Exploring serial crystallography for drug discovery

Abstract

Structure-based drug design is highly dependent on the availability of structures of the protein of interest in complex with lead compounds. Ideally, this information can be used to guide the chemical optimization of a compound into a pharmaceutical drug candidate. A limitation of the main structural method used today - conventional X-ray crystallography - is that it only provides structural information about the protein complex in its frozen state. Serial crystallography is a relatively new approach that offers the possibility to study protein structures at room temperature (RT). Here, we explore the use of serial crystallography to determine the structures of the pharmaceutical target, soluble epoxide hydrolase. We introduce a new method to screen for optimal microcrystallization conditions suitable for use in serial crystallography and present a number of RT ligand-bound structures of our target protein. From a comparison between the RT structural data and previously published cryo-temperature structures, we describe an example of a temperature-dependent difference in the ligand-binding mode and observe that flexible loops are better resolved at RT. Finally, we discuss the current limitations and potential future advances of serial crystallography for use within pharmaceutical drug discovery.

Keywords: drug discovery; fixed-target devices; microcrystals; room-temperature structures; serial crystallography; soluble epoxide hydrolase; temperature-dependent structural differences.

Figure 1

Structure of sEH. (Left) Structure of inhibitor-bound sEH solved at cryogenic temperature (PDB entry 5ake; Öster et al., 2015 ▸). The active site with the bound compound 4 (purple) is located in the C-terminal domain. (Right) Zoom-in of the active site where the compound (purple) interacts with the catalytic triad (Asp335, Tyr383 and Tyr466).

Figure 2

Microcrystallization and data collection workflow. Panel 1: production of crystal seeds from macro crystals using seed beads and vortexing. Panel 2: hybrid crystallization used for screening to find optimal batch crystallization conditions. Panel 3: batch crystallization with seeding and vortexing to induce nucleation. Panel 4: soaking of compound into the microcrystals. Panel 5: dispensing of soaked crystals onto the chip and sealing. Panel 6: fixed-target raster grid-scan data collection on ligand-soaked crystals at a synchrotron source.

Figure 3

Microcrystals of sEH. The effect of using different protein:precipitant/seed solution ratios during screening for optimal conditions using the hybrid crystallization method is shown. (a) A protein:precipitant/seed solution ratio of 1 is used. (b) A protein:precipitant/seed solution ratio of 2 is used. A ratio of 2 gives a lower number of crystals but a more homogeneous sample with fewer overlapping crystals.

Figure 4

RT structures of sEH in complex with compounds. The outer images show the different compounds that form complexes numbered according to Table S2. The F_oF_c omit difference electron density map (green) is contoured at +3.0σ in each case. The central panel displays an overlay of the seven RT sEH complex structures zoomed-in on the active site.

Figure 5

Temperature-dependent difference in compound binding mode. The RT structure from crystals soaked with compound 7 is compared with the RT apo structure and the cryo-T structure in complex with compound 7. (a) In the RT structure of sEH obtained from crystals soaked with compound 7, the active-site density is best fitted with a PEG fragment (PDB entry 8qwg). (b) RT apo structure with a PEG fragment bound in the active site (PDB entry 8qvm). (c) In the cryo-T structure from crystals soaked with compound 7 (PDB entry 5ai8; Öster et al., 2015 ▸), the compound is bound as well as a sulfate ion and a water molecule. For each structure, the 2F_oF_c electron density map is contoured at 1σ (blue) and the F_oF_c electron density map at +3.5σ (green).

Figure 6

Comparison of water molecules modelled at RT and at cryo-T. (Left) RT sEH structure (in complex with compound 3, PDB entry 8qvk) with water molecules in red. (Right) Corresponding cryo-T structure (PDB entry 5ake) with water molecules in black. The central panel displays an overlay of the water molecules.

Figure 7

B-factor comparison. A B-factor comparison is shown for one RT structure (PDB entry 8qwg) and its cryo-T counterpart (PDB entry 5ai8). At the top, the RT (left-hand side) and cryo-T (right-hand side) sEH structures are displayed colored according to B factors. At the bottom, the average B factors of main chain atoms are plotted according to residue number (RT structure – orange, cryo-T structure – blue).

Figure 8

Ordering of loops. An example of a loop (Pro65 to Ala95) with a higher degree of order in the RT structures compared with in the cryo-T structures. (a) 2F_oF_c electron density map associated with crystals soaked with compound 7 at RT (PDB entry 8qwg). (b) 2F_oF_c electron density map associated with the corresponding cryo-T structure (PDB entry 5ai8). The 2F_oF_c electron density maps are contoured at 1σ (blue).