Cryo-electron microscopy-based drug design

Abstract

Structure-based drug design (SBDD) has gained popularity owing to its ability to develop more potent drugs compared to conventional drug-discovery methods. The success of SBDD relies heavily on obtaining the three-dimensional structures of drug targets. X-ray crystallography is the primary method used for solving structures and aiding the SBDD workflow; however, it is not suitable for all targets. With the resolution revolution, enabling routine high-resolution reconstruction of structures, cryogenic electron microscopy (cryo-EM) has emerged as a promising alternative and has attracted increasing attention in SBDD. Cryo-EM offers various advantages over X-ray crystallography and can potentially replace X-ray crystallography in SBDD. To fully utilize cryo-EM in drug discovery, understanding the strengths and weaknesses of this technique and noting the key advancements in the field are crucial. This review provides an overview of the general workflow of cryo-EM in SBDD and highlights technical innovations that enable its application in drug design. Furthermore, the most recent achievements in the cryo-EM methodology for drug discovery are discussed, demonstrating the potential of this technique for advancing drug development. By understanding the capabilities and advancements of cryo-EM, researchers can leverage the benefits of designing more effective drugs. This review concludes with a discussion of the future perspectives of cryo-EM-based SBDD, emphasizing the role of this technique in driving innovations in drug discovery and development. The integration of cryo-EM into the drug design process holds great promise for accelerating the discovery of new and improved therapeutic agents to combat various diseases.

Keywords: cryo-electron microscopy; drug development; high-resolution; single particle analysis; structure-based drug design.

FIGURE 1

Workflow of drug design. The workflows of target-, phenotype-, ligand-, and structure-based drug designs are compared. The same color is used to describe the steps in the same category. For SBDD, the main focus of the review, we describe applicable tools in each step highlighted in the light blue box. In the SBDD section, the methods and tools applied in each step are listed in the boxes near each step.

FIGURE 2

Electron microscopy (EM) timeline. The critical events in the development of techniques and software (top part) and determination of structures of critical biomacromolecules (bottom part) in EM research are marked by year. References and additional information on events are as follows: first EM 3D structure (sample: bacteriophage T4, 35 Å) (De Rosier and Klug, 1968) and first field emission gun (FEG) (Crewe et al., 1968) in 1968; first EM (sample: human wart virus, 60 Å; tomato bushy stunt virus, 28 Å) in 1970 (Crowther et al., 1970), first cryo-EM (diffraction image using the hydrated and frozen condition) (sample: catalase) in 1974 (Taylor and Glaeser, 1974); first cryo-EM 3D structure (sample: Halobacterium halobium rhodopsin, 7 Å) in 1975 (Henderson and Unwin, 1975); particle image classification in 1981 (van Heel and Frank, 1981); aqueous solution vitrification in 1981 (Dubochet and McDowall, 1981); random conical tilt and angular reconstitution (sample: 50S ribosomal subunit of Escherichia coli, 30 Å) in 1987 (Radermacher et al., 1987a); bacterial rhodopsin (cryo-EM structure at the highest resolution from diffraction data, 3.5 Å) in 1990 (Henderson et al., 1990); 3D projection of matching and angular refinement (sample: 70S E. coli ribosome, 37 Å) (Penczek et al., 1994); first time resolved cryo-EM (using on-grid mixing method) (sample: droplets) in 1994 (Berriman and Unwin, 1994); the first single particle cryo-EM (sample: 50S and 30S with tRNA, 25 Å) in 1995 (Frank et al., 1995); SPIDER and WEB in 1996 (Frank et al., 1996); maximum likelihood 2D alignment in 1998 (Sigworth, 1998); EMAN in 1999 (Ludtke et al., 1999); first direct electron detector (DED) as active pixel sensor chip in 2004 (Xuong et al., 2004); time-resolved cryo-EM as ms level (using microfluidic mixing-spray) (sample: E. coli ribosome 70S) in 2009 (Lu et al., 2009); RELION (the most popular software) (Scheres, 2012) and cold field emission gun (CFEG) for cryo-EM (Ricolleau et al., 2012) in 2012; TRPV1 (first membrane channel, 3.4 Å) in 2013 (Cao et al., 2013; Liao et al., 2013); 20S proteosome (Thermoplasma acidophilum, 3.3 Å) in 2013 (Bai et al., 2013); 80S ribosome (Saccharomyces cerevisiae, 4.5 Å) in 2013 (Li et al., 2013); Volta phase plate (VPP) in 2014 (Danev et al., 2014); glutamate dehydrogenase (GDH; the first achievement at a resolution below 2 Å, 1.8 Å) in 2016 (Merk et al., 2016); cryoSPARC (software using stochastic gradient descent and branch-and-bound maximum likelihood optimization algorithms) in 2017 (Punjani et al., 2017); α1β3γ2L γ-aminobutyric acid [GABA]_A receptor (target–scaffold complex structure using cryo-EM, 3.2 Å) in 2019 (Laverty et al., 2019); apoferritin (cryo-EM structure at the highest resolution, 1.15 Å) in 2020 (Yip et al., 2020); cyclin-dependent kinase (CDK)-activating kinase (CAK: a target for fragment-based drug discovery using cryo-EM) in 2023 (Cushing et al., 2023); 70S ribosome (E. coli, 1.55 Å) in 2023 (Fromm et al., 2023). The events for technical advancement are highlighted using purple and the cryo-EM structures are highlighted green.

FIGURE 3

The annually released number and resolution distribution of EM maps in EM Data Bank (EMDB). The number of all released single-particle EM maps in the EMDB and structural models in the Protein Data Bank (PDB) from total samples are shown per year (A) and resolution range (B). Ligand–target complex samples are also described per year (C) and resolution range (D). The number of EM maps in EMDB with and without structural models in PDB is depicted in green and pink, respectively. All data for 2023 have been collected up to 2 August 2023.

FIGURE 4

Modern techniques for cryo-EM–based drug discovery. The newly emerged techniques of cryo-EM for drug discovery are scaffolds (A), rapid structure determination (B), functionalized grids (C), fragment-based drug design (D), and antibody design (E), described in Section 4.1, 4.2, 4.3, 4.4 and 4.5, respectively. Examples using each technique are displayed with individual EM maps and structural models. The following are representative examples: (A) α1β3γ2L GABA_A receptor (6HUJ:EMD-02779) (Fromm et al., 2023); (D) PKM2 (6TTI:EMD-10577, 6TTF:EMD-10575, 6TTH:EMD-10576, and 6TTQ:EMD-10584) (Saur et al., 2020); and (E) the trimer of the heterodimer of gp120 (BG505 SOSIP MD39) and gp41 (BG505 SOSIP MD3) with antigen-binding fragment (Fab) (Rh.33104 polyclonal antibody class C (7I8A:EMD-23227) (Antanasijevic et al., 2022a). Rapid structure determination is described using a scatter graph, comparing the resolution and the number of particles from 39 samples, collected for 1 h (n = 26) (green) and 4 h (n = 13) (pink) (B) (Cushing et al., 2023). The scaffold in (A), fragments-binding sites in (D) and antibody in (E) are marked using red open circles.