EXAMPLE OF AN
INDEPENDENT MODELING PROJECT PROPOSAL

Independent Modeling Project Proposal
Chad Bennett & Marty Pagel
Feb. 21, 1999

ABSTRACT
Lobatamide A and Salicylihalamide A have recently been isolated and identified, and have been shown to have similar antitumor activity. The low-energy conformers of these compounds will be determined using Energy Minimization and Simulated Annealing. These calculations will be repeated using the CVFF and ESFF force fields to determine if the choice of force field affects this molecular modeling experiment. The low-energy conformations will be used to identify the absolute chiralities of Lobatamide A, the identity of the pharmacophores of Lobatamide A and Salicylihalamide A, and whether differences in Lobatamide A, B, C affect pharmacorphoric conformations.

 

INTRODUCTION
Lobatamides A-F have recently been isolated from Southwestern Pacific Tunicates.¹ The structures of these Lobatamides have been identified, although the identity of several chiral centers have not been elucidated (Figure 1). These compounds have been shown to have very similar antitumor activity, with a mean panel GI₅₀ of ~1.6 nM using the standard National Cancer Institute (NCI) human tumor 60 cell-line screen test.

Figure 1: Lobatamide Structures

Salicylihalamide A and B have also been isolated from the sea sponge Haliclona species.² The structures of these Salicylihalamides have also been identified, and, unlike the Lobatamides, the identities of all chiral centers have been elucidated (Figure 2). Salicylihalamide A was shown to have antitumor activity, with a mean panel GI₅₀ of ~15 nM using the standard NCI human tumor 60 cell-line screen test.

Figure 2: Salicylihalamide Structures

Lobatamides and Salicylihalamides do not have any significant correlations to the profiles of known antitumor compounds contained in the NCI's standard agent database. Therefore, these compounds appear to be a new class of antitumor agents that could be used for lead compound optimization in the development of new anticancer drugs. These compounds have macrolides that are similar to fungal-derived lasiodiplodin and searalenone, although these fungal-derived macrolides lack the enamide side chain. Also, the enamide side chain of the Lobatamides and Salicylihahamides are similar to the enamine formamide residues in other classes of antitumor agents, indicating that these similar functional groups may be similar pharmacophores.

  

PURPOSES and INTENDED CONCLUSIONS of this PROJECT

This Project will attempt to identify the absolute chirality of Lobatamide A. This will reduce or eliminate the need to synthesize all enantiomeric forms of Lobatamide A during lead compound optimization studies.

This Project will attempt to identify the pharmacophore (the functional groups responsible for binding and biological activity) of Lobatamide A and Salicylihalimide A. In particular, the similarities in the Lobatamide and Salicylihalamide structures, and evidence presented in the background material, indicates that the eneamide and salicylic acid functional groups are likely to be part of the pharmacophore. These functional groups will be specifically examined during this analysis.

This Project will attempt to identify functional groups that are NOT important to the pharmacophore. In particular, the specific geometry of the oxime of Lobatamide A and Lobatamide B will be compared, as will the specific geometry of the side chain olefin nearest the macrilide of Lobatamide A and Lobatamide C. If there is no difference in the pharmacophoric conformations of these versions of Lobatamides, it will be concluded that a specific geometry about these double bonds is not necessary for activity, as indicated by the similar IG50 values for Lobatamide A, B, and C.

Each of these investigations will be dependent on the assumption that the mechanism of binding and biological activity of the Lobatamides and Salicylihalamide A are identical. Also, this investigation will assume that the low-energy conformers of these compounds, as modeled in a vacuum, are similar to the binding and biologically-active conformations of these compounds.

 

METHODS & PROCEDURES
Simulated annealing of Salicylihalamide A and each enantiomer of Lobatamide A will be performed using an identical protocol, using the CVFF force field and Discover_3. The simulations will then be repeated using the ESFF force field, for a total of 10 simulated anealing calculations. The simulations will contain no constraints or restraints.

The conformers of Salicylihalamide A resulting from calculations using the CVFF force field will be compared to results from the calculations using the ESFF force field. The conformers from each separate calculation will be combined, and a cluster graph of the combination of conformers will be made. If the cluster graph shows a substantial RMSD difference for the "CVFF" conformers vs the "ESFF" conformers (Figure 3), the Analysis module will be used to make graphs of angles and distances, to determine how the different forcefields causes differences in geometries.

Figure 3: Schematic of Cluster Graphs
Comparing Conformers from
"CVFF calculations" and "ESFF calculations"

Using the Analysis module, distances between functional group atoms and dihedral angles will be graphed to determine the geometrical characteristics of specific functional groups of the low-energy conformers. Functional groups that show consistent geometrical characteristics will be considered to be part of the pharmacophore. Atomic and molecular properties (e.g., electrostatic charge distribution on the conformers' surfaces) of these conformations will also be visualized to identify consistent pharmacophoric properties.

In particular, the distances between specific macrolide and side chain atoms will be graphed to determine if the side chain occupies a consistent position relative to the macrolide in all low-energy conformers. Graphs involving the dihedral angle of the bond linking the side chain to the macrolide will also be used to investigate the relative side chain-macrolide geometry.

The correct chiralities of Lobatamide A will be identified by comparing the pharmacophores of the low-energy conformations of Salicylihalamide A with the pharmacophores of the low-energy conformations of each enantiomer of Lobatamide A. The enantiomer with a pharmacophore that most resembles the pharmacophore of Salicylihalamide A, while retaining the consistent structural characteristics of Lobatamide A, B, and C (i.e., the consistent cis- and trans- double bonds of the macrolide), will be identified as the most likely enantiomer.

No constraints or restraints will be used during the calculations, and the dynamics will be conducted at a high temperature (1000K). Therefore, it is expected that the dihedral angles of double bonds will be able to isomerize during the high-temperature dynamics of the simulated annealing. Both isomers will substantially populate the final ensemble if there is no substantial energy penalty for either isomer in the final low-energy conformers. The dihedral angles of the double bonds will be graphed, to determine if the low-energy conformers of Lobatamide prefer particular isomers. It is expected that the double bonds that are the same in Lobatamides A, B, and C will show a consistent isomeric form in all low-energy conformers, but the double bonds that differ in Lobatamides A, B, and C will show different isomeric forms in the low-energy conformers.

 

MODELING REPORT
I plan to submit my Modeling Paper as a HTML file and several GIF image files. These files will be located in my mpagel_c687/Modeling_Report directory.

 

POSTER SESSION
I plan to display my poster in the Energy Minimization & Dynamics poster section. However, my poster could also be displayed in the Ligand Design poster section.

REFERENCES
¹McKee, T.C., Galinis, D.L., Pannell, L.K., Cardellina, J.H.II, Laakso, J., Ireland, C.M., Murray, l., Capon, R., & Boyd, M.R. The Lobatamides, Novel Cytotoxic Macrolides from Southwestern Pacific Tunicates J. Org. Chem., 1998, v63, 7805-7810.

²Erickson, K.L., Beutler, J.A., Cardellina, J.H.II, & Boyd, M.R. Salicylihalamides A and B, Novel Cytotoxic Macrolides from the Marine Sponge Haliclona sp., J. Org. Chem., 1997, v62, 8188-8192.

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