key: cord-1030605-qyud3ykr
authors: Van Drie, John H; Tong, Liang
title: Cryo-EM as a powerful tool for drug discovery
date: 2020-09-02
journal: Bioorg Med Chem Lett
DOI: 10.1016/j.bmcl.2020.127524
sha: 092c8322b77d946c9d0d2c792093e6eba1c2c086
doc_id: 1030605
cord_uid: qyud3ykr

The recent revolution in cryo-EM has produced an explosion of structures at near-atomic or better resolution. This has allowed cryo-EM structures to provide visualization of bound small-molecule ligands in the macromolecules, and these new structures have provided unprecedented insights into the molecular mechanisms of complex biochemical processes. They have also had a profound impact on drug discovery, defining the binding modes and mechanisms of action of well-known drugs as well as driving the design and development of new compounds. This review will summarize and highlight some of these structures. Most excitingly, the latest cryo-EM technology has produced structures at 1.2 Å resolution, further solidifying cryo-EM as a powerful tool for drug discovery. Therefore, cryo-EM will play an ever-increasing role in drug discovery in the coming years.

These are exciting times for our ability in drug discovery to exploit the 3D structure of the macromolecular target. Structure-based drug discovery (SBDD) began in the 1980s, when both X-ray crystallography and molecular modelling were first introduced into drug discovery; by the 1990s SBDD had matured into a routinely used approach. The first major success of SBDD was the discovery of HIV protease inhibitors against AIDS; this pandemic began in 1981 and became a huge threat by the mid-90s, when the introduction of both HIV reverse-transcriptase (RT) inhibitors and HIV protease inhibitors began to turn AIDS into a manageable disease ( Figure 1 ) [1] . Indeed, this is a success story for the pharmaceutical R&D enterprise as a whole, in terms of our ability to respond to a pandemic.

A revolution in structural biology has occurred in the past few years, due to dramatic advances in the technology of electron microscopy (EM) at cryogenic temperatures -cryo-EM. Not that long ago, EM was often disparaged as 'blob-ology', due to the low resolution of the structures; these were useless for drug discovery. Figure 2 shows how structural information has improved over the years for the ribosome, in parallel with advances in X-ray crystallography and EM.  Figure 2A shows the first EM structure of the ribosome from 1984 [2] .  Figure 2B shows the EM structure in 1995 [3] ;  Figure 2C the X-ray density in 1998 [4] ,  Figure 2D the X-ray structure from 2000 [5] ,  Figure 2E a 2008 X-ray structure showing atomic detail how the antibiotic linezolid binds to the ribosome [6] and finally  Figure 2F shows a recent cryo-EM structure [7] , which now displays the various components of the complex (mRNA, tRNA, the emerging polypeptide chain).

Improvements on multiple fronts in EM have brought EM structures to the resolution where they are now useful for drug discovery. The 2017 Nobel Prize in Chemistry was awarded to Dubochet, Frank and Henderson, in recognition of their contributions to these improvements that transformed the low-resolution EM into the high-resolution cryo-EM [8] . Some of these improvements were: the ability to rapidly capture the undistorted macromolecule in vitrified ice, the ability to deconvolute the multiple conformations and orientations of the molecule in that vitrified state, and improvements in the instrumentation, especially the sensitivity of the detectors.

Cryo-EM and X-ray crystallography are complementary techniques; each facilitates the other. Compared to X-ray crystallography, cryo-EM provides a number of advantages:

(1) capturing large complexes containing multiple proteins is routine, allowing the capture of a protein in its native milieu vs. the study of a protein in isolation in crystallography, (2) the protein is usually captured in a near-native state, something especially important in transmembrane proteins, like ion channels and G-protein-coupled receptors (GPCRs);

(3) conformational changes are routinely obtained, making clear the manner in which the target is a 'molecular machine'. This often exposes unanticipated drug binding sites, or leads to transformational insights into mechanism, or opportunities for selectivity;

(4) some important off-targets of drugs can now be incorporated into the SBDD approach, e.g. ion channels and transporters; and (5) the timeline for determining these structures can be more predictable, because crystallography requires a crystal, which is a serious bottleneck, while cryo-EM can work with the sample directly.

The primary disadvantage is that today the resolution of many cryo-EM structures is still only comparable to a low-resolution X-ray structure. However, this is changing rapidly (Figure 3 ), and some recent structures have 1.2 Å resolution [9, 10] , comparable to the best macromolecular X-ray structures. Figure 3 shows the growth in the number of publicly available cryo-EM structures and their improving resolution in the past decade. ( We are fortunate that most funding agencies and journal policies insist that published structures are made publicly available).

There are many reviews on cryo-EM structural biology [11] [12] [13] [14] [15] [16] [17] [18] [19] . What differentiates this review is our focus on examples of how cryo-EM structures have impacted drug discovery, in terms of both driving the discovery of new compounds and defining the mechanism of action of known drugs. Some examples are described in detail; others are summarized briefly in a later section. There are two major challenges to compiling such a review: the literature is growing at an astonishing pace, and many examples of uses of cryo-EM for SBDD are still proprietary, but are expected soon. In each case, we will highlight many of the points made above, in terms of the special advantages of cryo-EM, and will primarily highlight examples where crystallography has fallen short of what is required.

Brief description of cryo-EM technology: Structure determination by cryo-EM typically proceeds through the following steps:

1. Isolation and purification of the sample, from natural and/or recombinant sources. This step is similar to that needed for crystallography, but the amount of sample required for EM is much smaller. Typically, several microliters at ~1 mg/ml concentration would be sufficient.

Detailed description of selected cryo-EM structures: ATP-citrate lyase (ACLY). ACLY catalyzes the conversion of citrate and CoA to oxaloacetate and acetyl-CoA, which also requires the hydrolysis of ATP. It is a major source of acetyl-CoA in the cytoplasm, and a potential target for drug discovery against dyslipidemia and cancer [29, 30] . Bempedoic acid (ETC-1002) has been approved for the treatment of hypercholesterolemia [31] , validating ACLY as a drug discovery target. This compound has a linear C15 di-acid backbone and needs to be converted to the CoA ester in the liver for its inhibitory activity against ACLY, which likely occurs through binding to the active site.

A series of low nanomolar ACLY inhibitors has recently been disclosed by Nimbus Therapeutics, and they are competitive against the citrate substrate of the enzyme [32] . The structure of one of these inhibitors (NDI-091143) in complex with human ACLY was determined by cryo-EM at 3.67 Å resolution in the Tong lab ( Figures 4A, 5A ) [32] . Remarkably, the compound is bound in an allosteric cavity, in the hydrophobic core of the citrate binding domain of the enzyme and has no overlap with citrate ( Figure 5B) . A large conformational change in the domain is required for the compound to bind, which indirectly competes against citrate. Especially, Arg379 interacts with the citrate but clashes with the inhibitor. It moves by 12 Å upon inhibitor binding ( Figure 5B ). Ile344 moves by 6 Å upon inhibitor binding, and its new position clashes with citrate. The binding cavity does not exist without this conformational change. Free-energy perturbation calculations based on the binding mode of this compound can explain the binding affinities of other compounds in the series [32] , supporting the structural observations and indicating that a structure at this resolution could be useful for explaining the SAR of the compounds and drug design. In fact, extensive efforts at determining the binding mode of this compound by crystallography were not successful, and therefore cryo-EM enabled the breakthrough on this compound.

GPCRs. GPCRs (G-protein-coupled receptors) are one of the oldest targets for drug discovery. Structural data on GPCRs have exploded in the past 20 years, starting with the X-ray structures of rhodopsin [33] and the beta-2 adrenergic receptor [34] . Crystallography, cryo-EM, and NMR have all contributed to a wealth of knowledge on the details of structure and mechanism of existing drugs, which have led to the design of improved molecules [35] .

There are many examples of cryo-EM structures of GPCRs that we could describe in this review. We will discuss one here in detail, GLP-1R; others will be described in brief in the following section.

GLP-1R. GLP-1R is the receptor for the gastrointestinal peptide hormone GLP-1, a key component of insulin regulation. Peptides based on GLP-1 have long been on the market for treatment of diabetes [36] . Most other drugs have been discovered via peptidomimetic approaches, targeting the orthosteric site where GLP-1 binds. Activity in the past 10 years has focused on non-peptidic molecules originating from HTS hits, functioning as positive allosteric modulators ( Figure 6 ), but these molecules are sub-optimal in many ways. [37, 38, 39] . Upstream of GLP-1R, the "gliptin" drugs inhibit DPP-IV, the peptidase responsible for the cleavage and inactivation of GLP-1.

What makes this example of the cryo-EM structure of GLP-1R especially compelling is that it exemplifies all the advantages of cryo-EM: (1) the full structure highlights aspects that were not apparent in the X-ray structures of the individual components; (2) the key conformational change upon ligand binding and activation is now apparent; (3) this structure is significantly different than what had been anticipated via homology models, and (4) based on the state of the art, it is poised to drive the discovery of much improved drug candidates. The first cryo-EM structure of GLP-1R was published in 2017 [40] , Figure 7 .

In contrast to earlier work, this new cryo-EM structure shows the full seven transmembrane (TM) domain portion of the receptor, the N-terminal domain, the peptide ligand, and the G protein which transduces the signal. The structure, coupled to other biophysical and enzymatic data, clarifies how peptide binding at the extracellular surface is communicated to the intracellular receptor side: conformational changes in the transmembrane domain result in a sharp kink in the middle of TM helix 6, which pivots its intracellular half outward to accommodate the α5 helix of its G-protein. In that publication, the binding site of the known allosteric modulators could only be modelled, by homology to other GPCRs with modulators bound. In addition, it is thought that this structure may be a paradigm for the structure of other family B GPCRs, highlighting a general mechanism whereby a flexible N-terminal domain outside of the membrane folds to form the binding site for the endogenous ligand. This structure also allowed them to propose a mechanism of activation. Finally, the interaction with the Gprotein suggests a molecular basis for the "biased signaling" that is seen in the allosteric modulators, namely that the ratio of signaling in multiple downstream pathways differs from molecule to molecule.

A series of new GLP-1R allosteric modulators has appeared from vtv Therapeutics (the company formerly known as Trans Tech Pharma). The first of these compounds TTP054 was tested in Phase 2 clinical trials, but there is no evidence that it continued on phase 3 [38, 41] . However, newer analogs of these molecules are now in Phase 2, e.g. TTP054-201, and the cryo-EM structure of an analog has been published this year [42] , Figure 8 , This structure shows the same overall conformation as the earlier cryo-EM structure, but now shows the location of the ligand. Of course, others are in this race as well, e.g. Pfizer's PF-06882961, which has been disclosed briefly, but apparently not in conventional scientific literature.

In addition, GLP-1R has been implicated in some CNS disorders, like AD and PD [36] . Additional work on GPL-1R allosteric modulators driven by this cryo-EM structure may allow further optimization for these indications.

Ion channels. Ion channels control the conduction of signals in nerves and are one of the oldest drug targets. They have been a rich source for structural biologists as well, starting with the first major breakthrough in 1998 with the publication of the X-ray structure of the potassium channel by Rod MacKinnon and colleagues [43] . Three examples which most strongly exemplify the strengths of cryo-EM will be discussed in detail here: TRP channels, CFTR, and hERG. In each case, novel insights into mechanism have arisen by understanding the conformational changes in these molecular machines. In addition, they are the targets of important new drugs, and are being actively pursued by many drug discovery groups. Unfortunately, in each case there is little in the public literature that describes how these structures are being exploited in drug design.

TRP channels. The TRP channels (Transient Receptor Potential channels) mediate pain and temperature signals, and have been pursued recently as a target for neuropathic pain, migraine, and inflammatory pain; multiple drug candidates are being evaluated in clinical studies [44] TRPV1 is the receptor to which capsaicin binds (the active substance in hot chili peppers), and has been described as the "'poster child' for elucidating basic principles underlying TRP channel function and structure". Its structure was first determined via cryo-EM by the Julius lab at UCSF (Figure 9 ) [45, 46] . Cryo-EM is spurring an on-going revolution in this area [47] . It has enabled the determination of a large number of TRP channel structures (Table S1 ).

This initial cryo-EM structure was noteworthy for multiple reasons. It gave the first, approximate picture how capsaicin and related molecules bind; the second publication gave the atomic-level detail of the ligand-protein complex. These publications also showed multiple functional states, allowing hypotheses to be formed on the mechanism of activation ( Figure  10A ). While the overall architecture resembled other channels like the potassium channel, the mechanism of activation was startlingly different from what was known from other channels, pointing to an unusual allosteric coupling from the ligand-binding S1-S4 helices (homologous to the voltage-sensing domain in voltage-gated ion channels) to the pore-forming domains. These differences are critical to achieving selectivity between and among these families of channels and contributed to the ability to design out the side effects of the first TRPV1 antagonists discovered [48] . These experiences exemplify the power of cryo-EM in drug discovery: (1) multiple conformational states can readily be determined, often in one experiment, (2) these states lead to mechanistic hypotheses, which in turn lead to (3) insights into how to achieve selectivity for the channel of interest.

One example of a medicinal chemistry effort exploiting this structure is from a group in Seoul, S Korea, who have published extensively on a series of analogs designed using this cryo-EM structure ( Figure 10B ) [49, 50] They achieved sub-nM activity and excellent activity in a neuropathic pain model at 10 mg/kg.

Cystic Fibrosis is a fatal lung disease, which was found in 1989 to originate from a single mutation in one gene, expressing a protein which was later found to regulate chloride levels, given the name CFTR [51] [52] [53] . One mutation, F508del, accounts for the majority of patients, though disease mutations occur in many positions in CFTR. The first CF drugs directly targeting CFTR were discovered by Vertex Pharmaceuticals [54, 55] : ivacaftor, approved in 2012, followed by lumacaftor, and tezacaftor, which now form the standard-of-care as a triple combo, Trikafta, approved in 2019. The Vertex drugs were all discovered empirically, in heroic medicinal chemistry efforts [56] , exploiting a high-throughput cell-based assay. No knowledge of the receptor structure was exploited, except to select specific cell lines with certain mutations. Despite their great commercial success, Vertex has thus far attracted little competition, suggesting that this is a challenging area for drug discovery.

All efforts to crystallize CFTR, the first step to determining an X-ray structure, have failed. However, the lab of J. Chen at Rockefeller has used cryo-EM to determine increasingly useful structures of CFTR: first from zebrafish [57] ; then human [58] with some domains unresolved; then human, better-resolved with indications of the conformational changes upon channel activation [59] ; and, finally, in 2019, CFTR in complex with ivacaftor, and a drug candidate discovered by Galapagos ( Figure 11 ) [60] . These two structurally distinct compounds bind in the same pocket, at the interface between the CFTR and the lipid membrane. The binding pocket is part of the hinge for channel gating, and the presence of the compound may stabilize the channel pore in an open configuration, explaining its potentiating effect.

Persuasive homology models of human CFTR had been built [e.g. 61] but these did not prove useful. It is difficult to convey how astonishing this final structure of ligand-bound CFTR was to those active in CF, as all prior hints about possible ligand-binding sites pointed to regions in the nucleotide-binding domains near F508. Instead, both ivacaftor and GLPG-1837 bind to a kink in a transmembrane helix embedded in the membrane. None of the earlier structures showed either a clear kink in that helix, or a binding pocket there, and generally the notion of a binding pocket contacting the lipid bilayer is exceptional and poses special challenges to compound design.

It is in the early days, but it is reasonable to expect that this new structure will now permit structure-based optimization of such molecules. Further structures clarifying conformational changes, mechanism, etc. will undoubtedly be helpful.

As an aside, from the perspective of genomics-based small-molecule drug discovery, it is worth noting the length of time required: the mutation was identified in 1989, and not until 2012 was the first drug approved to treat the disease. And, of course, there are many other targets where disease-causing mutations have been known for a long time but thus far no treatment is available.

hERG channel. Unwanted inhibition of the hERG channel has been a bête noire of drug hunters since the discovery in the mid-1990s that this channel mediates the long-QT syndrome which often leads to fatal arrhythmias [62, 63] Homology modelling [64] has not shown to be especially useful in designing out the hERG liability. In 2017, a cryo-EM structure was published from the MacKinnon lab ( Figure 12 ) [65] , which broadly showed the same architecture as that homology model -a central volume surrounded by four hydrophobic pockets -but this new detail made it clear why this channel should be so promiscuous, why a basic amine is essential, and highlights how this channel's selectivity filter is subtly different from other known K channel structures. It is not yet evident in the medicinal chemistry literature that this structure has permitted structure-based optimization away from hERG affinity, or whether a structure with a ligand bound will be needed. It has, however, played a role in the steadily increasing ability of various software tools to estimate the long QT liability [66, 67] Viruses. Here, we will focus on one single virus, SARS-CoV-2, the cause of COVID-19, the scourge of this annus horribilis. The SARS-CoV-2 genome sequence was first published in January 2020 [68] . Since that time, over 200 X-ray structures and 20 cryo-EM structures have been published. From the simplest perspective, the key viral proteins are Drug discovery driven by these structures is still in its infancy, but the structures do highlight multiple opportunities to design inhibitors of the key steps of viral infection and propagation. Furthermore, one structure suggests the binding conformation of remdesivir, the only drug which has been authorized by the FDA for emergency use against the virus.

The spike protein. The virus gains entry into the cell via interactions between the spike proteins on the virus surface and a transmembrane protein which sits on the surface of the host cell, ACE2. The spike protein functions as a trimer and undergoes a conformational change and a series of steps to infiltrate into the cell. Many X-ray structures have appeared of the isolated spike protein, but especially compelling are the larger complexes which have been determined via cryo-EM, and ones which demonstrate the large conformational changes that occur upon activation ( Figure 13A , 13B) [69] The RNA-dependent RNA polymerase (RdRp). SARS-CoV-2 is an RNA virus, encoding its genome in strands of RNA, unlike humans whose genome is encoded in DNA. Hence to replicate its genome, it requires an enzyme that is not present in the host cell, an RNA-dependent RNA polymerase (RdRp). Some known antivirals are weak RdRp inhibitors, such as the nucleoside mimics remdesivir and sofusbuvir. A cryo-EM structure has been published which allows one to form reasonable hypotheses how these molecules may bind to RdRp ( Figure 13C , Figure 13D ) [70] . This structure probably provides the greatest opportunity for structure-based design of COVID-19 antivirals.

The RdRp is only one component of a much larger complex, the replicase complex. A recent preprint describes a cryo-EM structure of a large section of that replicase complex [71] ( Figure  13E ). Understanding the motions of this molecular machine, as RdRp 'walks' along the template RNA, may give insights into alternative ways of inhibiting the polymerase action. One new revelation from the structure is that there are extensions of the viral protein NSP-8 which interact with the paired RNA.

Another great opportunity lies ahead for cryo-EM in SARS-CoV-2 antivirals by looking at other mega-complexes involving host proteins, for example the enhanceosome, the target of interferon, a protein which is well-known for its antiviral activity against other viruses, and has even been demonstrated to show clinical efficacy against COVID-19 [72] .

Here, we describe several additional structures with drug-like small molecules bound (Table P-1) . Unlike the previous section, only brief descriptions will be given due to space considerations; the readers are referred to the original publications for detail. The cryo-EM density for some of the small molecules are also shown (Figure 4) , as an illustration of the quality of the cryo-EM reconstructions. A more complete listing of the published structures with bound drug-like small molecules is given in Table S1 . Only those structures with explicitly modeled small molecules are considered here. Structures with bound biochemical ligands (substrates, substrate analogs, cofactors, prosthetic groups) are generally not included in the consideration here, although goodquality EM density can be observed for them as well. In addition, cryo-EM structures have been obtained for other drug discovery targets, such as inflammasome, but no structures of complex with drug-like molecules are currently reported for them. Of course, there are likely a substantial number of structures of ligand complexes determined at pharmaceutical and biotech companies that have not yet been published or deposited in the PDB.

The analysis of deposited cryo-EM structures with bound drug-like ligands also indicates that the majority of them are for large complexes (ribosome, proteasome, spliceosome, microtubule, actin, virus, and others) and membrane proteins (receptor, channel, transporter, and others). This is a reflection of the fact that cryo-EM has made it possible (and/or more readily) to determine such structures, and the drug-like ligands probably also helped to stabilize some of the complexes for structural studies. In comparison, the number of structures of soluble enzymes is smaller, although several of them have been used to demonstrate the possibility of pushing cryo-EM to high resolution [73] [74] [75] . Nonetheless, small-molecule ligands can be observed for these targets [76] , and fragment-based drug discovery by cryo-EM has recently been reported [77] . Some of these proteins are too small for the current EM technology, which favors larger molecules and complexes. Structures of smaller proteins can be determined by EM for example by complexing it with an antibody. At the same time, constant improvement in EM technology has made it possible to determine the structures of ever smaller proteins. It is also likely that many of these structures are still being determined by X-ray crystallography, which is faster and easier if good quality crystals and compound soaking protocols are available. Routine use of high-throughput structure determination by cryo-EM, for example for lead optimization, will benefit from additional hardware and software improvement. However, if good conditions for preparing frozen grids of the sample are known, it may be envisioned that faster data collection and processing can be achieved to substantially increase the throughput of cryo-EM.

A large number of cryo-EM structures have been reported of ribosomes from various organisms bound to antibiotics, anti-protozoals, and other compounds (Table S1 ). These compounds include natural products (such as erythromycin, avilamycin, paromomycin, emetine, mefloquine) as well as synthetic molecules (such as linezolid and cadazolid). The binding modes of some of these compounds were originally determined by X-ray crystallography, but cryo-EM is now the dominant method for determining ribosome structures. For example, the binding mode of emetine is clearly defined by the cryo-EM map at 3.2 Å resolution ( Figure 4C ) [78] .

Serotonin receptor. The 5-HT3 serotonin receptors are pentameric ligand-gated ion channels (pLGIC) and they are targets for therapeutic agents against nausea, vomiting, and depression [79] . The cryo-EM structures of these receptors in complex with serotonin (5hydroxytryptamine, 5-HT) were reported recently [80, 81] . Serotonin binding causes extensive conformational changes, promoting a conductive, open state. The anti-emetic drug granisetron is bound in the same site as serotonin ( Figures 4D, 14A , B) [82] , but it stabilizes a closed state of the channel, distinct from the apo, resting state.

AMPA receptor. The ligand-gated ion channels AMPA-type ionotropic glutamate receptors (AMPARs) are associated with serious neurological diseases such as depression and Alzheimer's. A cryo-EM structure of the GluA2 receptor bound to the competitive antagonist ZK200775 (also known as fanapanel) was reported recently (Fig 4E) [83] . A crystal structure of this compound bound to the ligand binding domain (LBD) of the receptor is also available, at 1.25 Å resolution [84] .

GABA receptor. Type A GABA (-aminobutyric acid) receptors (GABA A receptors) are ligandgated chloride channels in the central nervous system and are associated with mental illnesses such as epilepsy, chronic pain, anxiety and insomnia [85] . These receptors contain many allosteric sites, and they are targets for benzodiazepines (such as diazepam/Valium and alprazolam/Xanax) and other therapeutic agents as well as for drug abuse. Competitive antagonists (such as flumazenil) are used clinically to reverse the effects of benzodiazepines, especially their overdose.

The binding modes of flumazenil [86] , Valium and Xanax (Figures 4F,15 ) [87] have been revealed by recent cryo-EM studies. Xanax and Valium share essentially the same binding modes, at the interface between the extracellular domains of two of its subunits. Binding of these compounds may facilitate channel activation by stabilizing the interactions between the subunits. The antagonist flumazenil binds deeper into this pocket and may not be able to stabilize the interactions between the subunits.

Sodium channel. Voltage-gated sodium channel 1.5 (Nav 1.5) produces the cardiac action potential for heartbeat, and is the target of many drugs, e.g. antiarrythmic drugs such as flecainide. The cryo-EM structure of flecainide in complex with Nav 1.5 (Fig 4G) [88] reveals the detailed interactions between the compound and the channel pore, next to the selectivity filter. Other voltage-gated sodium channels are sensitive to neurotoxins such as tetrodotoxin (TTX, from the fugu fish) and saxitoxin (a paralytic shellfish toxin). The molecular basis of their pore-blocking activity has also been revealed by cryo-EM studies [89] .

Potassium channel. ATP-sensitive potassium (KATP) channels in pancreatic -cells control insulin secretion in response to blood glucose levels. They are the targets of the type 2 diabetes drug sulfonylureas, such as glibenclamide (GBC). It is bound in a central cavity of the transmembrane domain of the peripheral sulfonylurea receptor (SUR) subunit (Figs 4H,16) [90] . The structure suggests a mechanism how binding of glibenclamide to the peripheral subunit can inhibit the central potassium channel. The carbamoylbenzoic acid drug repaglinide shares the same binding site [90] [91] .

Voltage-gated calcium channels. Voltage-gated calcium (Cav) channels are targets for widely used therapeutic agents. Cav1 antagonists such as dihydropyridines (DHP, with nifedipine as an example), benzothiazepines (BTZ, diltiazem), and phenylalkylamines (PAA, verapamil) are used in the clinic to treat hypertension, angina pectoris, and cardiac arrhythmia. BTZ and PAA compounds block the pore of the channel, while DHP compounds bind in an allosteric site, and some DHP compounds (Bay K 8644) are agonists. The binding modes of these compounds have been defined by cryo-EM studies (Figs 4I,17) [92, 93] .

Ryanodine receptor. Ryanodine receptors (RyRs) are ligand-gated calcium channels important in muscle function; they are a therapeutic target for arrhythmia, muscular dystrophy, neurodegenerative disease, and other myopathies [94] . They are exceptionally large, and hence have been resistant to crystallography. The first cryo-EM structures were determined in 2015 [95] , with multiple conformational states associated with gating and binding sites of modulators (e.g. caffeine) determined one year later ( Figure 18 ) [96] . Ca 2+ converts the original rigid apo state to a 'primed' (non-conducting) state, poised for activation, which then is activated for pore conduction by binding of ATP. The natural product ryanodine locks the channel in an ionconducting state. Intriguingly, dilation of the pore occurs by bowing and distortion of the S6 helix from a point midway through the membrane. The binding site and mechanism of competitive partial agonists nucleosides and nucleotides is now clear. A small molecule, ARM210, discovered based on these structures, is in clinical trials for RyR-mediated myopathies [97] .

Intasome. The HIV intasome mediates the integration of the viral genetic material into the host genome and is the target of integrase strand-transfer inhibitors (INSTIs), such as the secondgeneration drugs bictegravir and dolutegravir. Recent cryo-EM studies have revealed how these compounds, as well as other related molecules, are bound to integrase ( Figure 4J ) [98, 99] , coordinating two Mg 2+ ions in the active site. The structure of a resistant mutant in complex with bictegravir provides insights into the mechanism of resistance.

Prospects and summary: The recent revolution in cryo-EM has produced an explosion of structures at near-atomic or better resolution. Further developments in applying cryo-EM to smaller proteins, in preparing cryo-grids for data collection [100, 101] , and in automation of data collection and image analysis will further enhance the power and applicability of this technology. The establishment of national EM facilities should make it easier to access the top-end instruments for data collection and reconstruction at high resolution. Moreover, recent developments suggest that electron microscopes at 100 kV could be sufficient to produce high quality structures, which would reduce the cost of instruments and make them more widely available [102] .

In terms of impact on drug discovery, we expect cryo-EM has the greatest future potential in cases where the advantages of cryo-EM over other structural approaches are most needed:

1. Where ligand binding site is unknown, and where ligand-induced conformational changes are critical for understanding mechanism, like ion channels 2. In cytoplasmic proteins where the target may be one or two proteins in a large oligomeric structure. This includes not only the viral proteins mentioned above, but also cases like the inflammasome [103] , and the actin-myosin complex [104] , microtubules [105] and the ribosome [106] . 3. Where the SAR has seemed intractable and/or where optimization of molecules has not achieved the target profile, 4. where the targets are resistant to crystallization.

In focusing here on cryo-EM, we are not diminishing the impact of X-ray crystallography. The methods are complementary in many ways. For example, the first X-ray structure of the ribosome relied on information from earlier cryo-EM structures. [4, 5] . Fitting cryo-EM density of large complexes often relies on X-ray structures of individual proteins, for example [70, 78] . A recurring question is "in those cases where the same protein has been studied via X-ray crystallography and cryo-EM, how do those structures compare?". We are not aware of any systematic studies of this fascinating question. However, for those instances where a comparison can be made, in general the significant differences are in flexible loops on the surface, e.g. [69, 70] . These loops in solution may not have a well-defined structure anyway.

Another question is whether cryo-EM will be able to support the rapid iterative cycles that crystallography is often capable of in structure-based design. At this point, there is little in the published literature that allows us to properly address that. Private communications from those performing such work in pharma R&D organizations indicates that a wave of publications is imminent, which will hopefully shed light on this issue.

The new cryo-EM structures have provided unprecedented insights into the molecular mechanisms of complex biochemical processes. They have also had a profound impact on drug discovery, defining the binding modes and mechanisms of action of well-known drugs as well as driving the design and development of new compounds; they provide compelling opportunities for further optimization of existing molecules. Most excitingly, the latest cryo-EM technology has produced structures at 1.2 Å resolution, which demonstrates the power of this technology and should lead to resolution improvements in most cryo-EM structures. Therefore, we are convinced that cryo-EM will play an ever-increasing role in drug discovery in the coming years.

Funding: Research in the Tong laboratory is funded by the NIH (R35GM118093), Nimbus Therapeutics and Kintor Pharmaceuticals. JVD did not receive any specific grants from funding agencies in the public, commercial, or not-for-profit sectors.

TABLE S1. A complete listing of the published structures with bound drug-like small molecules. (C). 9 Å X-ray structure of the large subunit [4] ; (D). 2.4 Å X-ray structure of the large subunit [5] ; (E). a 3.5 Å X-ray structure of the antibiotic linezolid bound to the 50S subunit [6] ; (F). a cryo-EM structure of the full ribosome at 2.4-3.5 Å. [7] The nascent polypeptide chain is shown in magenta, and mRNA density is shown in red (it protrudes in the direction of the viewer). The P site tRNA is green, the small subunit in yellow, and large subunit in light blue. [42] reprinted with permission) Figure 9 . Structure of TRPV1 in complex with vanilloid ligand and spider toxin a.TRPV1 channel, bottom view. Note that S1-S4 domains flank and interact with S5-P-S6 pore modules from adjacent subunit, reminiscent of voltage-gated ion channels. b.Resiniferatoxin (RTX) c.Vanillotixin (DkTx, spider toxin) d. TRPV1 channel, bound to RTX (red sphere) and vanillotoxin (magenta). (from [46] , reprinted with permission) Figure 10 . a. Model for TRPV1 activation (from [46] reprinted with permission)) b. TRPV1 antagonist designed from this structure (from [49] ) Figure 11 . Structure of CFTR. Ivacaftor (green) bound to CFTR, viewed from the membrane. The first nucleotide binding domain (NBD1) is in yellow. NBD2 in dark blue. The transmembrane helices are light blue, except for the kinked helix TM8 to which ivacaftor binds is shown in orange. F508 is shown in purple. The two ATP molecules, not shown, would be sandwiched in between the NBD's. (from [60] , PDB code 6O2P) The cryo-EM structure shows that glibenclamide (red) is bound in a central cavity of the transmembrane domain of the K-channel, the peripheral sulfonylurea receptor (SUR) subunit (left viewed in membrane; right from extracellular orientation). ATP is in green. (from [90] , their Fig 1 reprinted with permission) . 

Declining Morbidity and Mortality among Patients with Advanced Human Immunodeficiency Virus Infection

Representation of three-dimensionally reconstructed objects in electron microscopy by surfaces of equal density

A model of protein synthesis based on cryo-electron microscopy of the E. coli ribosome

A 9 A˚ Resolution X-Ray Crystallographic Map of the Large Ribosomal Subunit

The Complete Atomic Structure of the Large Ribosomal Subunit at 2.4 Å Resolution

The Oxazolidinone Antibiotics Perturb the Ribosomal Peptidyl-Transferase Center and Effect TRNA Positioning

Mechanism of ribosome stalling during translation of a poly(A) tail

Advances in the Field of Single-Particle Cryo-Electron Microscopy over the Last Decade

Breaking the next Cryo-EM resolution barrier -Atomic resolution determination of proteins! bioRxiv 2020

Unravelling Biological Macromolecules with Cryo-Electron Microscopy

The development of cryo-EM into a mainstream structural biology technique

Cryo-Electron Microscopy: Moving Beyond X-Ray Crystal Structures for Drug Receptors and Drug Development

Resolution Advances in Cryo-EM Enable Application to Drug Discovery

Breaking Cryo-EM Resolution Barriers to Facilitate Drug Discovery

Cryo-EM in Drug Discovery: Achievements, Limitations and Prospects

Single Particle Electron Cryomicroscopy: Trends, Issues and Future Perspective

Building and Refining Protein Models within Cryo-Electron Microscopy Density Maps Based on Homology Modeling and Multiscale Structure Refinement

Membrane protein structural biology in the era of single particle cryo-EM

New tools for automated high-resolution cryo-EM structure determination in RELION-3

cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination

cisTEM, user-friendly software for single-particle image processing

New software tools in EMAN2 inspired by EMDatabank map challenge

Coot: model-building tools for molecular graphics

PHENIX: building a new software for automated crystallographic structure determination

Refinement of macromolecular structures by the maximum-likelihood method

Automated structure refinement of macromolecular assemblies from cryo-EM maps using Rosetta

UCSF ChimeraX: meeting modern challenges in visualization and analysis

ATP-citrate lyase: genetics, molecular biology and therapeutic target for dyslipidemia

ATP citrate lyase (ACLY) inhibitors: an anti-cancer strategy at the crossroads of glucose and lipid metabolism

Efficacy and safety of bempedoic acid in patients with hypercholesterolemia and statin intolerance

An allosteric mechanism for potent inhibition of human ATP-citrate lyase

Crystal Structure of Rhodopsin: A G Protein-Coupled Receptor

High-Resolution Crystal Structure of an Engineered Human β2-Adrenergic G Protein-Coupled Receptor

GPCR drug discovery: integrating solution NMR data with crystal and cryo-EM structures

Glucagon-Like Peptide-1 and Its Class B G Protein-Coupled Receptors: A Long March to Therapeutic Successes

An Overview, Advantages and Therapeutic Potential of Nonpeptide Positive Allosteric Modulators of Glucagon-Like Peptide-1 Receptor

Discovery of ( S )-2-Cyclopentyl-N -((1-Isopropylpyrrolidin2-Yl)-9-Methyl-1-Oxo-2,9-Dihydro-1 H -Pyrrido[3,4-b]Indole-4-Carboxamide (VU0453379): A Novel

Positive Allosteric Modulator (PAM)

Mechanisms of Action and Therapeutic Application of Glucagon-like Peptide-1

Cryo-EM Structure of the Activated GLP-1 Receptor in Complex with a G Protein

Drug Discovery Approaches Targeting the Incretin Pathway

plus company reports from vTv Therapeutics

Activation of the GLP-1 Receptor by a Non-Peptidic Agonist

The Structure of the Potassium Channel: Molecular Basis of K+ Conduction and

Clinical development of TRPV1 antagonists: targeting a pivotal point in the pain pathway

Structure of the TRPV1 Ion Channel Determined by Electron Cryo-Microscopy

TRPV1 Structures in Distinct Conformations Reveal Activation Mechanisms

TRP channel structural biology by cryo-electron microscopy

Transient receptor potential vanilloid type 1 antagonists: a patent review

Note they describe using a homology model of the human form

Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA

Identification of the cystic fibrosis gene: genetic analysis B Kerem

53. Identification of the cystic fibrosis gene: chromosome walking and jumping

Human In Silico Drug Trials Demonstrate Higher Accuracy than Animal Models in Predicting Clinical Pro-Arrhythmic Cardiotoxicity

hERG toxicity assessment: Useful guidelines for drug design. Garrido A

Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding

Antigenicity of the SARS-CoV-2 Spike Glycoprotein

Structure of the RNA-dependent RNA polymerase from COVID-19 virus

Structure of Replicating SARS-CoV-2 Polymerase

Triple combination of interferon beta-1b, lopinavir-ritonavir, and ribavirin in the treatment of patients admitted to hospital with COVID-19: an open-label, randomised, phase 2 trial

2.2 A resolution cryo-EM structure of beta-galactosidase in complex with a cell-permeant inhibitor

Breaking Cryo-EM Resolution Barriers to Facilitate Drug Discovery

Atomic Resolution Cryo-EM Structure of beta-Galactosidase. Structure

Using Cryo-EM to Map Small Ligands on Dynamic Metabolic Enzymes: Studies with Glutamate Dehydrogenase

Fragment-based drug discovery using cryo-EM

Cryo-EM structure of the Plasmodium falciparum 80S ribosome bound to the anti-protozoan drug emetine

Recent advances with 5-HT3 modulators for neuropsychiatric and gastrointestinal disorders

Cryo-EM reveals two distinct serotonin-bound conformations of full-length 5-HT3A receptor

Conformational transitions of the serotonin 5-HT3 receptor

Molecular mechanism of setron-mediated inhibition of full-length 5-HT3A receptor

Structures of the AMPA receptor in complex with its auxilliary subunit cornichon

TAK-137, an AMPA-R potentiator with little agonistic effect, has a wide therapeutic window

A half century of gamma-aminobutyric acid

Structure of a human synaptic GABAA receptor

GABA A receptor signalling mechanisms revealed by structural pharmacology

Structure of the Cardiac Sodium Channel

Structures of human Nav1.7 channel in complex with auxilliary subunits and animal toxins

Anti-diabetic drug binding site in a mammalian KATP channel revealed by cryo-EM

The structural basis for the binding of repaglinide to the pancreatic KATP channel

Molecular basis for ligand modulation of a mammalian voltage-gated Ca2+ channel

Cryo-EM structure of apo and antagonist-bound human Cav3.1

Structure of a mammalian ryanodine receptor

Ryanodine Receptor Structure and Function in Health and Disease

Structural Basis for Gating and Activation of RyR1

Structural basis of second-generation HIV integrase inhibitor action and viral resistance

Current outcomes when optimizing 'standard' sample preparation for singleparticle cryo-EM

High-yield monolayer graphene grids for near-atomic resolution cryoelectron microscopy

CryoEM at 100 keV: a demonstration and prospects

Regulation of signaling by cooperative assembly formation in mammalian innate immunity signalosomes by molecular mimics

Structure of the F-actin-tropomyosin complex

Nucleotide-dependent bending flexibility of tubulin regulates microtubule assembly

Mechanism of Premature Translation Termination on a Sense Codon

TRPV1 structures in nanodiscs reveal mechanisms of ligand and lipid action

Supplemental Information: A table of all current known cryo-EM structures of macromolecules with bound ligand(s)

Cryo-EM structure of the Plasmodium falciparum 80S ribosome bound to the anti-protozoan drug emetine

Drug sensing by the ribosome induces translational arrest via active site perturbation

Structure of the E. coli ribosome-EF-Tu complex at <3 angstrom resolution by Cscorrected cryo-EM

Mechanisms of ribosome stalling by SecM at multiple elongation steps

Structure of the hypusinylated eukaryotic translation factor eIF-5A bound to the ribosome

Ribosome-dependent activation of stringent control

A combined cryo-EM and molecular dynamcis approach reveals the mechanism of ErmBL-mediated translation arrest

Ensemble cryo-EM uncovers inchworm-like translocation of a viral IRES through the ribosome

Structures of the orthosomycin antibiotics avilamycin and evernimicin in complex with the bacterial 70S ribosome

Structural basis for linezolid binding site rearrangement in the Staphylococcus aureus ribosome

Decoding mammalian ribosome-mRNA states by translational GTPase complexes

Mefloquine targets the Plasmodium falciparum 80S ribosome to inhibit protein synthesis

Structural insights into species-specific features of the ribosome from the human pathogen Mycobacterium tuberculosis

Atomic resolution snapshot of Leishmania ribosome inhibition by the aminoglycoside paromomycin

Visualization of chemical modifications in the human 80S ribosome structure

cryoEM-guided development of antibiotics for drugresistant bacteria

Structural insights into the role of diphthamide on elongation factor 2 in mRNA reading-frame maintenance

Structural basis for antibiotic resistance mediated by the Bacillus subtilis ABCF ATPase VmlR

Ribosome-NatA architecture reveals that rRNA expansion segments coordinate N-terminal acetylation

Structural basis of translation inhibition by cadazolid, a novel quinoxolidinone antibiotic

Structural basis for selective stalling of human ribosome nascent chain complexes by a drug-like molecule

Exit tunnel modulation as resistance mechanism of S. aureus erythromycin resistant mutant

Cryo-EM reveals the conformation of a substrate analogue in the human 20S proteasome core

Structure and function based design of Plasmodium-selective proteasome inhibitors

Longrange allosteric regulation of the human 26S proteasome by 20S proteasome-targeting cancer drugs

Structure of human immunoproteasome with a reversible and noncompetitive inhibitor that selectively inhibits activated lymphocytes

Preclinical candidate for the treatment of visceral leishmaniasis that acts through proteasome inhibition

The cryo-EM structure of the SF3b spliceosome complex bound to a splicing modulator reveals a pre-mRNA substrate competitive mechanism of action

Structural basis of transcription inhibition by fidaxomicin (lipiarmycin A3)

Fidaxomicin jams Mycobacterium tuberculosis RNA polymerase motions needed for initiation via RbpA contacts

Structures of an RNA polymerase promoter melting intermediate elucidate DNA unwinding

Structure of the nucleotide exchange factor eIF2B reveals mechanism of memory-enhancing molecule

eIF2B-catalyzed nucleotide exchange and phosphoregulation by the integrated stress response

Near-atomic cryo-EM structure of PRC1 bound to the microtubule

Insights into the distinct mechanisms of action of taxane and nontaxane microtubule stabilizers from cryo-EM structures

Structural differences between yeast and mammalian microtubules revealed by cryo-EM

Mitotic phosphorylation by NEK6 and NEK7 reduces the microtubule affinity of EML4 to promote chromosome congression

Structural determinants of microtubule minus end preference in CAMSAP CKK domains

Near-atomic structure of jasplakinolide-stabilized malaria parasite F-actin reveals the structural basis of filament instability

Structural transitions of F-actin upon ATP hydrolysis at near-atomic resolution revealed by cryo-EM

Structural basis of the filamin A actin-binding domain interaction with F-actin

Hepatitis B virus core protein allosteric modulators can distort and disrupt intact capsids

A novel druggable interpromoter pocket in the capsid of rhino-and enteroviruses

Unexpected mode of engagement between enterovirus 71 and its receptor SCARB2

Cryo-EM structure of pleconaril-resistant rhinovirus-B5 complexed to the antiviral OBR-5-340 reveals unexpected binding site

Modulation of cardiac ryanodine receptor 2 by calmodulin

Cryo-EM reveals two distinct serotonin-bound conformations of full-length 5-HT3A receptor

Serotonin transporter-ibogaine complexes illuminate mechanisms of inhibition and transport

Conformational transitions of the serotonin 5-HT3 receptor

Molecular mechanism of setron-mediated inhibition of full-length 5-HT3A receptor

Structures of the M1 and M2 muscarinic acetylcholine receptor/G-protein complexes

Glycine receptor mechanism elucidated by electron cryo-microscopy

Activation of the GLP-1 receptor by a non-peptidic agonist

Structural mechanism of glutamate receptor activation and desensitization

Structural basis of kainate subtype glutamate receptor desensitization

Mechanisms of channel block in calcium-permeable AMPA receptors

Structural insights into the activation of metabotropic glutamate receptors

Structures of the AMPA receptor in complex with its auxilliary subunit cornichon

Structure of a human synaptic GABAA receptor

Cryo-EM structure of the benzodiazepine-sensitive alpha 1 beta 1 gamma 2A tri-heteromeric GABAA recetor in complex with GABA

Cryo-EM structure of the human alpha 5 beta 3 GABAA receptor

GABAA receptor signalling mechanisms revealed by structural pharmacology

Cryo-EM structure of the serotonin 5-HT1B receptor coupled to heterotrimeric Go

Structure of a signaling cannabinoid receptor 1-G protein complex

Structure of an endosomal signaling GPCR-G protein-beta-arrestin megacomplex

Structural principles of distinct assemblies of the human alpha 4 beta 2 nicotinic receptor

Agonist Selectivity and Ion Permeation in the alpha3beta4 Ganglionic Nicotinic Receptor

Human TRPML1 channel structures in open and closed conformations

Structure and gating mechanism of the transient receptor potential channel TRPV3

Structural basis for PtdInsP2-mediated human TRPML1 regulation

Structural basis of cooling agent and lipid sensing by the cold-activated TRPM8 channel

Structural insights into TRPM8 inhibition and desensitization

Symmetry transitions during gating of the TRPV2 ion channel in lipid membranes

Regulatory switch at the cytoplasmic interface controls TRPV channel gating

Structural Insights into Electrophile Irritant Sensing by the Human TRPA1 Channel

Molecular mechanism of TRPV2 channel modulation by cannabidiol

Structural basis for the modulation of voltagegated sodium channels by animal toxins

Structures of human Nav1.7 channel in complex with auxilliary subunits and animal toxins

Structure of the Cardiac Sodium Channel

Anti-diabetic drug binding site in a mammalian KATP channel revealed by cryo-EM

The structural basis for the binding of repaglinide to the pancreatic KATP channel

Mechanism of pharmacochaperoning in a mammalian KATP channel revealed by cryo-EM

Molecular basis for ligand modulation of a mammalian voltage-gated Ca2+ channel

Cryo-EM structure of apo and antagonist-bound human Cav3.1

Structural identification of a hotspot on CFTR for potentiation

Structural basis of small-molecule inhibition of human multidrug transporter ABCG2

Structure of a zosuquidar and UIC2-bound human-mouse chimeric ABCB1

Cryo-EM structures of a human ABCG2 mutant trapped in ATP-bound and substrate-bound states

Structural insights into substrate and inhibitor discrimination by human P-glycoprotein

Structure of the human LAT1-4F2hc heteromeric amino acid transporter complex

Structural basis for itraconazole-mediated NPC1 inhibition

X-ray and cryo-EM structures of inhibitor-bound cytochrome bc1 complexes for structure-based drug discovery

Structural basis of indisulam-mediated RBM39 recruitment to DCAF15 E3 ligase complex

2.3 A resolution cryo-EM structure of human p97 and mechanism of allosteric inhibition

An allosteric mechanism for potent inhibition of human ATP-citrate lyase

Cryo-EM structures of STING reveals its mechanism of activation by cyclic GMP-AMP

Cryo-EM structure of the complete E. coli DNA gyrase nucleoprotein complex

Structural basis of second-generation HIV integrase inhibitor action and viral resistance

Structural basis for strand-transfer inhibitor binding to HIV intasomes

Fragment-based drug discovery using cryo-EM