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Suresh V. Ambudkar, Ph.D.

Laboratory of Cell Biology Head, Transport Biochemistry Section Senior Investigator Building 37, Room 2120 National Cancer Institute 37 Convent Drive Bethesda, MD 20892-4256 Phone:   301-402-4178 Fax:   301-435-8188 E-Mail:   ambudkar@mail.nih.gov

Biography

Dr. Ambudkar obtained his Ph.D. from Madurai Kamaraj University, Madurai, India, and received his postdoctoral training in membrane bioenergetics with Dr. Barry Rosen at the University of Maryland. He continued his postdoctoral work on biochemistry of membrane transport proteins at the Johns Hopkins University School of Medicine. In July 1995, after 5 years as an assistant professor in the departments of medicine and physiology at Johns Hopkins, he joined the Laboratory of Cell Biology at the NCI. Current position-Chief, Transport Biochemistry Section

Research

Biochemistry of Multidrug Transporters

The ATP-binding cassette (ABC) transporter superfamily contains membrane proteins that utilize the energy of ATP hydrolysis to transport a variety of substrates such as ions, sugars, glycans, cholesterol, peptides, proteins, toxins, antibiotics, and amphipathic natural product anticancer drugs. This superfamily, which includes ~1100 members is defined by the homology within the ABC region, which includes the Walker A and B motifs found in all ATP-binding proteins, plus a dodecapeptide motif known as the ABC signature region or the linker region. The functional unit is comprised of two ABCs and twelve membrane-spanning domains, which are formed either as a single polypeptide chain or as homo or heterodimers. The genetic variation in ABC transporter genes is the cause or contributor to a wide variety of human diseases such as cancer, cystic fibrosis, Stargardt disease, age-related macular degeneration, adrenoleukodystrophy, rheumatoid arthritis, insulin-dependent diabetes, Dubin-Johnson syndrome, Tangier disease, familial high-density lipoprotein deficiency, progressive familial intrahepatic cholestasis, and Pseudoxanthoma elasticum.

The ATP-binding cassette (ABC) transporters such as P-glycoprotein (Pgp, ABCB1), the multidrug resistance-associated protein (MRP1, ABCC1), and the mitoxantrone-resistance protein (MXR also known as breast cancer resistance protein, BCRP, ABCP or ABCG2), which function as ATP-dependent efflux pumps, play an important role in the development of multidrug resistance in most cancers. There are 48 known ABC transport proteins in the human and at least 14 of these transporters are involved in the movement of a variety of amphipathic agents including anticancer agents, nucleotide analogs and cyclic nucleotides. Thus, some of these transporters also may contribute to the development of multidrug resistance in malignant cells. Our studies are directed toward understanding the mechanism of action of the multidrug resistance-linked ABC drug transporters. By using a baculovirus-insect cell expression system, a large amount (9-10 mg/ml) of biologically active Pgp has been prepared for biophysical and structural studies, as further understanding of the mechanism of these transporters would be accelerated by resolution of the structure of Pgp. Currently, we have also developed methods for large-scale purification of mutant Pgp variants that are trapped in a transition state during catalytic cycle. At present our collaborator, Dr. Di Xia has tested >25,000 conditions for generation of crystals of wild-type and mutant Pgps. In last couple of years we have directed our efforts towards understanding the catalytic cycle of ATP hydrolysis by Pgp, identification of rate-limiting step(s) and modulation of the ATPase activity by substrates and modulators. Similar studies have been initiated with MRP1, MRP4 and MRP8 to gain insight into the role of the two ATP sites in ATP hydrolysis by these transporters. In addition, we have characterized curcumin, a natural product modulator that inhibits the function of Pgp, MRP1 and ABCG2 -the major three ABC drug transporters. Curcumin, which is relatively nontoxic, might have a beneficial effect on cancer chemotherapy with respect to the possibility of long-term use without concerns regarding MDR1, MRP1 or ABCG2 activation. Similarly we also found that major dietary flavonoids such as quercetin and silymarin can modulate function of MRPs--1, 4, and 5. Such studies will provide an insight into the role of these transporters in the development of multidrug resistance in cancers and aid in the development of new therapeutic strategies.

1. Development of natural product modulators for the reversal of drug resistance-mediated by Pgp, MRP1 and ABCG2: Our previous studies demonstrated that curcumin; an active ingredient of turmeric powder which is consumed in many parts of world modulates the function of Pgp. The curcumin is a mixture of three curcuminoids—curcumin I, II, and III. We purified each curcuminoid from curcumin mixture by using solvent extraction and HPLC chromatography. Among curcuminoids, curcumin I is the most effective modulator of Pgp, MRP1 as well as ABCG2. We characterized the interaction of curcuminoids with these drug transporters and found that these curcuminoids also interact at the drug-substrate-binding sites on these transporters. It is interesting to note that curcumin is one of the few modulators so far known which inhibit the function of the three major ABC drug transporters. It remains to be seen whether these transporters have a common modulator-binding site.

2. Characterization of human MRP1 (ABCC1), MRP4 (ABCC4) and MRP5 (ABCC5): Multidrug Resistance Proteins 4 (MRP4/ABCC4) and 5 (MRP5/ABCC5) transport cyclic nucleoside monophosphates, nucleoside analog drugs, chemotherapeutic agents and prostaglandins. We have characterized the effect of mefloquine—a quinoline based antimalarial drug on the function of MRP4 and 5, which are also expressed in human red blood cells. Mefloquine appears to be a transport substrate for MRP1 and MRP4 but not for MRP5. Similarly we also found that major dietary flavonoids such as quercetin and silymarin can modulate function of MRPs--1, 4, and 5. Our results indicate that polyphenols interact directly with MRP1, MRP4, and MRP5, and that some of them i.e., quercetin and silymarin, may well prove to be substrates for MRPs. They modulate both transport function and ATPase activities of MRP1 and MRP4. Given the amounts of polyphenols ingested daily, it is likely that the transporters in vivo would be exposed to relatively high concentrations and become susceptible to modulation of both function and expression. This in turn could influence bioavailability, distribution and transport of various dietary toxins and chemotherapeutics handled by these transporters. Understanding the interactions of these polyphenols with MRPs may be useful for improving the efficacy of anticancer as well as antiviral drug therapies.

3. Characterization of the substrate-binding sites on Pdr5p, a major yeast drug transporter: We have been studying the yeast drug transporters, which are functional homologs of human ABC drug transporters. The yeast Pdr5p transporter has been overexpressed in a strain which lack major nine ABC transporters. We previously have shown that substrate size is an important factor in substrate-transporter interaction and that this transporter has at least three substrate-binding-sites. We observed that substrate site 1 requires substrates with three electronegative groups where as site 2 substrates should have at least a single hydrogen bond acceptor group. The presence of multiple sites with different requirements for substrate—Pdr5p interaction may explain the broad specificity of xenobiotic compounds transported by this protein.

4. Resolution of three-dimensional structure of human Pgp: The high-resolution structure of Pgp at various stages during the catalytic cycle will be essential to understand the transport mechanism. This is one of our major interests and we have invested considerable effort in the past to develop methods for obtaining pure and active Pgp in large amounts. We have developed methods for large-scale purification of human Pgp expressed in baculovirus-MDR1 infected insect cells by metal affinity (Talon) and anion exchange (DE52) chromatography. Recently, by making improvements in our methods, we have obtained pure Pgp at ~9 to 10 mg/ml concentration. In addition, we are now able to purify various mutant variants of Pgp that are trapped in a transition state during the catalytic cycle. Most importantly, even at such a high concentration, Pgp in detergent solution is mainly present as a monomer. At present our collaborator, Dr. Di Xia has tested >25,000 conditions for generation of crystals of wild-type and mutant Pgps. The optimization of conditions for generation of 3D-crystals is currently in progress.



Collaborators on this research include
Susan Bates, CCR, NCI, NIH
Margery Barrand, Univ. Cambridge, UK
Stephen Hladky, Univ. Cambridge, UK
Pornngarm Limtrakul, Chiang Mai University, Thailand; Rajendra Prasad, Jawaharlal Nehru University, India.
Di Xia, LCB, CCR, NCI

This page was last updated on 6/11/2008.