Progress 01/01/05 to 12/31/05
Outputs
Persistent mycobacteria have recently been shown to adapt to the adverse environment within the inflammatory macrophages by a metabolic shift in its carbon source to C2 substrates generated by beta-oxidation of fatty acids (McKinney et al., Nature). Under these conditions, glycolysis is decreased and the glyoxylate shunt is significantly unregulated to allow anaplerotic maintenance of the TCA cycle leading to net assimilation of carbon via gluconeogenisis. The glyoxylate shunt accomplishes this by converting isocitrate to succinate and glyoxylate by isocitrate lyase (ICL), followed by addition of a molecule of a molecule of acetyl-CoA to glyoxylate to form malate by malate synthase (MS). The requirement of glyoxylate shunt pathway to persistent infection makes ICL and MS attractive targets for drug design. In previous years, we solved the structure of the structure of the apo-form of isocitrate lyase. We have since determined the structure of numerous structures of
substrate/inhibitor structures. Over the past year, we have been involved in a great deal of analysis of these structures, in collaboration with GlaxoSmithKline. Recently, we have substantiated the results of Munoz-Elias et al for an additional role of ICL in TB. We have quantitated the methylisocitrate lyase activity as well as solved the structure of ICL bound with the products of the methylisocitrate lyase reaction, pyruvate and succinate. This new finding further implicates the importance of ICL1 as a drug target for TB. In a complementary project, we are working on the antimalarial drug development focused on inhibition of Plasmodium falciparum fatty acid biosynthesis. We have been researching the enzyme components of type II fatty acid biosynthesis for many years, in a variety of organisms, as they represent outstanding targets for drugs against bacteria. It has been shown that many first-line antibiotics used to treat infections, including tuberculosis, work by inhibiting
bacterial fatty acid synthesis type II (FAS-II) pathways, which are absent in humans. A FAS-II pathway was discovered within the apicoplast in Plasmodium falciparum an essential organelle that has inspired an active drug discovery project. The initial stages of this project focused on the known antibacterial triclosan, an inhibitor of bacterial fatty acid synthesis enzyme enoyl activity against PfENR, a key component of the P. falciparum FAS-II. The project's objective is to identify antimalarial compounds that are highly active specific, and chemically diverse against PfENR, through high-throughput screens of a 50,000 compound library and design of triclosan analogs. Additionally, we have pursued virtual screening methods to generate new leads against PfENR. We have since analyzed crystal structures in the presence of Jacobus Pharmaceutical compounds JPC-2136, JPC-2137, JPC-2141, JPC-2166, JPC-2305, as well as GlaxoSmithKline compounds SB643152, SB533394, SB389656, GSK626808,
GSK626814, GSK660582, GSK508982, and GSK585309.
Impacts
Mycobacterium tuberculosis infections are responsible for one in four avoidable adult deaths in developing countries. While there are a number of effective drugs available for treating tuberculosis (TB), current strategies are greatly complicated by the several months of chemotherapy required to eliminate persistent bacteria. In addition, widespread non-compliance has contributed to the emergence of multidrug-resistant (MDR) and (XDR) TB, extensively drug-resistant strains. There is a clear need for fast acting drugs that are capable of eliminating an infection in just a few weeks. Similarly, an estimated 300-500 million cases of malaria are reported annually, and Plasmodium falciparum is the most common cause of malaria in humans. Most of the effective chemotherapeutics have given rise to drug-resistance with few attractive anti-malarial agents on the horizon; new drugs with novel pharmacological properties must be identified and developed to combat the increasing
threat of malaria.
Publications
- Lee JE, Kim K, Sacchettini JC, Smith CV, Safe S. (2004) Vitamin D-interacting protein 150 (DRIP 150) coactivation of estrogen receptor alpha (ERALPHA) ZT-75 breast cancer cells is independent of LXXLL motifs. J Biol Chem.
- Xu M, Arulandu A, Struck DK, Swanson S, Sacchettini JC, Young R. (2005) Disulfide Isomerization After Membrane Release of Its SAR Domain Activates P1 Lysozyme. Science Vol 307 Jan. 7, 2005
- Crocetti E, Sacchettini JC, Caldarella A, Paci E. 2005.[Automatic coding of pathologic cancer variables by the search of strings of text in the pathology reports. The experience of the Tuscany Cancer Registry] Epidemiol Prev. Jan-Feb;29(1):57-60. Italian. PMID: 15948654 [PubMed - indexed for MEDLINE]
- Vilcheze C, Weisbrod TR, Chen B, Kremer L, Hazbon MH, Wang F, Alland D, Sacchettini JC, Jacobs WR Jr. 2005. Altered NADH/NAD+ ratio mediates coresistance to isoniazid and ethionamide in mycobacteria. Antimicrob Agents Chemother. Feb;49(2):708-20.
- Sridharan S, Razvi A, Scholtz JM, Sacchettini JC. 2005. The HPr proteins from the thermophile Bacillus stearothermophilus can form domain-swapped dimers. Journal of Molecular Biology. Feb 25;346(3):919-31.
- Johnson SM, Petrassi HM, Palaninathan SK, Mohamedmohaideen NN, Purkey HE, Nichols C, Chiang KP, Walkup T, Sacchettini JC, Sharpless KB, Kelly JW. 2005. Bisaryloxime ethers as potent inhibitors of transthyretin amyloid fibril formation. Journal of Medial Chemistry. Mar 10;48(5):1576-87.
- Colangeli R, Helb D, Sridharan S, Sun J, Varma-Basil M, Hazbon MH, Harbacheuski R, Megjugorac NJ, Jacobs WR Jr, Holzenburg A, Sacchettini JC, Alland D. 2005. The Mycobacterium tuberculosis iniA gene is essential for activity of an efflux pump that confers drug tolerance to both isoniazid and ethambutol. Molecular Microbiology. Mar;55(6):1829-40.
- Dey S, Hu Z, Xu XL, Sacchettini JC, Grant GA. 2005. -3-Phosphoglycerate dehydrogenase from Mycobacterium tuberculosis is a link between the Escherichia coli and mammalian enzymes. Journal of Biological Chemistry. Apr 15;280(15):14884-91.
- Dey S, Grant GA, Sacchettini JC. 2005. Crystal structure of Mycobacterium tuberculosis D-3-phosphoglycerate dehydrogenase: Journal of Biological Chemistry. Apr 15;280(15):14892-9.
- Gopal K, Romo TD, Sacchettini JC, Ioerger TR. 2005. Determining relevant features to recognize electron density patterns in x-ray protein crystallography. Journal of Bioinformatics Comput Biol. Jun;3(3):645-76.
- Gao T, Zhang X, Xia Y, Cho Y, Sacchettini JC, Golden SS, Liwang AC. 2005. 1H, 13C and 15N chemical shift assignments of the C-terminal, 133-residue pseudo-receiver domain of circadian input kinase (CikA) in Synechococcus elongatus. Journal of Biomolecular NMR. Jul;32(3):259.
- Gokulan K, Khare S, Ronning DR, Linthicum SD, Sacchettini JC, Rupp B. 2005. Cocrystal structures of NC6.8 Fab identify key interactions for high potency sweetener recognition: implications for the design of synthetic sweeteners. Biochemistry. Jul 26;44(29):9889-98.
- Brown AK, Sridharan S, Kremer L, Lindenberg S, Dover LG, Sacchettini JC, Besra GS. 2005. Probing the mechanism of the Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein synthase III mtFabH: factors influencing catalysis and substrate specificity. Journal of Biological Chemistry 2005 Sep 16;280(37):32539-47.
- McKee EW, Kanbi LD, Childs KL, Grosse-Kunstleve RW, Adams PD, Sacchettini JC, Ioerger TR. 2005. FINDMOL: automated identification of macromolecules in electron-density maps. Acta Crystallogr D Biol Crystallogr Nov;61(Pt 11):1514-20.
- Bhatt A, Kremer L, Dai AZ, Sacchettini JC, Jacobs WR Jr. 2005. Conditional depletion of KasA, a key enzyme of mycolic acid biosynthesis, leads to mycobacterial cell lysis. Journal of Bacteriology Nov;187(22):7596-606.
- Paricharttanakul NM, Ye S, Menefee AL, Javid-Majd F, Sacchettini JC, Reinhart GD. 2005. Kinetic and Structural Characterization of Phosphofructokinase from Lactobacillus bulgaricus.Biochemistry. Nov 22;44(46):15280-6.
- Freundlich JS, Anderson JW, Sarantakis D, Shieh HM, Yu M, Valderramos JC, Lucumi E, Kuo M, Jacobs WR Jr, Fidock DA, Schiehser GA, Jacobus DP, Sacchettini JC. 2005. Synthesis, biological activity, and X-ray crystal structural analysis of diaryl ether inhibitors of malarial enoyl acyl carrier protein reductase. Part 1: 4'-substituted triclosan derivatives. Bioorganic & Medicinal Chemistry Letters. Dec 1;15(23):5247-52.
- Trevino SR, Gokulan K, Newsom S, Thurlkill RL, Shaw KL, Mitkevich VA, Makarov AA, Sacchettini JC, Scholtz JM, Pace CN. 2005. Asp79 Makes a Large, Unfavorable Contribution to the Stability of RNase Sa. Journal of Molecular Biology Dec 9;354(4):967-78.
- Gaffin RD, Gokulan K, Sacchettini JC, Hewett TE, Klevitsky R, Robbins J, Sarin V, Zawieja DC, Meininger GA,Muthuchamy M. 2006. Changes in the End-to-End Interactions of Tropomyosin Affect Mouse Cardiac Muscle Dynamics.Am J Physiol Heart Circ Physiol. Feb 24; [Epub ahead of print PMID:16501024 [PubMed - as supplied by publisher]
- Freundlich JS, Yu M, Lucumi E, Kuo M, Tsai HC, Valderramos JC, Karagyozov L, Jacobs, WR Jr, Schiehser GA, Fidock DA, Jacobus DP, Sacchettini JC. 2006. Synthesis and biological activity of diaryl ether inhibitors of malarial enoyl acyl carrier protein reductase. Part 2: 2'-Substituted triclosan derivatives. Bioorg Med Chem Lett. Feb 6; [Epub ahead of print] PMID: 16466916 [PubMed - as supplied by publisher]
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Progress 01/01/04 to 12/31/04
Outputs
Persistent mycobacteria have recently been shown to adapt to the adverse environment within the inflammatory macrophages by a metabolic shift in its carbon source to C2 substrates generated by beta-oxidation of fatty acids (McKinney et al., Nature). Under these conditions, glycolysis is decreased and the glyoxylate shunt is significantly upregulated to allow anaplerotic maintenance of the TCA cycle leading to net assimilation of carbon via gluconeogenesis. The glyoxylate shunt accomplishes this by converting isocitrate to succinate and glyoxylate by isocitrate lyase (ICL), followed by addition of a molecule of acetyl-CoA to glyoxylate to form malate by malate synthase (MS). The requirement of glyoxylate shunt pathway to persistent infection makes ICL and MS attractive targets for drug design. In previous years, we solved the structure of the structure of the apo-form of isocitrate lyase. We have since determined the structure of numerous structures of
substrate/inhibitor structures. Over the past year, we have been involved in a great deal of analysis of these structures, in collaboration with GlaxoSmithKline. In a complementary project, we are working on the antimalarial drug development focused on inhibition of Plasmodium falciparum fatty acid biosynthesis. We have been studying the enzymes of fatty acid biosynthesis for many years as they represent outstanding targets for drugs against bacteria. It has been shown that many first-line antibiotics used to treat infections, including tuberculosis, work by inhibiting bacterial fatty acid synthesis type II (FAS-II) pathways, which are absent in humans. A FAS-II pathway was discovered in Plasmodium falciparum has lead to an active drug discovery project. Early stages of this project focused on the known antibacterial triclosan, an inhibitor of bacterial fatty acid synthesis enzyme enoyl ACP reductase (ENR) that also shows activity against PfENR, a key component of the P. falciparum
FAS-II system. The project's objective is to identify antimalarial compounds that are highly active and specific against PfENR, through high-throughput screens of a 50,000 compound library and design of triclosan analogs. We have since analyzed crystal structures in the presence of Jacobus Pharmaceutical compounds JPC-2136, JPC-2137, JPC-2141, JPC-2166, JPC-2305, as well as GlaxoSmithKline compounds SB643152, SB533394, SB389656, GSK626808, GSK626814, GSK660582, GSK508982, and GSK585309.
Impacts
The research is aimed at developing new drugs against persistent Mycobacterium tuberculosis. Drugs from this work will have the potential to significantly reduce the duration of chemotherapy which would have a great impact on TB worldwide
Publications
- Lee JE, Kim K, Sacchettini JC, Smith CV, Safe S. (2004) Vitamin D-interacting protein 150 (DRIP150) coactivation of estrogen receptor alpha (ERalpha ) in ZR-75 breast cancer cells is independent of LXXLL motifs. J Biol Chem.
- Purkey HE, Palaninathan SK, Kent KC, Smith C, Safe SH, Sacchettini JC, Kelly JW. (2004) Hydroxylated Polychlorinated Biphenyls Selectively Bind Transthyretin in Blood and Inhibit Amyloidogenesis: Rationalizing Rodent PCB Toxicity.Chem Biol. 2004 Dec;11(12):1719-28.
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Progress 03/09/98 to 03/08/04
Outputs
We have focused on the structural analysis and inhibitor identification of potential drug targets from Mycobacterium tuberculosis and Plasmodium falciparum, the deadliest of the human pathogens. Over the past year we concentrated on developing a drug target list and fine tuning our high throughput structure determinating pipeline. We believe that our target list represents the best protein drug targets for persistent TB. This list is available on our website (www.webTB.org) Target proteins are currently being put into our structure determination pipeline. We have also implemented a computer Grid based virtual screening computer that is producing astonishing results. The protein production and crystallization pipeline is functioning to produce diffraction quality crystals of many of these targeted proteins. The virtual screening facilities are being readied for use on the target protein list. For functional characterization, we have implemented computational, genetic,
and biochemical methods to characterize proteins of unknown function, identify novel inhibitors, and validate targets. It is clear that the results of our reseach program project will continue to increase our knowledge of Mtb pathogenisis, particularly regarding persistence, and lead to the discovery of chemical inhibitors of drug targets for future development. Major advances during this project's duration have been: 1) We have a new public website at http://www.webTB.org that provides exhaustive and fully searchable information on all Mtb proteins and genes and that shows detailed status information on research issues. Our previous web site was accessed over 100,000 times in 2006; it is consistently the #1 or #2 Google hit when searching the phrase structural genomics. We expect that the new website will become even more popular. 2) We have completed cloning of the entire M. tuberculosis genome. These Gateway entry clones are being readied for distribution from Colorado State
University. 3) We have a drug target list that we believe represents the most up-to-date information on the top persistence TB drug targets. The target list has been approved by the PI's and is now on the website. Allocation of the targets to the individual PI labs is underway. 4) We have completed 52 new M. tuberculosis structures in 2006, including the protein of unknown function Rv1404 and Rv2844, as well as the drug target HtraA2, PtpB and ATP sylfurylase.
Impacts
Mycobacterium tuberculosis infections are responsible for one in four avoidable adult deaths in developing countries. While there are a number of effective drugs available for treating tuberculosis (TB), current strategies are greatly complicated by the several months of chemotherapy required to eliminate persistent bacteria. In addition, widespread non-compliance has contributed to the emergence of multidrug-resistant (MDR) and (XDR) TB, extensively drug-resistant strains. There is a clear need for fast acting drugs that are capable of eliminating an infection in just a few weeks. Similarly, an estimated 300-500 million cases of malaria are reported annually, and Plasmodium falciparum is the most common cause of malaria in humans. Most of the effective chemotherapeutics have given rise to drug-resistance with few attractive anti-malarial agents on the horizon; new drugs with novel pharmacological properties must be identified and developed to combat the increasing
threat of malaria.
Publications
- Sridharan, S., Wang, L., Brown, A. K., Dover, L. G., Kremer, L., Besra, G. S., and Sacchettini, J. C. (2007) X-ray crystal structure of Mycobacterium tuberculosis beta-ketoacyl acyl carrier protein synthase II (mtKasB), Journal of molecular biology 366, 469-480.
- Vilcheze, C., Wang, F., Arai, M., Hazbon, M. H., Colangeli, R., Kremer, L., Weisbrod, T. R., Alland, D., Sacchettini, J. C., and Jacobs, W. R., Jr. (2006) Transfer of a point mutation in Mycobacterium tuberculosis inhA resolves the target of isoniazid, Nature medicine 12, 1027-1029.
- Wang, F., Cassidy, C., and Sacchettini, J. C. (2006) Crystal structure and activity studies of the Mycobacterium tuberculosis beta-lactamase reveal its critical role in resistance to beta-lactam antibiotics, Antimicrobial agents and chemotherapy 50, 2762-2771.
- Zhao, X., Yu, H., Yu, S., Wang, F., Sacchettini, J. C., and Magliozzo, R. S. (2006) Hydrogen peroxide-mediated isoniazid activation catalyzed by Mycobacterium.
- Gould, T. A., van de Langemheen, H., Munoz-Elias, E. J., McKinney, J. D., and Sacchettini, J. C. (2006) Dual role of isocitrate lyase 1 in the glyoxylate and methylcitrate cycles in Mycobacterium tuberculosis, Molecular microbiology 61, 940-947.
- Morbidoni, H. R., Vilcheze, C., Kremer, L., Bittman, R., Sacchettini, J. C., and Jacobs, W. R., Jr. (2006) Dual inhibition of mycobacterial fatty acid biosynthesis and degradation by 2-alkynoic acids, Chemistry & biology 13, 297-307.
- Owens, R. M., Hsu, F. F., VanderVen, B. C., Purdy, G. E., Hesteande, E., Giannakas, P., Sacchettini, J. C., McKinney, J. D., Hill, P. J., Belisle, J. T., Butcher, B. A., Pethe, K., and Russell, D. G. (2006) M. tuberculosis Rv2252 encodes a diacylglycerol kinase involved in the biosynthesis of phosphatidylinositol mannosides (PIMs), Molecular microbiology 60, 1152-1163.
- Romo, T. D., Sacchettini, J. C., and Ioerger, T. R. (2006) Improving amino-acid identification, fit and C(alpha) prediction using the Simplex method in automated model building, Acta crystallographica 62, 1401-1406.
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Progress 01/01/03 to 12/31/03
Outputs
Project 1 - Drug Discovery for Persistent Tuberculosis The adaptation of Mycobacterium tuberculosis to the adverse environment within inflammatory macrophages to respire on C2 substrates generated by b-oxidation of fatty acids and the glyoxylate shunt is now well established. In fact, several other intracellular pathogens have now been shown to also rely on their glyoxylate shunt for virulence. Therefore, the enzymes of the glyoxylate shunt which convert isocitrate to succinate and glyoxylate by the enzyme isocitrate lyase (ICL), followed by addition of a molecule of acetyl-CoA to glyoxylate to form malate by malate synthase (MS) represent excellent drug targets for persistent MTB. Our focus has been on developing ICL and MS as targets for drug discovery. The structure of the apo-form of isocitrate lyase has been determined as well as the structure of malate synthase complexed with relevant substrates/inhibitors. We are now analyzing the results of a high throughput
screen performed in collaboration with GlaxoSmithKline. Project 2-Antimalarial drug development focused on inhibition of Plasmodium falciparum fatty acid biosynthesis It has been known for many years that the enzymes of fatty acid biosynthesis represent outstanding targets for drugs against bacteria. Indeed, several first-line antibiotics used to treat infections, including Tuberculosis, work by inhibiting bacterial fatty acid synthesis type II pathways, which are absent in humans. The discovery that Plasmodium falciparum used a FASII system for fatty acid biosynthesis has opened the door to developing antimalarial drugs that are specific to this pathway. This FASII project began with the known antibacterial triclosan, an inhibitor of bacterial fatty acid synthesis enzyme enoyl ACP reductase (ENR) that also shows activity against PfENR, a key component of the P. falciparum FASII system. The project's objective is to identify antimalarial compounds that are highly active and specific
against PfENR. Crystals of PfENR in complex with either JPC-2001-A1 or isoniazid (INH), an inhibitor that targets the enoyl-ACP reductase from M. tuberculosis, were produced via vapor diffusion hanging drop experiments. Crystallization experiments were conducted through incubation of PfENR, NAD, and inhibitor, in a 1:3:3 molar ratio. The crystal structure of PfENR in complex with isoniazid, an inhibitor that targets the enoyl-ACP reductase from M. tuberculosis, has been determined to 2.5 A resolution. Isoniazid covalently attaches to the NADH cofactor, in a site distinct from triclosan binding. The isoniazid portion of the INH-NADH adduct ring stacks with Tyr267, unlike triclosan which interacts with Tyr277. This ring stacking interaction provides another possibility for increasing the potency of triclosan analogs. Studies are underway to solve the structure of PfENR with JPC-2001-A1 and several other inhibitors.
Impacts
The research is aimed at developing new drugs against persistent Mycobacterium tuberculosis. Drugs from this work will have the potential to significantly reduce the duration of chemotherapy which would have a great impact on TB worldwide
Publications
- Reddy, V., Swanson, S. M., Segelke, B., Kantardjieff, K. A., Sacchettini, J. C., Rupp, B. (2003) Effective electron-density map improvement and structure validation on a Linux multi-CPU web cluster: The TB Structural Genomics Consortium Bias Removal Web Service. Acta Crystallographica 59, 2200-2210.
- Adams, P. D., Gopal, K., Grosse-Kunstleve, R. W., Hung, L. W., Ioerger, T. R., McCoy, A. J., Moriarty, N. W., Pai, R. K., Read, R. J., Romo, T. D., Sacchettini, J. C., Sauter, N. K., Storoni, L. C., Terwilliger, T. C. (2003) Recent developments in the PHENIX software for automated crystallographic structure determination. Journal of Synchrotron Radiation 11, 53-55.
- Cho, Y., Sharma, V. and Sacchettini, J. C. (2003) Crystal structure of ATP Phosphoribosyltransferase from Mycobacterium tuberculosis. Journal of Biological Chemistry, 278, 8333-8339.
- Zhang, F., Lucke, C., Baier, L. J., Sacchettini, J. C. and Hamilton, J. A. (2003) Solution structure of human intestinal fatty acid binding protein with a naturally-occurring single amino acid substitution (A54T) that is associated with altered lipid metabolism. Biochemistry 42, 7339-7347.
- Smith, C. V., Huang, C. C., Miczak, A., Russell, D. G., Sacchettini, J. C., Honer zu Bentrup, K. (2003) Biochemical and structural studies of malate synthase from Mycobacterium tuberculosis. Journal of Biological Chemistry 278, 1735-1743.
- Gokulan, K. Rupp, B., Martin S. Pavelka, Jr., M. S., Jacobs Jr., W. R., and Sacchettini, J. C. (2003) Crystal Structure of Mycobacterium tuberculosis Diaminopimelate Decarboxylase, an Essential Enzyme in Bacterial Lysine Biosynthesis. Journal of Biological Chemistry 278, 18588-18596.
- Sharma, V., Arockiasamy, A., Ronning, D.R., Savva, C. G., Holzenburg, A., Braunstein, M., Jacobs, Jr., W. R. and Sacchettini, J. C. (2003) Crystal structure of M. tuberculosis SecA, a preprotein translocating ATPase. Proceedings of the National Academy of Science U.S.A. 100, 2243-2248.
- Kuo, M. R., Morbidoni, H. R., Alland, D., Sneddon, S. F., Gourlie, B. B., Staveski, M. M., Leonard, M., Gregory, J. S., Janjigian, A. D., Yee, C., Kreiswirth, B., Iwamoto, H., Perozzo, R., Jacobs, Jr., W. R., Sacchettini, J. C. and Fidock, D. A. (2003) Targeting tuberculosis and malaria through inhibition of enoyl reductase: compound activity and structural data. Journal of Biological Chemistry 278, 20851-20859.
- Takano, K., Scholtz, J.M., Sacchettini, J.C., Pace, C.N. (2003) The contribution of polar group burial to protein stability is strongly context dependent. Journal of Biological Chemistry 279, 31790-31795.
- Kremer, L., Dover, L.G., Morbidoni, H.R., Vilcheze, C., Maughan, W.N., Baulard, A., Tu, S.C., Honore, N., Deretic, V., Sacchettini, J.C., Locht, C., Jacobs, W. R., Jr, Besra, G.S. (2003) Inhibition of InhA activity, but not KasA activity, induces formation of a KasA-containing complex in mycobacteria. Journal of Biological Chemistry 278, 20547-20554.
- Zhang, F., Lucke, C., Baier, L. J., Sacchettini, J. C., Hamilton, J. A. (2003) Solution structure of human intestinal fatty acid binding protein with a naturally-occurring single amino acid substitution (A54T) that is associated with altered lipid metabolism. Biochemistry 42, 7339-7347.
- Terwilliger, T. C., Park, M. S. Waldo, G. S., Berendzen, J. Hung, L. W., Kim, C. Y., Smith, C. B. Sacchettini, J. C., Bellinzone, M., Bossi, R., DeRossi, E., Mattevi, A., Milano, A., Riccardi, G., Rizzi, M., Roberts, M. M., Coder, A. R., Fossate, G., Mascagni, P., Coates, A. R., Wood, S. P., Goulding, C. W., Apostol, M. I., Anderson, D. H., Gill, H. S., Eisenberg, D. S., Taneja, B., Mande, S., Pohl, E., Lamzin, V., Tucker, P., Wilmanns, M., Colovos, C., Meyer-Klaucke, W., Munro, A. W., McLean, K. J., Marshall, K. R., Leys, D., Yang, J. K., Yoon, H. J., Lee, B I., Lee, M. G., Kwak, J. E., Han, B. W., Lee, J. Y., Baek, S. H., Suh, S. W., Komen, M. M., Arcus, V. L., Baker, E. N., Lott, J. S., Jacobs, W., Alber, T., Rupp, B. (2003) The TB structural genoimcs consortium: a resource for Mycobacterium tuberculosis biology. Tuberculosis 83, 223-249.
- Eicken, C., Pennella, M. A., Chen, X., Koshlap, K. M., VanZile, M. L., Sacchettini, J. C., Giedroc, D. P. (2003) A metal-ligand-mediated intersubunit allosteric switch in related SmtB/ArsR zinc sensor proteins. Journal of Molecular Biology 333, 683-695.
- Razavi, H., Palaninathan, S. K., Powers, E. T., Wiseman, R. L., Purkey, H. E., Mohamedmohaideen, N. N., Deechongkit, S., Chiang, K. P., Dendle, M. T. A., Sacchettini, J. C., Kelly, J. W. (2003) Benzoxazoles as Transthyretin Amyloid Fibril Inhibitors: Synthesis, Evaluation, and Mechanism of Action. Angewandte Chemie International Edition 42, 2758-2761.
- Green, N. S., Palaninathan, S. K., Sacchettini, J. C., Kelly, J. W. (2003) Synthesis and characterization of potent bivalent amyloidosis inhibitors that bind prior to transthyretin tetramerization. Journal of the American Chemical Society 125, 13404-13414.
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Progress 01/01/02 to 12/31/02
Outputs
The Sacchettini laboratory has solved several new protein structures this year primarily in lipid and amino acid metabolism. However, underpinning our target selection is that targets will have some relevance to virulence and persistence of TB, host immune response, drug discovery and vaccine development. Our longstanding interest in enzymes of lipid metabolism and mycobacterial persistence converged in the project on mycolic acid methyl transferases. These enzymes catalyze modifications of cell wall mycolic acids essential in TB. One member of this family has been shown to be important in maintaining a chronic infection. We have overexpressed 7 of the 8 members of this mycolic acid methyltransferase family from M. tuberculosis, crystallized 5 and completed and reported the structures of 3. In the coming year we will work to solve all 8 structures as a basis to study the importance of these enzymes in virulence, and begin to focus on other enzymes in this important
pathway. We have also focused on enzymes of the histidine biosynthetic pathway. This work is linked with our long term interest in nucleotide synthesis, which is an important component of histidine synthesis. We have focused on 2 enzymes in this essential pathway HisG and HisI, a cyclase and a PRTase, the first and third enzymes in the pathway. HisG is a well-studied enzyme which can be inhibited to block biosynthesis of histidine. While we have yet to demonstrate that this pathway is made up of enzymes that represent good drug targets, there is every expectation that this will be the case. The structures of both enzymes are nearly complete.
Impacts
The research is aimed at developing new drugs against persistent Mycobacterium tuberculosis. Drugs from this work will have the potential to significantly reduce the duration of chemotherapy which would have a great impact on TB worldwide.
Publications
- Yang, D., Shipman, L. W., Roessner, C. A., Scott, A. I. and Sacchettini, J. C. (2002) Structure of the Methanococcus jannaschii mevalonate kinase a member of the GHMP kinase superfamily. Journal of Biological Chemistry 277, 9462-9467.
- Huang, C.C., Smith, C. V., Glickman, M., Jacobs, Jr., W. R., and Sacchettini, J. C. (2002) Crystal structures of mycolic acid cyclopropane synthases from Mycobacterium tuberculosis. Journal of Biological Chemistry 277, 11559-11569.
- Sacchettini, J. C. and Kelly, J. W. (2002) Therapeutic strategies for human amyloid disease. Nature Reviews Drug Discovery 1, 267-275.
- Perozzo, R., Kuo, M., bir Singh Sidhu, A., Valiyaveettil, J.T., Bittman, R., Jacobs, Jr., W. R., Fidock, D.A. and Sacchettini, J.C. (2002) Structural elucidation of the specificity of the antibacterial agent triclosan for malarial enoyl ACP reductase. Journal of Biological Chemistry 277, 13106-13114.
- Eicken, C., Sharma, V., Klabunde, T., Lawrenz, M. B., Hardham, J. M., Norris, S. J. and Sacchettini, J. C. (2002) Crystal structure of the variable surface antigen VlsE of Borrelia burgdorferi. Journal of Biological Chemistry 277, 21691-21696.
- Riley-Lovingshimer, M. R., Ronning, D. R., Sacchettini, J. C. and Reinhart, G. R. (2002) Reversible Ligand-Induced Dissociation of a Tryptophan-Shift Mutant of Phosphofructokinase from Bacillus stearothermophilus. Biochemistry 41, 12967-12974.
- Ioerger, T. R. and Sacchettini, J. C. (2002) The TEXTAL system: Artificial intelligence techniques for automated protein model-building. Methods in Enzymology, in press.
- Larsen, M. H., Vilcheze, C., Kremer, L., Besra, G. S., Parsons, L., Salfinger, M., Heifets, L., Hazbon, M. H., Alland, D., Sacchettini, J. C. and Jacobs, Jr., W. R. (2002) Overexpression of inhA, but not kasA, confers resistance to Isoniazid and Ethionamide in Mycobacterium smegmatis, M. bovis BCG and M. tuberculosis. Molecular Microbiology 46, 453-466.
- Zhang, F., Lucke, C., Baier, L. J., Sacchettini, J. C. and Hamilton, J. A. (2002) NMR structure of the threonine 54 substituted human intestinal fatty acid binding protein: implications for altered biological activity. Submitted to Journal of Biological Chemistry.
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Progress 01/01/01 to 12/31/01
Outputs
Persistent mycobacteria have recently been shown to adapt to the adverse environment within the inflammatory macrophages by a metabolic shift in its carbon source to C2 substrates generated by beta-oxidation of fatty acids. Under these conditions, glycolysis is decreased and the glyoxylate shunt is significantly upregulated to allow anaplerotic maintenance of the TCA cycle leading to net assimilation of carbon via gluconeogenesis. The glyoxylate shunt accomplishes this by converting isocitrate to succinate and glyoxylate by isocitrate lyase, followed by addition of a molecule of acetyl-CoA to glyoxylate to form malate by malate synthase. The requirement of glyoxylate shunt pathway to persistent infection makes isocitrate lyase and malate synthase attractive targets for drug design. Isocitrate lyase was crystallized with the inhibitors 3-nitropropionate or 3-bromopyruvate. The structure of the ternary complex of rotein (C191S mutant) with 3-nitropropionate and
glyoxylate was determined at a resolution of 2.25A using molecular replacement techniques. Clear density was observed for glyoxylate, 3-nitropropionate and Mg2+ in NCS-averaged difference maps contoured at 2 sigma level. The planar glyoxylate was modeled such that its coordinates to a Mg2+ ion. The inhibitor 3-nitropropionate occupies the succinate-binding pocket and adopts a conformation that allows most catalytic residues to be ideally placed for the C-C bond formation. Inhibition of isocitrate lyase by 3-bromopyruvate is accomplished via dehalogenation of the inhibitor to form a covalent adduct with active site nucleophile, Cys191. The pyruvyl moiety occupies the site where the second carboxylate of succinate was located, however, the orientation of the carboxylate group differs in the two cases perhaps as a result of the covalent linkage with Cys191 in the case of 3-bromopyruvate modified isocitrate lysase. Both inhibitor complexes trap the enzyme in a catalytic conformation with
the active site completely inaccessible to solvent. Structure-based improvement of these inhibitors should yield antimycobacterials effective against an essential metabolic phase in the infection cycle of these bacteria. We have determined the 3-D structure of malate synthase - the second enzyme of the glyoxylate shunt pathway. The gene encoding malate synthate was identified from the TB genome and subsequently cloned and expressed in E. coli. The purified protein crystallized and the structure has been solved using multiple anomalous dispersion techniques using crystals of enzyme obtained after incorporation of selenomethionine. The current structure has been refined to a high resolution of 2.1A with bound substrates. We now have structures of ICL and MS with bound substrates and inhibitors. This work is part of a larger collaboration between scientists of the program project and Glaxo-SmithKline in the U.K. Glaxo has provided us with a number of inhibitors from a directed screen
against the enzymes. In addition, we have screened several databases of available chemicals that resemble substrates and have identified 2 new inhibitors. We have analyzed these inhibitors structurally and are studying TB activity.
Impacts
The research is aimed at developing new drugs against persistent Mycobacterium tuberculosis. Drugs from this work will have the potential to significantly reduce the duration of chemotherapy which would have a great impact on TB worldwide.
Publications
- Eicken, C., Sharma, V., Klabunde, T., Owens, R. T., Pikas, D. S., Hook, M. and Sacchettini, J. C. (2001) Crystal structure of Lyme disease antigen outer surface protein C from Borrelia burgdorferi. Journal of Biological Chemistry 276, 10010-10015.
- Sacchettini, J. C., Baum, L. G. and Brewer, C. F. (2001) Multivalent protein-carbohydrate interactions. A new paradigm for supermolecular assembly and signal transduction. Biochemistry 40, 3009-3015.
- Thomson, C. T., Kalergis, A. M., Sacchettini, J. C. and Nathenson, S. G. (2001) A structural difference limited to one residue of the antigenic peptide can profoundly alter the biological outcome of the TCR/MHC-peptide interaction. Journal of Immunology 166, 3994-3997.
- Shipman, L. W., Li, D., Roessner, C. A., Scott, A. I. and Sacchettini, J. C. (2001) Crystal structure of precorrin-8x methyl mutase. Structure 9, 587-596.
- Jiang, X., Smith, C. S., Petrassi, H. M., Hammarstrom, P., White, J. T., Sacchettini, J. C. and Kelly, J. W. (2001) An engineered transthyretin monomer that is nonamyloidogenic, unless it is partially denatured. Biochemistry 40, 11442-11452.
- Holmes, J.H., 4th, Lieberman, J.M., Probert, C.B., Marks, W.H., Hill, M.E., Paull, D.L., Guyton, S.W., Sacchettini, J. and Hall, R.A. (2001) Elevated intestinal fatty acid binding protein and gastrointestinal complications following cardiopulmonary bypass: a preliminary analysis. Journal of Surgical Research 100, 192-196.
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Progress 01/01/00 to 12/31/00
Outputs
Most of our efforts over the past year have been on preparing recombinant expression systems for large scale production and subsequent crystallization of perforin, granulysin, and the human CD1b and CD1c proteins. These proteins are inherently difficult to produce and is most likely the reason why we have no structural information and little biochemical information on any of these proteins. The human CD1 system consists of five nonpolymorphic genes CD1A, CD1B, CD1C, CD1D and CD1E, four of which, CD1A-D, encode functional proteins. There is significant sequence homology between CD1 and MHC I proteins. Several CD1 proteins have been shown to display tuberculosis lipid antigens. No group I CD1 protein structures have been solved yet. Such a structure would help in our understanding of the structural basis of nonpeptide antigen presentation and recognition by the immune system. We have primarily focused on the human CD1b and CD1c protein. CD1c and b2 microglobulin genes
have been cloned into different plasmids and the two proteins overexpressed to high levels as inclusion bodies. Efforts are now underway to fold the complex with antigenic TB lipids provided by our collaborator, Dr. Del Besra, University of New Castle upon Tyne, United Kingdom. Plasmid constructs containing the genes for both the proteins have also been made in order to potentially simplify the production and purification method coupled to our M. smegmatis expression system. Both human and mouse perforin were cloned into a pET vector, and attempts were made at expressing it in E. coli. The protein proved not to express at visible levels, and so a different host was used. The perforins were then cloned into the BacNBlue (A) vector, where it was used for co-transfection into insect Sf9 cells. Cultures were grown, harvested, and perforin products confirmed on SDS-PAGE with Western blot analysis. The protein was produced at low levels so that it would not be time/cost effective to purify
in this manner for crystallographic studies. Work has begun on producing a PFN gene lacking the amino terminal signal sequence in order to determine if this protein expresses efficiently. Aside from using a different host system for expression, we also incorporated a chaperone protein to improve the yield of PFN from E. coli. The gene for calreticulin was put into a cloning vector. Attempts are underway to place it into an expression vector, and to see if co-expression of PFN and calreticulin will improve protein yield. PCR screens were performed on an activated T cell library for a copy of the 500 bp gene that codes for human granulysin. PCRs have not been successful to date so we are try a different protocol for isolating genes from a phage library.
Impacts
(N/A)
Publications
- O'Hagan, R. C., Schreiber-Agus, N., Chen, K., David, G., Engelman, J. A., Schwab, R., Alland, L., Thomson, C., Ronning, D. R., Sacchettini, J. C., Meltzer, P. and DePinho, R. A. 2000. Gene-target recognition among members of the myc superfamily and implications for oncogenesis. Nature Genetics 24, 113-119.
- Wolfrum, C., Borchers, T., Sacchettini, J. C. and Spener, F. 2000. Binding of fatty acids and peroxisome proliferators to orthologous fatty acid binding proteins from human, murine, and bovine liver. Biochemistry 39, 1469-1474.
- Duncan, K. and Sacchettini, J. C. 2000. Approaches to tuberculosis drug development. In Molecular Genetics of Mycobacteria (eds Hatfull, G. F. and Jacobs, Jr., W. R.) 297-307 (American Society of Microbiology, Washington, D. C.
- Holton, T., Ioerger, T.R., Christopher, J.A. and Sacchettini, J.C. 2000. Determining protein structure from electron density maps using pattern matching. Acta Crystallographica, 56, 722-734.
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Progress 01/01/99 to 12/31/99
Outputs
Critical elements of specificity in cell mediated immunity are dictated by the interactions between an antigenic peptide, the MHC class I molecule, and a CD8+T cell receptor (TCR). The aims of this application are to use protein crystallography to solve the three-dimensional structures of representatives of each of these key components of the cellular immune response. These structures will enable us to visualize and identify those features of MHC class I molecules involved in selecting and capturing peptides for presentation as studies will be combined through collaborations in genetics, immunology and molecular biology to provide a more thorough understanding of the class I mediated cellular immune response. The studies proposed in this application will emply x-ray crystallography to study antigen presentation. Our structural studies will address the selection and binding of foreign peptide epitopes by the class I major histocompatibility complex (MHC) as well as the
MHC-peptide complex recognition by the T-cell receptor (TCR). Specifically, we aim to use three-dimensional structural analysis in a continued effort to understand the role of murine class I molecules in antigen presentation and TCR recognition. Our goals are to understand at a molecular level the interactions which dictate peptide binding and presentation by MHCs and to identify, using x-ray crystallography, the features of the T-cell receptor involved in recognizing these peptides in the context of the MHC. Protein crystallography is being used to understand the structural basis of the cellular immune response. This project studies the interactions between proteins and their ligands or substrates.
Impacts
(N/A)
Publications
- Lucke, C., Fushman, D., Ludwig, C., Hamilton, J.A., Sacchettini, J.C. and Ruterjans, H. (1999) A comparative study of the backbone dynamics of two closely related lipid binding proteins: bovine heart fatty acid binding protein and porcine ileal lipid binding protein. Mol. Cell. Biochem. 192, 109-121.
- Balendiran, G.K., Molina, J.A., Xu, Y., Torres-Martinez, J. Stevens, R. Focia, P.J., Eakin, A.E., Sacchettini, J.C. and Craig III, S.P. (1999) Ternary complex structure of human HGPRTase, PRPP, and Mg and the inhibitor HPP reveals the involvement of the flexible loop in substrate binding. Prot. Sci. 8, 1023-1031.
- Rozwarski, D.A., Vilcheze, C., Sugantino, M., Bittman, R. and Sacchettini, J.C. (1999) Crystal structure of the Mycobacterium tuberculosis enoyl-ACP reductase (InhA) in complex with NAD and a C16 fatty acyl substrate. J. Biol. Chem. 274, 15582-15589.
- Kurokawa, H., Dewan, J. C., Mikami, B., Sacchettini, J. C. and Hirose, M. (1999) Crystal structure of hen apo-ovotransferrin. J. Biol. Chem. 274, 28445-28452.
- Eicken, C., Krebs, B. and Sacchettini, J. S. (1999) Catechol oxidase structure and activity. Current Opinion in Structural Biology. 9, 677-683.
- Ronning, D. R., Klabunde, T., Sacchettini, J.C., Besra, G.S., Vissa, V.D. and Belisle, J.T. (2000) Crystal structure of the secreted form of antigen 85C reveals potential targets for mycobacterial drugs and vaccines. Nature Structural Biology 7, 141-143.
- Petrassi, H. M., Kladunde, T., Sacchettini, J. C., Kelly, J. W. (2000) Structure-based design of N-phenyl phenoxazine transthyretin amyloid fibril inhibitors. J. Am. Chem. Soc. 122, in press.
- Klabunde, T., Petrassi, H. M., Oza, V. B., Raman, P., Kelly, J. W. and Sacchettini, J. C. (2000) Rational design of potent human transthyretin amyloid disease inhibitors. Nature Structural Biology 7, in press.
- Holton, T., Ioerger, T.R., Christopher, J.A. and Sacchettini, J.C. (1999) TEXTAL: Using pattern matching in determining protein structure from electron density maps. Acta Crystallographica. In press.
- Sacchettini, J. C., Morbidoni, H. R., Fidock, D., Alland, D., Kuo, M. R., Iwamoto, H., Cox, J. S. and Jacobs, Jr., W. R. (1999) Triclosan kills isoniazid-resistant Mycobacterium tuberculosis and chloroquine-resistant Plasmodium falciparum. Nature. Submitted.
- Sharma, V., Sharma, S., Hoener zu Bentrup, K., McKinney, J. D., Russell, D. G., Jacobs, Jr., W. R. and Sacchettini, J. C. (1999) Structure of M. tuberculosis isocitrate lyase: A lynchpin to survival within inflammatory phagocytes. Science. Submitted.
- Hoener zu Bentrup, K., Miczak, A., McKinney, J. D., Jacobs, Jr., W. R., Sacchettini, J. C. and Russell, D. G. (1999) Survival of Mycobacterium tuberculosis in inflammatory macrophages requires the glyoxylate shunt enzyme isocitrate lyase. Science. Submitted.
- Balendiran, G. K., Schnutgen, F., Scapin, G., Borchers, T., Xhong, N., Lim, K., Godbout, R., Spener, F. and Sacchettini, J. C. (2000) Structural and thermodynamic analysis of fatty acid binding to recombinant human brain fatty acid binding protein. In preparation.
- Schnutgen, F., Borchers, T., Xhong, N., Godbout, R., Sacchettini, J.C. and Spener, F. (2000) Human brain-type fatty acid binding protein shows high affinity for w3 fatty acids but not for w6 fatty acids. In preparation.
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Progress 01/01/98 to 12/31/98
Outputs
This project focuses on deriving structural information about proteins of the immune response, using protein crystallography and molecular biology approaches. MHCs, or major histocompatability complexes, are surface proteins which present a large variety of peptides, though certain restrictions on length and sequence, limit this set. The murine class I MHC molecule H-2Db predominantly binds peptides between nine and eleven residues in length, with an asparagine at position five. To examine how H-2D binds longer peptides, we have now determined the structure of the H-2D molecule with an immunodominant, 10 residue peptide derived from the SV40 large T antigen, LT206-215, SAINNYAQKL. MHC molecules in complex with different length peptides demonstrates that these molecules have altered conformations in the helical region of the a 2 subdomain. Our conclusion is that the H-2D molecule imposes a greater degree of structural uniformity on its bound peptides than has
previously been seen. In a second study, the binding of octapeptide VSV52-59, RGYVYQGL, VSV8, derived from the nucleocapsid of the vesicular stomatitis virus to the H-2K molecule was studied. Previous work showed that a variant peptide, RGYVYEGL, E6, with a glutamic acid for glutamine replacement at position 6 of the VSV8 peptide, elicits a number of biological responses that are quite different from those arising from the wild type VSV8 peptide. Our structure reveals that the only significant difference between the H-2K E6 structure and the previously determined H-2K VSV8 is limited to the area of the position 6 of the peptide. Thus, our findings help to confirm that substitutions of peptide residues that result in major alterations of biological functions are not likely to be associated with large changes in the structure of the pMHC.
Impacts
(N/A)
Publications
- Lim, K., Owens, S.M., Arnold, L., Sacchettini, J.C. and Linthicum, D.S., 1998. Crystal structure of monoclonal 6B5 Fab complexed with phencyclidine. J. Biol. Chem., 273, 28576-28582. Degano, M., Almo, S.C., Sacchettini, J.C. and Schramm, V.L., 1998. Trypanosomal nucleoside hydroxylase. A novel mechanism from the structure of a transition-state inhibitor. Biochemistry. 37, 6277-6285.
- Peterson, S.A., Klabunde, T., Lashuel, H.A., Purkey, H., Sacchettini, J.C. and Kelly, J.W., 1998. Inhibiting transthyretin conformational changes that lead to amyloid fibril formation. PNAS, 95, 12956-12960.
- Klabunde, T., Eicken, C., Sacchettini, J.C., Krebs, B., 1998. Crystal structure of a plant catechol oxidase containing a dicopper center. Nature Struct.Biol., 5, 1084-1090.
- Klabunde, T., Sharma, S., Telenti, A., Jacobs, W.R., Jr., and Sacchettini, J.C. , 1998. Crystal structure of GyrA intein from Mycobacterium xenopi reveals structural basis of protein splicing. Nature Struct. Biol., 5, 31-36.
- Rozwarski, D.A., Grant, G.A., Barton, D.H.R., Jacobs, W.R., Jr. and Sacchettini, J.C. , 1998. Modification of the NADH of the isoniazid target, InhA from Mycobacterium tuberculosis. Science, 279, 98-102.
- Wang, J.H., Lim, K., Smolyar, A., Teng, M.K., Liu, J.H., Tse, A., Liu, J., Hussey, R.E., Chishti, Y., Thomson, C.T., Sweet, R.M., Nathenson, S.G., Chang, H.C., Sacchettini, J.C., and Reinherz, R.L. , 1998 Atomic structure of the complex between a T cell receptor, TCR heterodimer and an anti-TCR Fab fragment derived from a mitogenic antibody. EMBO J., 17, 10-26.
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