The Shair Group at Harvard University

Research Overview. The Shair research group is working in two main areas: organic synthesis and chemical biology. Most projects involve syntheses of naturally occurring complex molecules that challenge the state-of-the-art of organic synthesis. We have chosen target molecules that are structurally unique and that have interesting, unstudied biological properties. We choose molecules that are different from other complex molecules that have been synthesized since this enables us to explore new areas of organic chemistry, especially with respect to reactivity and selectivity. We are particularly interested in developing cascade reactions for each of our synthesis targets, in order to achieve the most efficient and rapid syntheses possible.

We are also attracted to molecules that have unique biological properties. Our syntheses, in many cases, are the only means of accessing additional material and designed analogs to uncover the molecule’s cellular target(s) and mechanism(s). We also have a collaboration with Professor Tom Kirchhausen’s lab in the Cell Biology Department at Harvard Medical School on the discovery and use of small molecules to probe vesicular traffic and Golgi organization in cells.

Current Projects:

1. Palau’amine: This marine natural product is interesting to us because it has an unprecedented structure comprising two guanidines, a highly nitrogenous skeleton, and a stereochemically dense array of unusual functionality (for instance a secondary stereocenter bearing a chlorine atom).

Others and we have recognized that the biosynthesis of palau’amine is probably derived from the oxidative dimerization of two molecules of oroidin-like molecules, common precursors of many other marine natural products. We are using this observation and other approaches to develop a synthesis of palau’amine. A longer-term goal of this project is to uncover the cellular target(s) and mechanism of palau’amine. It has been reported that palau’amine potently inhibits the activation of T-lymphocytes in a mixed lymphocyte reaction by an unknown mechanism.

2. Cortistatin A. In 2006, the unique natural product cortistatin A was reported as an extremely potent inhibitor of the migration and proliferation of HUVECs (Human Umbilical Vascular Endothelial Cells); IC50 = 180 pM (VEGF-stimulated HUVECs proliferation). It was also shown that cortistatin A is 3000 times more potent at inhibiting the proliferation of HUVECs versus other cancer and normal cell lines, suggesting that cortistatin A may be a highly selective angiogenesis inhibitor. Cortistatin A has an unprecedented structure, with a particularly challenging [3.2.1]oxabicyclooctene ring system. We are currently working on a distinct metal-catalyzed cascade reaction to construct cortistatin A.

3. Lomaiviticins: Lomaiviticins A and B are marine-derived natural products with unprecedented structures. They are C2-symmetric dimers that comprise two diazoquinonefluorenones and heavily oxidized cyclohexyl rings.

A significant challenge of any synthesis of the lomaiviticins is the C-C bond connecting the two polycyclic units. This C-C bond is part of a 1,4-diketone and links the two units via two stereogenic centers. In addition, the highly oxidized cyclohexyl rings are poised for elimination and aromatization resulting in a significant synthesis challenge. We are currently developing a synthesis of the polycyclic monomers of the lomaiviticns and a strategy for linking them steroselectively to afford lomaiviticns A and B. In addition, we are interested in using our synthesis to explore the potent growth inhibitory activity of these unique molecules. Our synthesis will enable us to construct designed analogs of lomaiviticns A and B to determine the origin of their activities in cells.

4. Cephalostatin, Ritterazine and OSW-1: Cephalostatin 1 and ritterazine B are natural products that were isolated from deep-sea marine organisms in small quantites (<100mg). OSW-1 was isolated from bulbs of the African lily ornithogalum saundersiaein 400mg batches. Each of these compounds are single-digit nanomolar inhibitors of cell growth in the NCI-60 cell line panel; cephalostatin 1 GI50=1.8nM (average of 60 cell lines), ritterazine B GI50=3.2nM (average of 60 cell lines), OSW-1 GI50=0.78nM (average of 60 cell lines). COMPARE analysis between the three natural products suggests that they may have related or identical mechanisms. COMPARE profile of these compounds does not match the profile of any other known anticancer molecules, suggesting they have unique mechanisms.

Our first goal in this project is to develop a short, efficient, and flexible synthesis of cephalostatin 1. One challenge of the synthesis, if a steroid-based starting material is used, is the need for a remote oxidation of an angular methyl group on the left-hand side of the molecule. A second challenge is that the right-hand spiroketal is in a non-thermodynamic configuration requiring a kinetic spiroketalization.

Our second goal of this project is to identify the cellular target(s) and mechanism of cephalostatin 1. The potency, and more importantly importantly, unique mechanism of cephalostatin 1 suggest that by uncovering its cellular target(s) we may discover new approaches to inhibiting the growth of and killing tumor cells selectively. An efficient and flexible synthesis of cephalostatin 1 will allow us to make a range of designed molecules to enable target identification studies.

OSW-1 is a natural product that is structurally unrelated to cephalostatin, but it has been reported that its pattern of cytotoxicity in the NCI-60 cell line panel closely resembles that of cephalostatin 1. We are also trying to elucidate the cellular target(s) and mechanism of OSW-1 to determine whether it is identical to cephalostatin 1. If so, how can structurally distantly related molecules perturb the same target? If they have different targets, why do they have highly similar (and statistically relevant) cytotoxicity patterns? We are using a combination of chemical, biochemical, genetic, and cell biological approaches to identify the cellular target(s) of OSW-1 and cephalostatin 1.

5. Catalytic Enantioselective Enolate-Based Reactions Inspired by Fatty Acid and Polyketide Biosynthesis: During fatty acid and polyketide biosynthesis, Nature uses malonic acid half thioesters (MAHTs) as thioester enolate precursors. The primary C-C bond-forming reaction in these biosyntheses is a decarboxylative Claisen condensation between MAHTs and thioesters. These reactions occur in cells, in the presence of protic functionality and in the presence of oxygen and water. We have (Lalic, G.; Aloise, A. D.; Shair, M. D. J. Am. Chem. Soc. 2003, 125, 2852 and Magdziak, D.; Lalic, G.; Lee, H. M.; Fortner, K. C.; Aloise, A. D.; Shair, M. D. J. Am. Chem. Soc. 2005, 127, 7284) adapted MAHTs to Cu(II)(bisoxazoline)-catalyzed enantioselective decarboxylative aldol reactions that maintain their compatibility with protic functionality. We are currently expanding the scope of these reactions, especially for use in complex molecule synthesis.

6. Small Molecule Approaches to Studying Vesicular Traffic and Golgi Organization: In 1999, we began a collaboration with the laboratory of Professor Tomas Kirchhausen of the CBR Institute for Biomedical Research and Department of Cell Biology at Harvard Medical School to discover compounds that would be useful in studying vesicular traffic. Vesicular traffic is the regulated movement of vesicles in cells, usually containing protein cargo, which is responsible for movement of proteins and membranes through the secretory pathway. For instance, it is the process by which proteins synthesized in the endoplasmic reticulum that are destined for secretion are moved to the plasma membrane. Our approach has been to use high-throughput synthesis to construct libraries of compounds that are then screened in phenotypic image-based assays to determine whether any library members perturb normal exocytosis through the secretory pathway.

Exocytosis: In 2001 (Pelish, H. E.; Westwood, N. J.; Feng, Y.; Kirchhausen, T.; Shair, M. D. J. Am. Chem. Soc. 2001, 123, 6740), we reported our discovery of secramine A, a small molecule uncovered using the approach described above that inhibited vesicular traffic out of the Golgi apparatus. Recently (Pelish, H. E.; Peterson, J. R.; Salvareeza, S. B.; Rodriquez-Boulan, E.; Nazef, N.; Annis, D. A.; Chen, J.-L.; Stamnes, M.; Feng, Y.; Shair, M. D.; Kirchhausen, T. Nature Chemical Biology, 2006, 2, 39-46.), we reported that the effect of secramine A on vesicular traffic is due to inhibition of Cdc42, a small Rho-family GTPase. The inhibition also requires the presence of RhoGDI1, a guanine nucleotide dissociation inhibitor protein. Secramine A is the first chemical inhibitor of Cdc42, a protein that is central to many cellular processes, especially those involving regulation of actin. Since secramine A does not bind in the nucleotide binding site of Cdc42, it reveals a new means of inhibiting small GTPase proteins and it may become a valuable tool to study Cdc42, RhoGDI, and the cellular processes they mediate.

Golgi Organization: We are also working to discover small molecules that will help us understand how and why the Golgi is organized into stacked cisternae and whether the Golgi apparatus can self-organize on its own. We believe that cell-permeable, reversible small molecules and imaging are useful tools to decipher how the Golgi is constructed. We are interested in addressing the questions of what proteins are responsible for holding the cisternae together to form stacks of the Golgi apparatus. In addition, we are studying whether (and how) an organelle as complex as a Golgi apparatus with 1200 proteins and at least five separate membrane compartments can self-organize. Updates on this area will follow.


	Chemistry and Chemical Biology 12 Oxford St. Cambridge, MA 02138 Contact: Anita Pearson 617.384.9274 Contact the Shair Group		Research Overview. The Shair research group is working in two main areas: organic synthesis and chemical biology. Most projects involve syntheses of naturally occurring complex molecules that challenge the state-of-the-art of organic synthesis. We have chosen target molecules that are structurally unique and that have interesting, unstudied biological properties. We choose molecules that are different from other complex molecules that have been synthesized since this enables us to explore new areas of organic chemistry, especially with respect to reactivity and selectivity. We are particularly interested in developing cascade reactions for each of our synthesis targets, in order to achieve the most efficient and rapid syntheses possible. We are also attracted to molecules that have unique biological properties. Our syntheses, in many cases, are the only means of accessing additional material and designed analogs to uncover the molecule’s cellular target(s) and mechanism(s). We also have a collaboration with Professor Tom Kirchhausen’s lab in the Cell Biology Department at Harvard Medical School on the discovery and use of small molecules to probe vesicular traffic and Golgi organization in cells. Current Projects: 1. Palau’amine: This marine natural product is interesting to us because it has an unprecedented structure comprising two guanidines, a highly nitrogenous skeleton, and a stereochemically dense array of unusual functionality (for instance a secondary stereocenter bearing a chlorine atom). Others and we have recognized that the biosynthesis of palau’amine is probably derived from the oxidative dimerization of two molecules of oroidin-like molecules, common precursors of many other marine natural products. We are using this observation and other approaches to develop a synthesis of palau’amine. A longer-term goal of this project is to uncover the cellular target(s) and mechanism of palau’amine. It has been reported that palau’amine potently inhibits the activation of T-lymphocytes in a mixed lymphocyte reaction by an unknown mechanism. 2. Cortistatin A. In 2006, the unique natural product cortistatin A was reported as an extremely potent inhibitor of the migration and proliferation of HUVECs (Human Umbilical Vascular Endothelial Cells); IC50 = 180 pM (VEGF-stimulated HUVECs proliferation). It was also shown that cortistatin A is 3000 times more potent at inhibiting the proliferation of HUVECs versus other cancer and normal cell lines, suggesting that cortistatin A may be a highly selective angiogenesis inhibitor. Cortistatin A has an unprecedented structure, with a particularly challenging [3.2.1]oxabicyclooctene ring system. We are currently working on a distinct metal-catalyzed cascade reaction to construct cortistatin A. 3. Lomaiviticins: Lomaiviticins A and B are marine-derived natural products with unprecedented structures. They are C2-symmetric dimers that comprise two diazoquinonefluorenones and heavily oxidized cyclohexyl rings. A significant challenge of any synthesis of the lomaiviticins is the C-C bond connecting the two polycyclic units. This C-C bond is part of a 1,4-diketone and links the two units via two stereogenic centers. In addition, the highly oxidized cyclohexyl rings are poised for elimination and aromatization resulting in a significant synthesis challenge. We are currently developing a synthesis of the polycyclic monomers of the lomaiviticns and a strategy for linking them steroselectively to afford lomaiviticns A and B. In addition, we are interested in using our synthesis to explore the potent growth inhibitory activity of these unique molecules. Our synthesis will enable us to construct designed analogs of lomaiviticns A and B to determine the origin of their activities in cells. 4. Cephalostatin, Ritterazine and OSW-1: Cephalostatin 1 and ritterazine B are natural products that were isolated from deep-sea marine organisms in small quantites (<100mg). OSW-1 was isolated from bulbs of the African lily ornithogalum saundersiaein 400mg batches. Each of these compounds are single-digit nanomolar inhibitors of cell growth in the NCI-60 cell line panel; cephalostatin 1 GI50=1.8nM (average of 60 cell lines), ritterazine B GI50=3.2nM (average of 60 cell lines), OSW-1 GI50=0.78nM (average of 60 cell lines). COMPARE analysis between the three natural products suggests that they may have related or identical mechanisms. COMPARE profile of these compounds does not match the profile of any other known anticancer molecules, suggesting they have unique mechanisms. Our first goal in this project is to develop a short, efficient, and flexible synthesis of cephalostatin 1. One challenge of the synthesis, if a steroid-based starting material is used, is the need for a remote oxidation of an angular methyl group on the left-hand side of the molecule. A second challenge is that the right-hand spiroketal is in a non-thermodynamic configuration requiring a kinetic spiroketalization. Our second goal of this project is to identify the cellular target(s) and mechanism of cephalostatin 1. The potency, and more importantly importantly, unique mechanism of cephalostatin 1 suggest that by uncovering its cellular target(s) we may discover new approaches to inhibiting the growth of and killing tumor cells selectively. An efficient and flexible synthesis of cephalostatin 1 will allow us to make a range of designed molecules to enable target identification studies. OSW-1 is a natural product that is structurally unrelated to cephalostatin, but it has been reported that its pattern of cytotoxicity in the NCI-60 cell line panel closely resembles that of cephalostatin 1. We are also trying to elucidate the cellular target(s) and mechanism of OSW-1 to determine whether it is identical to cephalostatin 1. If so, how can structurally distantly related molecules perturb the same target? If they have different targets, why do they have highly similar (and statistically relevant) cytotoxicity patterns? We are using a combination of chemical, biochemical, genetic, and cell biological approaches to identify the cellular target(s) of OSW-1 and cephalostatin 1. 5. Catalytic Enantioselective Enolate-Based Reactions Inspired by Fatty Acid and Polyketide Biosynthesis: During fatty acid and polyketide biosynthesis, Nature uses malonic acid half thioesters (MAHTs) as thioester enolate precursors. The primary C-C bond-forming reaction in these biosyntheses is a decarboxylative Claisen condensation between MAHTs and thioesters. These reactions occur in cells, in the presence of protic functionality and in the presence of oxygen and water. We have (Lalic, G.; Aloise, A. D.; Shair, M. D. J. Am. Chem. Soc. 2003, 125, 2852 and Magdziak, D.; Lalic, G.; Lee, H. M.; Fortner, K. C.; Aloise, A. D.; Shair, M. D. J. Am. Chem. Soc. 2005, 127, 7284) adapted MAHTs to Cu(II)(bisoxazoline)-catalyzed enantioselective decarboxylative aldol reactions that maintain their compatibility with protic functionality. We are currently expanding the scope of these reactions, especially for use in complex molecule synthesis. 6. Small Molecule Approaches to Studying Vesicular Traffic and Golgi Organization: In 1999, we began a collaboration with the laboratory of Professor Tomas Kirchhausen of the CBR Institute for Biomedical Research and Department of Cell Biology at Harvard Medical School to discover compounds that would be useful in studying vesicular traffic. Vesicular traffic is the regulated movement of vesicles in cells, usually containing protein cargo, which is responsible for movement of proteins and membranes through the secretory pathway. For instance, it is the process by which proteins synthesized in the endoplasmic reticulum that are destined for secretion are moved to the plasma membrane. Our approach has been to use high-throughput synthesis to construct libraries of compounds that are then screened in phenotypic image-based assays to determine whether any library members perturb normal exocytosis through the secretory pathway. Exocytosis: In 2001 (Pelish, H. E.; Westwood, N. J.; Feng, Y.; Kirchhausen, T.; Shair, M. D. J. Am. Chem. Soc. 2001, 123, 6740), we reported our discovery of secramine A, a small molecule uncovered using the approach described above that inhibited vesicular traffic out of the Golgi apparatus. Recently (Pelish, H. E.; Peterson, J. R.; Salvareeza, S. B.; Rodriquez-Boulan, E.; Nazef, N.; Annis, D. A.; Chen, J.-L.; Stamnes, M.; Feng, Y.; Shair, M. D.; Kirchhausen, T. Nature Chemical Biology, 2006, 2, 39-46.), we reported that the effect of secramine A on vesicular traffic is due to inhibition of Cdc42, a small Rho-family GTPase. The inhibition also requires the presence of RhoGDI1, a guanine nucleotide dissociation inhibitor protein. Secramine A is the first chemical inhibitor of Cdc42, a protein that is central to many cellular processes, especially those involving regulation of actin. Since secramine A does not bind in the nucleotide binding site of Cdc42, it reveals a new means of inhibiting small GTPase proteins and it may become a valuable tool to study Cdc42, RhoGDI, and the cellular processes they mediate. Golgi Organization: We are also working to discover small molecules that will help us understand how and why the Golgi is organized into stacked cisternae and whether the Golgi apparatus can self-organize on its own. We believe that cell-permeable, reversible small molecules and imaging are useful tools to decipher how the Golgi is constructed. We are interested in addressing the questions of what proteins are responsible for holding the cisternae together to form stacks of the Golgi apparatus. In addition, we are studying whether (and how) an organelle as complex as a Golgi apparatus with 1200 proteins and at least five separate membrane compartments can self-organize. Updates on this area will follow.