UM Chemistry/Research/Baker Research Group

Current Group Members

• Krista Bearden, Ph.D. Student
• Irene Wanjala, Ph.D. Student
• Trucchi Pham, Ph.D., Research Scientist

Research in the Baker laboratory utilizes liquid chromatography mass spectrometry (LC-MS) as a primary tool to answer questions relevant to understanding the initiation, progression and treatment of cardiovascular disease and cancer. Work from numerous laboratories over the last 20 years has proven an important role for phospholipid growth factors (lysophosphatidic acid, LPA and sphingosine 1-phosphate, S1P) in these human diseases. LC-MS is applied in two main ways in our work. First, we utilize stable-isotope dilution techniques for the quantitative analysis of bioactive lipids in various biological fluids. A current example of this work entails analysis of the synthetic pathways that contribute to the formation of LPA and alkyl glycerophosphate (AGP) levels in oxidized human lipoproteins. We have previously shown that these products stimulate the earliest events associated with cardiovascular disease. Second, we utilize a library of LPA, AGP and S1P photoaffinity analogs in tandem with LC-MS to determine protein targets that mediate biological activity of these compounds. Current examples of this work include identification of transport proteins (in plasma/serum and across cellular membranes) and the characterization of ligand-receptor interactions (GPCR and PPARγ). Recently, we have developed a collaboration with the Parrill and Webster groups that aims to elucidate the structure of the recently described autocrine motility factor autotoxin (ATX), an important anticancer target. From this work we plan rational design, synthesis and analysis of novel ATX inhibitors as cancer chemotheraputics.

Quantitative Analysis of Bioactive Lipid Mediators

A major focus of the lab revolves around the determination of bioactive lipid concentrations in biological samples. To this end we have employed stable isotope dilution and liquid chromatography mass spectrometry (LC-MS) to measure lipid concentrations in plasma, serum and tumor fluids from healthy volunteers and cancer patients. Likewise, we have analyzed bioactive lipid levels in order to better understand the pathways leading to their synthesis.We are currently studying the formation of bioactive lipids in human lipoprotein fractions which is funded through the National America Heart Association.

Quantitative Analysis of Protein targets of Bioactive Lipid Mediators

It has been long appreciated that bioactive lipids are transported in the plasma as albumin complexes. Likewise, these lipids elicit most of their cellular response via integral membrane receptors of the G protein coupled receptor super family, whereas some responses are mediated through nuclear hormone receptors. Finally, the concentrations of these lipids are controlled via biosynthetic (autotaxin) and hydrolytic enzymes (lipid phosphate phosphohydrolase, LPP). Currently, if one wants to measure the absolute concentrations of these important protein targets, they are dependent on mRNA quantitation (Q-PCR) or Western blot. Unfortunately mRNA levels do not always correlate with expressed protein level. Likewise, Western blots are semi-quantitative and are dependent on high quality antibodies (which are lacking for these targets). We are currently developing methodology to combine stable isotope dilution with cell fractionation and nanospray ionization LC-MC for the absolute quantitation of protein targets of bioactive lipids.

Photoaffinity Labeling

In an effort to better understand interactions between bioactive lipids and their protein targets we are designing, characterizing and applying photoaffinity analogs. We have designed several analogs of lysophosphatidic acid that include (trifluoromethyl)phenyl diazirine, aryl azide and benzophenone photoreactive groups. When exposed to intense UV light, these compounds generate reactive intermediates that form covalent adducts with the interacting protein targets. We are currently utilizing these reagents to identify novel lipid transport proteins, and to examine the specific interactions that govern lipid-protein binding and activation.

Examination of the Structure and Function of Autotaxin

Autotaxin (ATX) is the terminal enzyme in the biosynthesis for the majority of the bioactive lipid lysophosphatidic acid (LPA) produced in vivo. This LPA is sufficient to explain the cancer promoting effects of ATX. Despite the fact that ATX was first identified in the early 1990's, very little is known about the structure of this important cancer chemotherapeutic enzyme. We have recently partnered with the Parill and Webster groups in our department to critically evaluate the structure and function of ATX via an integrated interdisciplinary approach including protein expression and purification, CD and NMR characterization, photoaffinity labeling and subsequent LC-MS analysis, homology modeling, binary QSAR and database mining, high throughput screening and quantum mechanical modeling. This project is the subject of grant proposals that have been submitted to both NIH and NSF. ATX inhibitor identification and characterization is currently funded by the Elsa Pardee Foundation.

Major Laboratory Instrumentation

• ThermoFisher Scientific LCQ Advantage 3-Dimentional Ion Trap
• ThermoFisher Scientific LTQ-XL Linear Ion Trap
• BioTek Synergy-2 Plate Reader

1. Parrill, A.L., Echols, U., Nguyen, T., Pham, T.T., Hoeglund, A., and Baker, D.L. 2008. Virtual screening approaches for the identification of non-lipid autotaxin inhibitors. Bioorg. Med. Chem. In press.
2. Baker, D.L., Fujiwara, Y., Pigg, K.R., Tsukahara, R., Kobayashi, S., Uchiyama, A., Murakami-Murofushi, K., Koh, E., Bandle, R.W., Byun, H., Bittman, R., Murphy, M., Fiedler A., Mills, G.B., and Tigyi, G., 2006. Carba Analogs of Cyclic Phosphatidic Acid Are Selective Inhibitors of Autotaxin and Cancer Invasion. J Biol. Chem. 281(32):22786-22793.
3. Tsukahara, T., Tsukahara, R., Yasuda, S., Makarova, N., Valentine, W.J., Yuan, H., Baker, D.L., Li, Z., Bittman,R., Parrill, A.L., and Tigyi, G., 2006. Ether Analogs of Lysophosphatidic Acid are Endogenous High-Affinity Partial Agonists of PPARg1. J Biol. Chem. 281(6):3398-3407.
4. Li, Z., Baker, D.L., Tigyi, G., and Bittman, R., 2006. Synthesis of Photoactivatable Analogues of Lysophosphatidic Acid and Covalent Labeling of Plasma Proteins. J Org. Chem. 71(2):629-635.
5. Durgam, G.G., Tsukahara, R., Makarova, N., Fujiwara, Y., Pigg, K.R., Baker, D.L., Sardar, V., Parrill, A.L., Tigyi, G., and Miller, D.D., 2006. Synthesis and pharmacological evaluation of second-generation phosphatidic acid derivatives as lysophosphatidic acid receptor ligands. Bioorg. Med. Chem. Lett. 16(3):633-640.
6. Gududuru, V., Zeng, K., Tsukahara, R., Makarova, N., Fujiwara, Y., Pigg, K.R., Baker, D.L., Tigyi, G., and Miller, D.D., 2006. Identification of Darmstoff analogs as selective agonists and antagonists of lysophosphatidic acid receptors. Bioorg. Med. Chem. Lett. 16(2):451-456.
7. Fujiwara, Y., Sardar, V., Tokumura, A., Baker, D.L., Murakami-Murofushi, K., Parrill, A., and Gabor Tigyi, G., 2005. Identification of residues responsible for ligand recognition and regioisomeric selectivity of LPA receptors expressed in mammalian cells. J Biol. Chem. 280(41):35038-35050.
8. Zhang, C., Baker, D.L., Yasuda, S., Makarova, N., Johnson, L.R., Balazs, L., McIntyre, T.M., Xu, Y., Prestwich, G.D., Byun, H-S., Bittman, R., and Tigyi, G., 2004. Lysophosphatidic acid induces neointima formation through PPAR activation. J. Exp. Med. 199(6): 763-774.
9. Yue, J., Yokoyama, K., Balazs, L., Baker, D.L., Pilquil, C., Brindley, D.N., and Tigyi, G., 2004. Transgenic over expression of LPP1 results in multiple phenotypic abnormalities that are independent of LPA signaling. Cell. Signaling 16, 385-399.
10. Rother, E., Brandl, R., Baker, D.L., Tigyi, G., and Siess, W., 2003. Inhibition of platelet activation induced by lysophosphatidic acid, mildly oxidized LDL and plaque lipid core by subtype-selective antagonists of lysophosphatidic acid receptors. Circulation 108: 741-747.
11. Baker, D.L., Morrison, P., Miller, B., Riely, C.A., Tolley, B., Bonfrer, J.M.G., Westermann, A.M., Moolenaar, W.H., and Tigyi, G.J., 2002. Lack of a Diagnostic Correlation Between Plasma Lysophosphatidic Acid Concentration and Ovarian Cancer. JAMA 287: 3081-3082.
12. Sano, T., Baker, D.L., Wada, A., Yatomi, Y., Kobayashi, T., Igarashi, Y., and Tigyi, G.J., 2002. Multiple mechanisms linked to platelet activation generate lysophosphatidic acid and sphingosine-1-phosphate in blood. J. Biol. Chem. 277:21197-21206.
13. Yokoyama, K., Baker, D.L., Virag, T., Liliom, K., Byun, H., Tigyi, G., and Bittman , R., 2001. Stereochemical properties of lysophosphatidic acid signaling and metabolism. Biochim. Biophys. Acta 1582: 296-309.
14. Sardar, V.M., Bautista, D.L., Fischer, D.J., Yokoyama, K., Nusser, N., Virag, T., Wang, D., Baker, D.L., Tigyi, G., and Parrill, A.L., 2001. Molecular basis for lysophosphatidic acid receptor antagonist selectivity. Biochim. Biophys. Acta 1582:310-318.
15. Fischer, D.J., Nusser, N., Virag, T., Yokoyama, K., Wang, D., Baker, D.L., Bautista, D., Parrill, A.L., and Tigyi, G.J., 2001. Short-chain phosphatidates are subtype-selective antagonists of lysophosphatidic acid receptors. Mol. Pharm. 60:776-784.
16. Baker, D.L., Desiderio, D.M., Miller, D.D., and Tigyi, G.J., 2001. Direct quantitative analysis of lysophosphatidic acid molecular species by stable isotope dilution electrospray ionization liquid chromatography mass spectrometry. Anal. Biochem. 292:287-295.
17. Liliom, K., Sun, G., Bunemann, M., Virag, T., Nusser, Baker, D.L., Wang, D., Fabian, M.J.N., Brandts, B., Bender, K., Eickel, A., Malik, K.U., Miller, D.D., Desiderio, D.M., Tigyi, G., and Pott, L., 2001. Sphingosylphosphorylcholine is a naturally occurring lipid mediator in blood plasma: a possible role in regulating cardiac function via sphingolipid receptors. Biochem. J. 355:189-197.
18. Parrill, A.L., Wang, D., Bautista, D.L., Van Brocklyn, J.R., Lorincz, Z., Fischer, D.J., Baker, D.L., Liliom, K., Spiegel S., and Tigyi, G., 2000. Identification of Edg1 receptor residues that recognize sphingosine 1-phosphate, J. Biol. Chem. 275:39379-39384.
19. Bautista, D.L., Baker, D.L., Wang, D., Fischer, D.J., van Brocklyn, J., Spiegel, S., Tigyi, G., and Parrill, A.L., 2000. Dynamic modeling of EDG1 receptor structural changes induced by site-directed mutations. Journal of Molecular Structure - Theochem. 529:219-224.
20. Baker, D.L., Krol, E.S., Jacobson, N., and Liebler, D.C., 1999. Reactions of β-carotene with cigarette smoke oxidants. Identification of carotenoid oxidation products and evaluation of the prooxidant/antioxidant effect. Chem. Res. Toxicol. 12:535-543.