Cryo-EM Success in Pharma

Want to learn how your drug discovery can benefit from cryo-EM? Learn from your peers’ successes. The benefits of this method have been proven in publications from pharmaceutical companies, showing how cryo-EM delivers previously unattainable structural insights needed to accelerate drug discovery.

The following recent publications provide proof of how cryo-EM delivered critical structural insights in different disease areas for a wide range of drug targets. In several cases, cryo-EM provided the insight that was unattainable with other methods, thus accelerating the path to drug discovery.

After you read about your peers' successes below, you might wonder if cryo-EM is a worthwhile investment for you and your company. One way to gauge the viability of cryo-EM is to have the business case and return on investment (ROI) calculated for your specific situation and use case.

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Recent cryo-EM publications:

Amgen: Cryo-EM clarifies synergistic activity of anti-tumor antibodies

Cryo-EM structure is vital for the development of potent bispecific antibodies.


Human epidermal growth factor receptor 2 (HER2) is overexpressed in 20–30% of breast cancer tumors and is associated with a more aggressive disease, higher recurrence rate, and increased mortality. Trastuzumab and pertuzumab are HER2 receptor blockers and have become the standard of care for the treatment of HER2-positive breast cancer. Adding these monoclonal antibodies to the treatment regimen of HER2-positive breast cancer has changed the paradigm for treatment in that form of cancer. The effectiveness of their synergistic activity has been well validated in research as well as in clinical practice.

Amgen: Cryo-EM clarifies synergistic activity of anti-tumor antibodies
Cryo-EM structure of HER2-trastuzumab-pertuzumab. HER2 (blue-green-red), trastuzumab Fab (magenta), and pertuzumab Fab (orange). No interaction is shown between trastuzumab and pertuzumab. Images of PDB-entry 6OGE created with CCP4mg by Hans Raaijmakers.
Cryo-EM Structure of HER2-trastuzumab-pertuzumab Complex; Hao Y, Yu X, Bai Y, McBride HJ, Huang X (2019), PLoS ONE 14(5): e0216095, Published: May 1, 2019.
Objective
Gain structural insight into the synergistic activity of the ternary complex HER2-trastuzumab-pertuzumab.
Benefits of cryo-EM
Instrumental in gaining better understanding of synergistic activity.
Key insights for the design of bispecific molecules with potentially greater clinical efficacy.
Cryo-EM insights complement X-ray crystallography.

Genentech (Roche): Structural basis of selective sodium channel inhibition

Cryo-EM complements crystallography in unraveling selective sodium channel blockers.


Voltage-gated sodium (Nav) channels are targets of disease mutations, toxins, and therapeutic drugs. Mutations in Nav channel subtypes are associated with migraines, epilepsy, pain, and cardiac and muscle paralysis syndromes. Channel blockers lack subtype selectivity and have not been well understood.

Genentech (Roche): Structural basis of selective sodium channel inhibition
Cryo-EM structures clearly show structural differences of ProTx2 bound to activated (blue, magenta) vs. deactivated (black) receptor. Image based on PDB-entry 6n4r and 6n4q created with Pymol by Hans Raaijmakers.
Structural Basis of Nav1.7 Inhibition by a Gating-Modifier Spider Toxin; Xu et al., 2019, Cell 176, 702–715, Published: February 7, 2019.
Objective
Determine key structural templates to design selective Nav channel antagonists using spider protoxin-II (ProTx2).
Benefits of cryo-EM
Cryo-EM analysis independently validated the crystallographic structural model of the receptor site.
Crystallographic structure took many years to solve. Soaking in compounds was not successful, so cryo-EM was the ultimate choice.
Cryo-EM structure supports mechanistic interpretation of ProTx2 complex shifting the activation of Nav and pharmacologically stabilizing the closed-channel state.

Novartis: Cryo-EM enables development antibody screen against polyomaviruses

Cryo-EM reveals structure of viral epitope, enabling development of new therapies.

Human polyomaviruses (BK and JC) are responsible for common, persistent infection in kidneys during childhood. This dormant infection presents minimal clinical manifestations. However, the virus re-activates under immunodeficient conditions, causing nephropathy and hemorrhagic cystitis. A new, high-throughput, functional antibody screen was developed to examine the response to polyomavirus. This approach enabled the isolation of monoclonal antibodies that neutralize BK and JC virus subtypes.

Novartis: Cryo-EM enables development antibody screen against polyomaviruses
Cyro-EM Structure of BK polyomavirus-like particle in complex with single chain antibody. Images of PDB-entry 6GG0 created with Pymol by Hans Raaijmakers.
Human Memory B Cells Harbor Diverse Cross-Neutralizing Antibodies against BK and JC polyomaviruses; Lindner et al., 2019, Immunity 50, 668–676, 2019, Published: February 26, 2019.
Objective
Identify complex binding site of the virus capsid protein, which was not possible with crystallography due to the complex’s quaternary structure.
Benefits to cryo-EM
Cryo-EM reveals the quaternary nature of viral epitope and unravels potent modality for inhibiting polyomavirus infection in kidney transplant recipients and other immunocompromised patients.

Pfizer: Structure of the human frataxin-bound iron-sulfur cluster assembly complex

Cryo-EM provides insight in clinical mutations of Friedreich’s ataxia.


Iron-sulfur clusters (ISC), located in the mitochondria matrix, are five-protein complexes containing the cysteine desulfurase that is activated by frataxin (FXN). Deficiency in FXN leads to Friedreich’s ataxia (FRDA), a genetic neurodegenerative disorder that results in cardiological symptoms, loss of limb sensation, locomotion symptoms, impaired speech, and neurological damage.

Pfizer: Structure of the human frataxin-bound iron-sulfur cluster assembly complex
In the complex, each FXN binds to a cavity formed by the interface of homodimer and one ISC. Colored by B-factor, blue (b=10) white (b=40) red (b=70) and ISCU surface with frataxin. Image of PDB-entry 6NZU created with Pymol by Hans Raaijmakers.
Structure of the human frataxin-bound iron-sulfur cluster assembly complex provides insight into its activation mechanism; Nicholas G. Fox, et al., Nature Communications 10, Article number: 2210 (2019), Published: May 17, 2019.
Objective
Determine complex structure to find out how FXN stabilizes ISC; guide FRDA clinical mutations.
Benefits of cryo-EM
Structure of FXN-bound human complex is one of very few reported cryo-EM structures of < 200 kDa and < 3.5 Å resolution for both membrane and soluble proteins.

Cryo-EM is applied here to a clinically-relevant target that remain intractable for X-ray crystallography.

GSK: Preclinical candidate for Black Fever by parasite enzyme inhibition

Cryo-EM structures reveal new, potent, selective inhibitor of parasite enzyme.


Visceral Leishmaniasis (VL), or Black Fever, is one of the most severe tropical diseases. It is responsible for up to 40,000 deaths annually. Present treatments have serious drawbacks, such as prolonged treatment duration, low tolerability and high teratogenicity, significant geographical variations in effectiveness, and high costs of treatments and cold storage. A new GSK preclinical candidate inhibits chymotrypsin-like activity over the human enzyme while selectively inhibiting the parasite enzyme.

Leishmania tarentolae proteasome 20S subunit
Leishmania tarentolae proteasome 20S subunit. Alpha type subunits in yellow, Beta type subunits in green, endopeptidase in orange, GSK3494245 in blue. Images based on PDB-entry 6qm7 created with Pymol by Hans Raaijmakers.
Preclinical candidate for the treatment of visceral leishmaniasis that acts through proteasome inhibition; Wyllie et al., PNAS May 7, 2019 116 (19) 9318-9323, published: April 8, 2019.
Objective
Identify low-cost, safe, effective, oral, short-course drug for VL based on inhibition of parasite enzyme.
Benefits of cryo-EM
High-resolution cryo-EM structures revealed previously undiscovered binding site for inhibitiors of chymotrypsin-like activity.

Nimbus Therapeutics: Enhanced “druggability” of ATP-citrate lyase

Cryo-EM reveals allosteric site for enhanced ACLY “druggabilty”.


ATP-citrate lyase (ACLY) is a central metabolic enzyme that catalyzes the ATP-dependent conversion of citrate and coenzyme A (CoA) to oxaloacetate and acetyl-CoA1. ACLY is an interesting target for anticancer drugs because cancer cells depend on its activity for proliferation. ACLY is also a target against dyslipidaemia and hepatic steatosis. The structure-activity relationship of targets is explained through binding modes of ACLY tetramers.

3D structure of ACL tetramer at 3.7 Å using cryo-EM. ADP in yellow, inhibitor in Tyrian purple
3D structure of ACL tetramer at 3.7A using cryo-EM. ADP in yellow, inhibitor in Tyrian purple. Image based on PDB-entry 6o0h created with Pymol by Hans Raaijmakers.
An allosteric mechanism for potent inhibition of human ATP-citrate lyase; Jia Wei, et al,; Nature 568, 566–570 (2019), Published: April 3, 2019.
Objective
Study previously undiscovered allosteric ACLY binding site as target for enhanced “druggability” of inhibitors.
Benefits of cryo-EM
First ever high-resolution ACLY structure.
Cryo-EM, along with computational insights, determined full tetrameric ALCY structure with small molecule "druggable" inhibitor.

NIBR*: A small-molecule inhibitor of C5 complement protein

Cryo-EM enabled development of new small-molecule inhibitor for enhanced therapy.


Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, acquired, life-threatening disease leading to the premature death of all blood cells. The human complement component 5 protein (C5) is a validated drug target in an anti-C5 antibody approved therapy (Soliris made by Alexion).

 

Atomic-resolution model derived from cryo-EM structure in the presence of bound inhibitor.
Atomic-resolution model derived from cryo-EM structure in the presence of bound inhibitor. Left: Cryo-EM structure of the C5- Cobra Venom Factor (CVF) complex at 3.35 Å resolution. Complement C5 alpha chain in green, beta chain in orange, cobra venom factor in grey, inhibitor in blue. Right: Electron microscopy density map (blue mesh) for bound inhibitor with binding site residues shown as green sticks. Images of PDB-entry 6i2x created with Pymol by Hans Raaijmakers.
A small-molecule inhibitor of C5 complement protein; Keith Jendza et al., Nature Chemical Biology, VOL 15, 666–668, July 2019.
Objective
Obtain first small-molecule inhibitor of C5 complement protein for new, more potent therapy.
Benefits of cryo-EM
Identified and optimized the mechanism of action for the first small-molecule inhibitor of C5 complement protein.
Growing crystals suitable for X-ray crystallography was unsuccessful. Cryo-EM had to be used in experiments to narrow in on binding region.

Pfizer: Stalling of ribosome nascent chain complexes by a drug-like molecule

Cryo-EM illuminates how stalling ribosome by small molecules opens new therapeutic strategy.


Utilizing small molecules that inhibit protein synthesis by selectively stalling the ribosome is a new strategy for therapeutic development. Structures of human ribosome nascent chain (RNC) complexes, stalled by drug-like molecule PF846, selectively block the translation of a small number of proteins by an unknown mechanism. It was revealed that PF846 binds in the ribosome exit tunnel and alters the path of the nascent polypeptide chain.

Cryo-EM structure of the stalled RNC showing ribosome
Cryo-EM structure of the stalled RNC showing Ribosome, 40S light blue, 60S grey mRNA in red, tRNA in green and blue, nascent chain in magenta with the inhibitor in blue. Image of PDB-entry 6ole created with Pymol by Hans Raaijmakers.
Structural basis for selective stalling of human ribosome nascent chain complexes by a drug-like molecule; Wenfei Li, et al., Nature Structural & Molecular Biology, Vol 26, 501-509, Published: June 2019.
Objective

Determine the structures of RNC complexes.

Benefits of cryo-EM
High-resolution cryo-EM structures reveal how a small molecule selectively stalls the translation of the human ribosome.
Structural foundation for developing therapeutic small molecule inhibitors.
Ribosomal complexes are difficult to study via X-ray crystallography due to their size, complexity and conformational flexibility.

Astex: Fragment-based drug discovery (FBDD) using cryo-EM

Cryo-EM utilized for FBDD is making transformative impact on the pharmaceutical industry.


The use of cryo-EM in fragment-based drug discovery (FBDD) is reviewed based on in-house method development. Cryo-EM reveals details of molecular interactions between fragments and protein at sufficient resolution. The current reproducibility, quality, and throughput are compatible with FBDD. The first cryo-EM structure of the oncology target PKM2 was solved.

Cryo-EM structure of the oncology target PKM2.
The first cryo-EM structure of the oncology target PKM2, a pyruvate kinase 2 (PKM2) and bound ligands. PKM2 is significantly up-regulated in tumor cells which makes it a potential target in cancer drug discovery.
Fragment-based drug discovery using cryo-EM; Saur, D.T.D. et al., Drug Discovery Today (2019).
Objective

To demonstrate that cryo-EM can be used for structure-based drug design with throughput necessary to support fragment-based (FBDD) screening.

Benefits of cryo-EM
X-ray crystallography requires protein crystallization which for target classes like membrane proteins and multiprotein complexes is not possible.
Cryo-EM maps can be used for structure-based drug design.
Cryo-EM insights complement X-ray crystallography.

Genentech: Structure of the secretory immunoglobulin A (sIgA) core

Cryo-EM determines dimeric, tetrameric and pentameric sIgA framework for potential antibody therapy.


Secretory immunoglobulin A (sIgA) is the immune system’s first line of defense against mucosal pathogens. IgAs are transported across the epithelium, as dimers and higher-order polymers, by the polymeric immunoglobulin receptor (pIgR). Major focus of biotechnology has been to engineer antibodies to target disease-relevant tissues and therapeutic IgAs may allow delivery to mucosal tissues inaccessible to traditional IgG-based therapeutics.

Cryo-EM structure of dimeric sIgA1, tetrameric and pentameric sIgA2m2.
Cryo-EM structure of dimeric sIgA1 (left), tetrameric (middle) and pentameric sIgA2m2 (right). Transparent maps overlaid with the model are shown. Images of PDB-entries 6ue7 (dimer), 6ue8 (tetramer) and 6ue9 (pentamer) were created with Pymol by Hans Raaijmakers.
Structure of the secretory immunoglobulin A core; N. Kumar et al., Science 10.1126/science.aaz5807 (2020).
Objective

To determine the architecture of sIgA-Fc dimers, tetramers, and pentamers at atomic resolution using cryoEM and propose a mechanism of JC-templated IgA oligomerization and describe pIgR recognition.

Benefits of cryo-EM
Despite extensive studies by other techniques sIgA structures have remained elusive.
Cryo-EM enables detailed structural characterization of sIgA is critical for the development of future therapeutics and vaccines.

Genentech: Structure of CD20 in complex with therapeutic mAb - rituximab

Cryo-EM reveals CD20 structure complex with mAb for therapy of cancer and auto-immune disorders.


Rituximab (MabThera, Rituxan), a chimeric IgG1 monoclonal antibody specifically targets the CD20 surface antigen expressed on normal and neoplastic Blymphoid cells. Rituximab is currently used in the treatment of both follicular and aggressive B-cell non-Hodgkin lymphomas. Despite demonstrated clinical effectiveness, in vivo mechanisms of Rituximab remain unknown and could differ by subtype of lymphoma, with unwanted side effects.

CD20: rituximab complex by cryo-EM.
CD20: rituximab complex by cryo-EM. The structural details revealed the interactions between CD20, a B cell membrane protein dimer, and two Fabs (heavy chain in purple and light chain in pink) in a glyco-diosgenin (GDN) micelle. Image of PDB-entry 6vja was created with Pymol by Hans Raaijmakers.
Structure of CD20 in complex with the therapeutic monoclonal antibody rituximab; L. Rougé et al., Science 10.1126/science.aaz9356 (2020).
Objective

To determine unknown CD20 structure complex with mAb by cryo-EM for improved therapy of malignancies and auto-immune disorders.

Benefits of cryo-EM
Cryo-EM provides new insight in previously unknown structure and function of CD20 as clinically-validated target.
Cryo-EM enabled deciphering binding mode of antibody to the receptor is essential to discover and develop more efficient antibody-based drugs.

Roche: Cryo-EM structure of the rhodopsin-Gαi-βγ complex

Cryo-EM reveals antibody stabilized complex structure of rhodopsin and Gi heterotrimer.


GPCRs modulate cell physiology by activating diverse intracellular transducers, prominently heterotrimeric G proteins. Despite surge in structural data the understanding of GPCR-mediated signal transduction in many aspects, including the existence of transient interactions, remain elusive. The cryo-EM structure of the signaling complex between bovine rhodopsin and a Gi protein heterotrimer, stabilized using an antibody Fab fragment was determined.

Cryo-EM structure of the rhodopsin-Gi-Fab16 complex.
Cryo-EM structure of the rhodopsin-Gi-Fab16 complex. EM density map of the complex (rhodopsin – blue, Gai – green, Gb – yellow, Gg – magenta, Fab16 – white). Image of PDB-entry 6qno was created with Pymol by Hans Raaijmakers.
Cryo-EM structure of the rhodopsin-Gai-bg complex reveals binding of the rhodopsin C-terminal tail to the gb subunit; Tsai et al. eLife 2019; 8:e46041, published June 2019.
Objective

Comparison of the GPCR-G protein binding interface of the residue-residue contacts between the receptor and the a5 helix in all the available GPCR/G protein complexes by means of cryo-EM.

Benefits of cryo-EM
Cryo-EM structure is in excellent agreement with the crystal structure of the same rhodopsin mutant bound to a mini-Go protein helices and the receptor.
Only EM density map provides structural evidence for the interaction between the C-tail of the receptor and the Gb subunit.

Merck: Cryo-EM structure of the insulin receptor – insulin complex

Cryo-EM structure of insulin receptor – insulin complex enables development of more effective therapeutics.


The insulin receptor (IR) is a dimeric protein that plays crucial role in controlling glucose homeostasis, regulating lipid, protein and carbohydrate metabolism, modulating brain neuro- transmitter levels. IR dysfunction has been associated with many diseases, including diabetes, cancer and Alzheimer’s Disease. Until recently, the binding mode of insulin to its receptor was unknown.

Electron density map obtained from cryo-EM (wireframe).
Electron density map obtained from cryo-EM (wireframe). The corresponding receptor-insulin complex structure is overlaid. Structure of insulin receptor in complex with insulin in color and superimposed the insulin structure in grey. Image of PDB-entry 6ce9 was created with Pymol by Hans Raaijmakers.
Structure of the insulin receptor–insulin complex by single-particle cryo-EM analysis; Scapin, G et al., Nature 556, p. 122-125 April 5, 2018.
Objective

Determination of cryo-EM structure of the insulin receptor – insulin complex enables knowledge of binding mechanism, leading to  development of more effective therapeutics.

Benefits of cryo-EM
Single particle analysis (SPA) enabled to ascertain the precise interaction between insulin and the insulin receptor, opening up new avenues in drug discovery.

Thermo Fisher Scientific & Monash U.: Cryo-EM structure of a class B GPCR–G-protein complex

Cryo-EM enables finding new safer and better oral medicines for Type 2 diabetes.


Class B G protein-coupled receptors are major targets for treatment of chronic disease, including diabetes and obesity. Glucagon-like peptide-1 receptor (GLP1R) is an important target for treatment of Type 2 diabetes. Most GLP1R agonists are injectable drugs and therapeutic potential of this receptor is far from exhausted. Structures of active receptors revealed that peptide agonists engage deep within the receptor core.

Cryo-EM density map of a GPCR complex: glucagon-like peptide-1 receptor (GLP1R).
Cryo-EM density map of a GPCR complex: glucagon-like peptide-1 receptor (GLP1R). Non-peptide agonist (TT-OAD2) bound to the Glucagon-Like peptide-1 (GLP-1) Receptor. Image of PDB-entry 6ORV was created with Pymol by Hans Raaijmakers.
Phase-plate cryo-EM structure of a class B GPCR–G-protein complex; Liang, YL et al., Nature v. 546, p. 118-123 June 2017.
Objective

Using cryo-EM identify a new agonist pocket for non-peptides.

Benefits of cryo-EM
Cryo-EM structure of a novel non-peptide agonist bound to the GLP-1 receptor identified an unpredicted non-peptide agonist binding pocket.
Learn from our collaborators how to prepare ideal GPCR cryo-EM specimen. This robust workflow delivers a high success rate for achieving sub-2.5A reconstructions of GPCRs.

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