDs at 34-37C but not at 4C. When Pgp is occluded with ATP-g-S, it exhibits reduced affinity for transport substrates. Collectively, these data provide evidence for the ATP-driven dimerization and ADP-driven dissociation of the NBDs and although, two ATP molecules may initiate dimerization, only one is driven to an occluded pre-hydrolysis intermediate state. It appears that in a full-length ABC transporter like Pgp, the occluded nucleotide conformation at one of the NBDs provides the power-stroke at the transport-substrate site. In collaboration with John Golin (The Catholic Univ. of America), we characterized the ATPase activity of the yeast Pdr5p transporter that effluxes a variety of xenobiotic compounds. Pdr5p-specific ATPase activity shows complete, concentration-dependent inhibition by clotrimazole, which is known to be a potent transport substrate, however both GTPase and UTPase activities are relatively resistant to this drug. Our results indicate that this inhibition is noncompetitive and caused by the interaction of clotrimazole with the transporter at a site that is distinct from the ATP-binding domains. We propose that Pdr5p increases its transport substrate specificity by using more than one nucleotide as an energy source. 2. Development of potent natural product and other non-toxic modulators/inhibitors of ABC transporters: To develop modulator(s) that will inhibit multiple transporters we screened synthetic compounds as well as natural products. The ABCG2 transporter confers resistance to multiple chemotherapeutic agents. One approach to combat MDR mediated by this transporter is the development of inhibitors/modulators that block its function at non-toxic concentrations. We found that napthoquinones, vitamin K3 and its structural analogue plumbagin are substrates of ABCG2. Thus, ABCG2 may have a role in the regulation of vitamin K3 levels in the body. In addition, a thiosemicarbazone, NSC73306, which kills specifically Pgp expressing cells, was found to be a potent modulator of ABCG2. Thus, NSC73306 exhibits due mode of action that can be exploited to overcome drug resistance by eliminating Pgp-overexpressing cells and by acting as a potent modulator to resensitize ABCG2 expressing cancer cells to chemotherapeutics. In collaboration with Drs. Susan Bates, Curtis Henrich, Michael Dean and James McMahon (CCR and Molecular Targets Development Program, NCI) we have screened with a high-throughput assay a library of ~7400 natural products and synthetic compounds to identify potent new inhibitors of ABCG2. We are also studying tyrosine kinase inhibitors for their potential use as inhibitors of ABC drug transporters. We found that the newly developed tyrosine kinase inhibitor AMN107 (nilotinib), and its parent drug imatinib (Gleevec) modulates activity of Pgp and ABCG2 by interacting at the drug-substrate binding sites instead of ATP sites as is the case with tyrosine kinase inhibitors. In collaboration with Dr. Zhe-Sheng Chen (St. Johns Univ.), we have demonstrated that Erlotinib (Tarceva), which is an EGFR tyrosine kinase inhibitor, reverses Pgp and ABCG2-mediated resistance by directly inhibiting the efflux function of these transporters. 3. Resolution of three-dimensional structure of human Pgp: The resolution of the three-dimensional structure of Pgp is an ongoing project and for this we have developed a purification scheme that has yielded total protein 7.5-10 mg of > 99% homogeneously pure Pgp at 10-12 mg/ml concentration. For improving the crystallization of Pgp, we have initiated another approach where in the Fab of the conformation-sensitive monoclonal antibody, UIC2 is incubated along with the purified Pgp during crystallization. We have optimized the conditions to generate Fab of UIC2 with protein concentration in the range of 5-7 mg/ml. Additional experiments demonstrate that Fab of UIC2 binds to Pgp in detergent solution under similar conditions that are used for generations of crystals indicating that it is feasible to generate co-crystals of Pgp and UIC2-Fab. At present we are in the process of generating 400-500 mg of UIC2 from the hybridoma cell line HB1287. This will allow us to prepare 150-200 mg of Fab for testing new crystallization conditions. 4. Molecular mechanism of drug resistance in single- and multi-step selection with anticancer agents in cancer cells: To understand the mechanism of multidrug resistance (MDR) under clinical conditions, we have begun to examine how treatment regimens affect the expression of ABC drug transporters in single- and multi-step selection with anticancer drugs such as doxorubicin. We established single-step doxorubicin-selected MCF-7 sublines using very low concentrations, 14 or 21 nM. We have found that ABCC2, ABCC4 and ABCG2 were overexpressed at the mRNA level in these single-step selected sublines. Yet, only ABCC4 and ABCG2 were overexpressed at the protein level. Both 14 and 21 nM single-step doxorubicin-selected sublines exhibit nearly 5-fold resistance to doxorubicin compared to parental MCF-7 cells. However, as ABCC4 does not confer resistance to doxorubicin it is most likely that ABCG2 is the major transporter responsible for the development of resistance. We also observed by using chromatin immunoprecipitation assay that the upregulation of ABCG2 is facilitated by histone hyperacetylation. Interestingly, stem cell markers including CD44 and CD24 are not enriched in single-step clones of breast cancer cell line MCF-7 selected with low concentration (21 nM) of doxorubicin. These results indicate that the MDR phenotype arises following lose-dose, single-step exposure to doxorubicin, and further suggest that overexpression of ABCG2 by epigenetic changes may mediate early stages of MDR development. In another related study in collaboration with Lyuba Varticovski (CCR, NCI), we sought to determine whether cancer stem cells occur in BRCA1-associated breast cancer and contribute to therapeutic response. We observed that Brca-1-deficient mouse mammary tumors harbor heterogeneous cancer stem cell populations, and CD44+/CD24- cells represent a population that correlates with human breast cancer stem cells. 5. Characterization of Single nucleotide polymorphisms and haplotypes in ABCB1: In collaboration with Dr. Michael Gottesman we showed that in MDR1, synonymous SNPs in the context of a haplotype, with two synonymous (3435C>T & 1236C>T) and one non-synonymous (2677G>T) SNP, were associated with altered substrate and inhibitor specificity. Further recent work in collaboration with Drs. Gottesman and Nussinov (SAIC, Frederick Inc., and CCR, NCI) suggests that long-enough ribosomal pause time-scales may lead to alternate folding pathways and distinct minima on the folding free energy surface. While the conformational and functional differences between the native and alternate states may be minor, the MDR1 case illustrates that the barriers may nevertheless constitute sufficiently high hurdles in physiological time-scales, leading to kinetically trapped states with altered structures and functions. Different folding pathways leading to conformationally-similar trapped states may be due to swapping of (fairly symmetric) segments.