Drug discovery is a cornerstone of advancing human health, traditionally relying on serendipitous findings and natural product screenings. Landmark discoveries like penicillin, sulfonamides, paclitaxel, and artemisinin illustrate this history. However, the late 20th century marked a turning point with breakthroughs in molecular biology, synthetic chemistry, structural biology, and computational technologies, fundamentally reshaping drug research and development. Today’s drug design is a more targeted and efficient process, demanding collaborative efforts across diverse scientific fields. Emerging drug screening platforms, leveraging high-throughput compound library screening and innovative technologies such as structure-based drug design (SBDD), fragment-based drug discovery (FBDD), DNA-encoded chemical libraries, proteolysis targeting chimeras (PROTAC), and drug repurposing, are becoming increasingly vital in modern drug development.
Structural biology’s contribution to drug discovery is undeniable. It provides direct, atomic-level insights into drug targets, influencing every stage of preclinical drug development, from target identification to lead compound optimization. Understanding the active or regulatory sites of target proteins at an atomic level makes rational drug design a reality. Currently, X-ray crystallography, nuclear magnetic resonance (NMR), and cryogenic electron microscopy (cryo-EM) are the three dominant structural biology techniques. X-ray crystallography excels at delivering atomic-level structural data, particularly for crystallizable macromolecules, but it often struggles with larger protein complexes and membrane proteins. NMR, while capable of analyzing protein structures in solution and protein dynamics, is limited to smaller proteins. This is where cryo-EM steps in, especially for macromolecules exceeding 100 kD, offering high-resolution structures, often beyond 3 Å, for proteins larger than 135 kD.
Cryo-EM is becoming increasingly powerful, even for smaller proteins, thanks to technological advancements. It allows for the study of macromolecular complexes and membrane proteins in their near-native solution state, requiring less protein and bypassing the need for crystallization. Furthermore, time-resolved cryo-EM can capture proteins in different conformations during reactions, providing a dynamic view crucial for understanding protein function and drug interactions.
The genesis of cryo-EM can be traced back to the invention of electron microscopy in 1932, initially used in materials science. The application to protein structure analysis began thirty years later with electron diffraction of protein crystals. Cryo-EM as we know it today, tailored for structural biology, emerged in 1982 with the development of single-particle 3D reconstruction algorithms and plunge-freezing techniques for biomacromolecules. Early cryo-EM faced resolution limitations, struggling to surpass 4 Å resolution until 2013. However, advancements in algorithms, hardware, and the introduction of direct electron detectors have revolutionized its resolution capabilities. The 2017 Nobel Prize in Chemistry recognized Jacques Dubochet, Joachim Frank, and Richard Henderson for their pivotal contributions to cryo-EM development. By 2020, the Protein Data Bank contained over 1700 cryo-EM structures solved at resolutions better than 4 Å. The highest resolution achieved by cryo-EM has reached an astonishing 1.2 Å, truly attaining atomic resolution.
The cryo-EM workflow encompasses several key stages: sample preparation, cryo-EM grid preparation and imaging, data collection and preprocessing, 3D map reconstruction, and subsequent model building and structure analysis. Even in its earlier, lower-resolution forms, cryo-EM, when combined with X-ray crystallography, played a role in drug development since the 1990s, particularly in target identification and validation. This combined approach integrated high-resolution crystal structures with lower-resolution cryo-EM density maps to study challenging macromolecular complexes. With cryo-EM now achieving atomic resolution, its significance in drug research and development has surged. This review will explore how cryo-EM is accelerating drug discovery across various modalities, including SBDD, FBDD, PROTAC, antibody drug development, and drug repurposing. The synergistic potential of cryo-EM with artificial intelligence (AI) will also be discussed, highlighting future avenues for cryo-EM advancements in drug discovery.
