O07_02

Relationship between cyclic peptide structure in solution and membrane permeability

Takashi MATSUMOTO *, Takashi SATO

Rigaku Corporation
( * E-mail: t-matumo@rigaku.co.jp)

Cyclic peptide drugs are attracting attention as a new modality that have complemental characteristics of small molecule drugs and biopharmaceuticals and are thought to have the potential to penetrate the limitations associated with each. In developing cyclic peptide drugs, three-dimensional structural information is essential for optimizing their interactions with target molecules and improving their functions. Solution NMR-based structural analysis has been the mainstream method, but sometimes, it becomes challenging because functional association and/or aggregation cause severe changes in inherent flexibility and relaxation time. On the other hand, crystal structures give clear-cut atomic coordinates. Still, packing artifacts are known to occur in crystal structures, especially for flexible peptide molecules; thus, there is always a question of whether the obtained fine structure reflects its functional states.
Small-angle X-ray scattering (SAXS) has been used to obtain protein structural information in the solution. However, the small-angle data represents long-range information such as the outer shapes of the proteins and/or protein-protein interactions and the lack of more detailed information in a finer distance range. To see more detail, such as the displacement of a loop region in a domain structure, we may need higher angle datasets that contain shorter-range information than the established SAXS dataset. However, the scattering signal decreases exponentially in solution through the scattering angle since the target molecules are vigorously moving in the solution, and those motions randomize distance vectors between fine structures.
Even in such a challenging landscape, if we can obtain the scattering data in good quality up to a sufficient resolution to observe the detailed characteristics and conformational movements, it will help to understand the function and conformational characters in the solution, which we have not known yet. We are developing technologies that enable that observation and abbreviating MAXS (middle angle x-ray scattering), which employs scattering data in a broader range up to higher q area to obtain more detailed structural information of proteins and especially open the doors to the analysis of peptides in solution, that possesses molecular size under the order of domain flexibility.
Here, we present solution conformations by two low molecular weight cyclic peptides, Polymyxin B and Cyclosporine A, by X-ray solution scattering. Those peptides were measured in well-dispersed conditions in pure water and anhydrous ethanol, respectively, and a detailed conformation of those peptides has been obtained utilizing MAXS data.
A comparison of the conformations of those peptides in solution and crystal states revealed changes in the structural nature and the usability of X-ray scattering for those cyclic peptides. We are extending the analysis to more complex systems where other spectroscopic methods have severe limitations.