NLGase

In 2024, the range of diseases treated by globally approved new drugs was diverse, with cancer and rare diseases leading in the total number of new drug approvals. Several new drugs were also approved in the fields of anti-infectives and cardiovascular diseases. Among the approved small molecule drugs, 8 were designated as "first-in-class" therapies with breakthrough therapy status. These drugs not only possess unique mechanisms of action but also provide significant therapeutic improvements over previous treatments. As the development technologies for peptide and nucleic acid-based drugs have matured, their proportion among FDA-approved new drugs has stabilized. In 2024, the approved nucleic acid-based drugs included the first telomerase inhibitor, Rytelo (imetelstat). This drug targets and binds to the RNA template of telomerase, inhibiting its activity. It received FDA approval in June 2024 for the treatment of adult patients with myelodysplastic syndromes (MDS). A major challenge for peptide-based drugs is their susceptibility to degradation in the human body, making it difficult to maintain their efficacy. However, advancements in peptide modification technologies have led to longer half-lives for these drugs, offering patients long-acting therapies with reduced treatment burdens. Yorvipath, a long-acting parathyroid hormone prodrug approved in 2024, links the parathyroid hormone prodrug to a polyethylene glycol carrier to control the hormone's release in the body. It received FDA approval in August 2024 as the first treatment for adult hypoparathyroidism. In 2024, CBER approved 8 cell and gene therapies. These therapies not only represent technological innovations but also provide the first approved treatments for patients with various types of diseases. Amtagvi (lifileucel): On February 16, 2024, the U.S. FDA granted accelerated approval to Iovance Biotherapeutics' tumor-infiltrating lymphocyte (TIL) therapy, Amtagvi (lifileucel), for the treatment of advanced melanoma. Amtagvi is the first approved TIL therapy and the first T-cell therapy approved for solid tumors, marking another milestone in cell therapy. Lenmeldy (atidarsagene autotemcel): On March 18, 2024, the FDA approved Lenmeldy (atidarsagene autotemcel, arsa-cel), a gene therapy developed by Orchard Therapeutics, for the treatment of pediatric patients with metachromatic leukodystrophy (MLD) who meet specific criteria. This is the first FDA-approved gene therapy for this rare genetic disorder. Tecelra (afamitresgene autoleucel): On August 1, 2024, the FDA approved Adaptimmune Therapeutics' engineered T-cell therapy, Tecelra (afami-cel), for the treatment of adult patients with advanced MAGE-A4+ synovial sarcoma of certain HLA types who have previously received chemotherapy. Tecelra is the first engineered T-cell therapy approved for solid tumors and the first effective treatment for synovial sarcoma in over a decade. Kebilidi (eladocagene exuparvovec): On November 13, 2024, the FDA granted accelerated approval to Kebilidi (eladocagene exuparvovec), a gene therapy developed by PTC Therapeutics, for the treatment of pediatric and adult patients with aromatic L-amino acid decarboxylase deficiency (AADCD), regardless of disease severity. This is the first FDA-approved gene therapy administered via direct injection into the brain. Ryoncil (remestemcel): On December 18, 2024, the FDA approved Ryoncil (remestemcel), developed by Mesoblast, for the treatment of steroid-refractory acute graft-versus-host disease (SR-aGVHD) in pediatric patients aged 2 months and older. The FDA's press release highlighted that this is the first FDA-approved mesenchymal stromal cell (MSC) therapy. In addition, the FDA approved Moderna's respiratory syncytial virus (RSV) vaccine, Mresvia, marking the first approval of an mRNA vaccine for a disease other than COVID-19, demonstrating the potential of mRNA technology. Top 10 Potential Blockbuster Therapies of 20241. Rezdiffra (resmetirom): The First FDA-Approved Therapy for Metabolic Dysfunction-Associated Steatohepatitis (MASH) Rezdiffra (resmetirom), developed by Madrigal Pharmaceuticals, is a "first-in-class," once-daily, oral thyroid hormone receptor (THR)-β selective agonist designed to target the underlying causes of MASH. Thyroid hormone plays a central role in liver function by activating β receptors in hepatocytes, influencing a range of health parameters, from serum cholesterol and triglyceride levels to the pathological accumulation of fat in the liver. Rezdiffra received FDA approval in March 2024, becoming the first approved therapy for MASH and marking a significant milestone in this field. 2. Winrevair (sotatercept): A "First-in-Class" Therapy for Pulmonary Arterial Hypertension Winrevair is a "first-in-class" activin receptor type IIA (ActRIIA) fusion protein. It combines a modified extracellular domain of ActRIIA with the Fc region of an antibody. This therapy blocks the binding of activin to its cell membrane receptors, thereby reducing activin-mediated signaling. Merck acquired this innovative therapy through its approximately $11.5 billion acquisition of Acceleron Pharma in 2021. Winrevair received FDA approval in March 2024 for the treatment of pulmonary arterial hypertension. Merck believes it has the potential to become a cornerstone therapy for this condition. 3. Anktiva: A "First-in-Class" IL-15 Receptor Agonist for Non-Muscle Invasive Bladder Cancer (NMIBC) Anktiva, developed by ImmunityBio's Altor BioScience, consists of an IL-15 mutant (IL-15N72D) bound to an IL-15 receptor α/IgG1 Fc fusion protein. Compared to natural IL-15, Anktiva exhibits superior pharmacokinetic properties in patients, persists longer in lymphoid tissues, and demonstrates enhanced anti-tumor activity. In April 2024, Anktiva received FDA approval for use in combination with Bacillus Calmette-Guérin (BCG) to treat adult patients with BCG-unresponsive NMIBC accompanied by carcinoma in situ (CIS). In clinical trials, patients treated with Anktiva achieved a complete response rate of 62%. 4. mResvia: The First mRNA Respiratory Syncytial Virus (RSV) Vaccine Moderna's mRNA-based RSV vaccine, mResvia (mRNA-1345), received FDA approval in May 2024 for protecting adults aged 60 and older from lower respiratory tract disease (RSV-LRTD) caused by RSV infection. This is Moderna's second FDA-approved mRNA vaccine. It consists of an mRNA sequence encoding a stabilized prefusion F glycoprotein, delivered using the same lipid nanoparticle (LNP) technology as Moderna's COVID-19 vaccine. 5. Rytelo (imetelstat): The First Telomerase Inhibitor In June 2024, the U.S. FDA approved Geron's "first-in-class" telomerase inhibitor, Rytelo (imetelstat), for the treatment of adult patients with low- to intermediate-1-risk myelodysplastic syndromes (MDS). This approval marked the arrival of the first telomerase-targeted therapy, nearly 40 years after the discovery of telomerase in 1984. Telomeres are protective caps at the ends of chromosomes that naturally shorten with each cell division. In MDS, abnormal bone marrow cells often express telomerase, which rebuilds telomeres, leading to uncontrolled cell division. Preclinical and clinical data show that Rytelo inhibits telomerase activity, curbing the uncontrolled proliferation of cancerous stem and progenitor cells and inducing apoptosis in malignant cells, demonstrating disease-modifying activity. 6. Ohtuvayre (ensifentrine): The First Inhaled Therapy with a Novel Mechanism of Action for COPD Maintenance Treatment in Over 20 Years Ohtuvayre, developed by Verona Pharma, is a "first-in-class" dual phosphodiesterase 3/4 (PDE3/4) inhibitor. It received FDA approval in June 2024 as a maintenance treatment for adult patients with chronic obstructive pulmonary disease (COPD). Ohtuvayre is the first inhaled COPD therapy to combine bronchodilator and non-steroidal anti-inflammatory effects. Additionally, it represents the first inhaled therapy with a novel mechanism of action for COPD maintenance treatment in over two decades. Ohtuvayre works by inhibiting PDE3, leading to relaxation of airway smooth muscles and bronchodilation, while its inhibition of PDE4 reduces inflammation by decreasing the release of pro-inflammatory cytokines. 7. Kisunla (donanemab): The First Alzheimer’s Disease (AD) Therapy Allowing Treatment Discontinuation Upon Meeting Criteria Kisunla, developed by Eli Lilly, is an anti-amyloid monoclonal antibody that specifically binds to the N3pG subtype of amyloid. By targeting this subtype, Kisunla selectively binds to amyloid plaques in the brain, facilitating their clearance. In July 2024, it received FDA approval for the treatment of adult patients with early symptomatic AD, including those with mild cognitive impairment (MCI) or mild dementia stage AD, confirmed by the presence of amyloid pathology. According to the press release, Kisunla, administered monthly, is the first amyloid plaque-targeting therapy with evidence supporting treatment discontinuation after amyloid plaque clearance, potentially reducing treatment costs and the frequency of infusions. 8. Ebglyss (lebrikizumab): A New First-Line Biologic Therapy for Atopic Dermatitis Ebglyss, co-developed by Almirall and Eli Lilly, is an anti-IL-13 antibody. It received FDA approval in September 2024 for the treatment of moderate-to-severe atopic dermatitis (AD) in adults and adolescents (aged 12 years and older, weighing at least 40 kg) whose disease remains uncontrolled despite topical treatments. IL-13 is a key cytokine in AD, driving type 2 inflammation in the skin, which leads to impaired skin barrier function, itching, skin thickening, and infections. Ebglyss's targeted mechanism of action, along with its demonstrated efficacy and safety in both short- and long-term use, makes it a compelling option for patients with moderate-to-severe AD inadequately controlled by topical therapies. Its once-monthly maintenance dosing offers convenience for patients. 9. Cobenfy (xanomeline, trospium chloride): The First Novel Mechanism Drug for Schizophrenia in Decades Cobenfy received FDA approval in September 2024 for the treatment of adult patients with schizophrenia. The FDA press release highlighted that Cobenfy is the first antipsychotic drug targeting cholinergic receptors, diverging from the long-standing standard therapies that target dopamine receptors. This approval marks the first novel mechanism drug for schizophrenia in decades. Cobenfy combines two active ingredients, xanomeline and trospium chloride, designed to activate muscarinic acetylcholine receptors in the brain while minimizing peripheral effects. By stimulating M1 and M4 muscarinic receptors, xanomeline alleviates negative symptoms such as apathy and reduced social drive, improves cognitive function, and helps manage other psychiatric symptoms like hallucinations and delusions. Originally developed by Karuna Therapeutics, Cobenfy was acquired by Bristol Myers Squibb in December 2023 through a merger agreement. 10. Attruby (acoramidis): The First Approved Product with Labeling Indicating Near-Complete Stabilization of Transthyretin (TTR) In November 2024, the FDA approved Attruby (acoramidis), developed by BridgeBio Pharma, for the treatment of adults with transthyretin-mediated amyloidosis with cardiomyopathy (ATTR-CM) to reduce cardiovascular mortality and cardiovascular-related hospitalizations. The press release noted that Attruby is the first approved product with labeling indicating near-complete stabilization of TTR. The drug is designed to mimic the protective function of the TTR T119M mutation, maintaining the normal tetrameric conformation of TTR and preventing the formation of toxic amyloid deposits. In a Phase 3 clinical trial, Attruby reduced all-cause mortality and recurrent cardiovascular-related hospitalizations by 42% compared to placebo at 30 months of treatment. How to obtain the latest research advancements in the field of biopharmaceuticals? In the Synapse database, you can keep abreast of the latest research and development advances in drugs, targets, indications, organizations, etc., anywhere and anytime, on a daily or weekly basis. Click on the image below to embark on a brand new journey of drug discovery!

In addition, Azafaros will host a satellite symposium on nizubaglustat on Day 2 of the symposium, discussing nizubaglustat’s profile and plans for global Phase 3 studies Six presentations will showcase nizubaglustat’s therapeutic potential for Lysosomal Storage Disorders, including Niemann-Pick disease type C (NPC) and GM1/GM2 gangliosidoses WORLDSymposium™ 2025 takes place from February 3 to 7 in San Diego, California Leiden, Netherlands, November 19, 2024 - Azafaros B.V., a clinical-stage biotechnology company focused on rare diseases and lysosomal storage disorders, today announced that advancements with its lead asset, nizubaglustat, will feature prominently on the agenda at the upcoming WORLDSymposium™ 2025, the pre-eminent conference on lysosomal diseases. An oral presentation will highlight the results of the company’s Phase 2 RAINBOW study. Additionally, five poster presentations (one being a collaboration with patient organisations) will showcase nizubaglustat’s promise as an investigational therapy for NPC and GM1/GM2 gangliosidoses. A satellite symposium will further discuss the clinical path for the development and profile of nizubaglustat, and outline plans for pivotal Phase 3 clinical trials next year. Azafaros' presence at the conference offers a timely platform to engage key stakeholders regarding our upcoming global Phase 3 studies for GM1 and GM2 gangliosidoses, as well as NPC. These studies, set to be conducted across major regions including the United States, Europe, Latin America, and other select countries, are anticipated to commence in 2025. Oral Presentation: The RAINBOW study, a Phase 2 investigation of nizubaglustat in Niemann-Pick Type C and GM2 gangliosidosis, will be presented in an oral session on February 6. The data underscore nizubaglustat’s potential as a therapeutic option, reinforcing Azafaros’ commitment to addressing critical unmet needs in lysosomal storage disorders. Satellite Symposium on Nizubaglustat and Phase 3 Development Azafaros will host a satellite symposium on February 4 at 12:15, creating an opportunity to engage key opinion leaders, healthcare professionals and patient advocacy organizations in Azafaros’ clinical roadmap. This forum will provide insights into the results from the RAINBOW study and outline the company’s Phase 3 global development plans for nizubaglustat. Poster Presentations: February 5 Seizures: Significant co-morbidity for patients and families living with Niemann-Pick Type C: Co-authored with the UK Niemann-Pick patient group, this poster explores the significant burden seizures impose on patients and families. Nizubaglustat, a novel GCS and NLGase inhibitor, significantly increases survival in a mouse model of GM1 Gangliosidosis: Preclinical data demonstrate that nizubaglustat significantly extends survival in a GM1 mouse model, providing promising insights into its potential therapeutic effects. February 6 Gangliosidoses: Understanding disease evolution in GM1 and GM2: Data from PRONTO, the largest prospective natural history study in these diseases, enhance the understanding of disease progression in these rare lysosomal conditions. Application of a natural history study on an investigational drug in clinical development: This analysis compares data from the RAINBOW and PRONTO studies, illustrating the role of natural history studies in guiding drug development. “Fact Checking" with the Patient Community: An Industry-Advocacy Collaboration for Optimizing Trial Design: This poster is the result of a collaboration between Azafaros and two US organisations, National Tay-Sachs & Allied Diseases Association (NTSAD) and the National Niemann-Pick Disease Foundation (NNPDF), as well as two UK organisations, the CATS Foundation and NPUK. “We are honoured to present our research at such a prominent conference as the WORLDSymposium™ 2025” said Stefano Portolano, Chief Executive Officer at Azafaros. “These presentations, including the Phase 2 RAINBOW study data, reflect the promise of our late-stage asset nizubaglustat to provide a potential new therapeutic treatment to address a large unmet need for patients with certain types of lysosomal storage disorders. We look forward to highlighting important nizubaglustat data and collaborating with the medical community and patient associations dedicated to improving the lives of those impacted by these rare diseases.” About nizubaglustat Nizubaglustat is a small molecule, orally available and brain penetrant azasugar with a unique dual mode of action, developed as a potential treatment for rare lysosomal storage disorders with neurological involvement, including GM1 and GM2 gangliosidoses and Niemann-Pick disease type C (NPC). Nizubaglustat has received the following designations and support: United States Food and Drug Administration (FDA) Rare Pediatric Disease Designations (RPDD) for the treatment of GM1 and GM2 gangliosidoses and NPC. Orphan Drug Designations (ODD) for GM2 gangliosidosis (Sandhoff and Tay-Sachs Diseases) and NPC. Fast Track Designation and IND clearance for GM1 and GM2 gangliosidoses and NPC European Medicines Agency (EMA) Orphan Medicinal Product Designation (OMPD) for the treatment of GM2 gangliosidosis. UK Medicines and Healthcare Products Regulatory Agency (MHRA) Innovation Passport for the treatment of GM1 and GM2 gangliosidoses. About GM1 and GM2 gangliosidoses GM1 gangliosidosis and GM2 gangliosidosis (Tay-Sachs and Sandhoff diseases) are lysosomal storage disorders caused by the accumulation of GM1 or GM2 gangliosides respectively, in the central nervous system (CNS), resulting in progressive and severe neurological impairment and premature death. These diseases mostly affect infants and children, and no disease-modifying treatments are currently available. About Niemann-Pick disease type C (NPC) Niemann-Pick disease type C is a progressive, life-limiting neurological lysosomal storage disorder caused by mutations in the NPC1 or NPC2 gene and aberrant endosomal-lysosomal trafficking, leading to the accumulation of various lipids, including gangliosides in the CNS. The onset of disease can happen throughout the lifespan of an affected individual, from prenatal life through adulthood. About Azafaros Azafaros is a clinical-stage company founded in 2018 with a deep understanding of rare genetic disease mechanisms using compound discoveries made by scientists at Leiden University and Amsterdam UMC and is led by a team of highly experienced industry experts. Azafaros aims to build a pipeline of disease-modifying therapeutics to offer new treatment options to patients and their families. By applying its knowledge, network and courage, the Azafaros team challenges traditional development pathways to rapidly bring new drugs to the rare disease patients who need them. Azafaros is supported by a syndicate of leading Dutch and Swiss investors including Forbion, BioGeneration Ventures (BGV), BioMedPartners, Asahi Kasei Pharma Ventures, and Schroders Capital. For further information: Azafaros B.V. Email: info@azafaros.com www.azafaros.com

On May 16th, researchers led by Professor Brian K. Shoichet from the University of California, San Francisco, and Professor Bryan L. Roth from the University of North Carolina at Chapel Hill, published a research paper titled "AlphaFold2 Structures Guide Prospective Ligand Discovery" in the journal Science. The researchers conducted a prospective docking of extensive compound libraries against the unoptimized AlphaFold2 (AF2) models of the σ2 receptor and the 5-HT2A receptor. They tested hundreds of new molecules and compared the results to those obtained from docking against experimental structures. High and comparable hit rates and affinities were observed for both experimental structures and AF2 models. Cryo-electron microscopy was used to determine the structure of an efficient 5-HT2A ligand identified in the AF2 docking. This revealed adaptive residue adjustments similar to those predicted by AF2. Although AF2 models may capture conformations different from experimental structures, these conformations are still low-energy states and are crucial for ligand discovery. This expands the scope of structure-based ligand discovery. Research BackgroundAlphaFold2's breakthroughs in protein structure prediction have demonstrated unprecedented capabilities, enabling atomic-level accuracy in structural predictions and scalable operations. As of now, the AlphaFold database contains protein structure information of the human proteome and proteins from 47 other organisms, encompassing over 200 million proteins and nearly all potential therapeutic protein targets. These structures have shown immense value in applications such as structural biology, protein design, protein-protein interactions, target prediction, protein function prediction, and the study of biological mechanisms. However, the impact of AF2 structures on structure-based ligand discovery remains unclear. In ligand binding sites, high fidelity to low-energy conformations is considered critical. Even relatively minor errors, which might be inconsequential for other applications, can interfere with ligand recognition and conformation prediction. Retrospective studies suggest that docking experiments with unoptimized AF2 models struggle with identifying known ligands and predicting their conformations compared to benchmark molecules. However, they perform better in direct comparisons with experimental structures. A drawback of such studies is their susceptibility to historical bias: known ligands may influence the conformations taken by experimental structures when determining complex structures, and experimental structures may, in turn, affect the process of new ligand exploration. Research ProtocolFirst, the researchers compared the AF2 model's structural prediction of the σ2 receptor (EXPERA) and the 5-HT2A receptor (GPCRs). Because the AF2 model made these predictions before the crystal structures of these receptors were published, this helps to minimize potential bias. For the σ2 receptor, the AF2 model accurately predicted the side-chain conformations of all positive site residues, with a root mean square deviation (RMSD) from the crystal structure of only 1.1 Å. Only a few residues had individual RMSD values greater than 1.5 Å. Regarding the 5-HT2A receptor, while most binding site residues were well predicted (with RMSD from the cryo-EM structure being less than 2 Å), two residues had RMSDs ranging from 2.5 to 3.1 Å, adopting different rotamers. Subsequently, the researchers used DOCK3.8 software to sample millions of potential ligand poses and scored each molecular pose based on physical fitness, typically targeting a predefined and fixed binding site. In DOCK3.8, the fit of ligands to the protein is assessed by calculating two types of interactions: electrostatic interactions using a probe charge model based on the Poisson-Boltzmann equation, and van der Waals interactions using the AMBER force field, with corrections for ligand desolvation and conformational strain. For the crystal structure of the σ2 receptor, the researchers previously screened 490 million dockable molecules from the ZINC20 database. Among the top 300,000 ranked molecules (0.06% of the ranked database), 138 high-scoring molecules were selected for radioligand competition assays, of which 70 displaced over 50% of the known ligand [³H]-DTG at a concentration of 1μM—a hit rate of 51%. The best 21 active molecules had Ki values ranging from 1.8 nM to low μM, with 19 having Ki < 50 nM, and 6 having Ki < 5 nM. Meanwhile, the researchers docked the same library of 490 million molecules against the AF2 model of the σ2 receptor and selected 119 new molecules from the top 30,000 dockings for synthesis and testing. Of these, 64 molecules displaced more than 50% of specific [³H]-DTG σ2 binding at 1 μM concentration, resulting in a hit rate of 54%. Although this rate is slightly higher than the 51% observed in the crystal structure docking test, there was no significant difference between the two hit rates based on a z-test. The Ki values of the top 18 hits from the AF2 test ranged from 1.6 nM to 84 nM, with 13 having Ki values less than 50 nM and 2 less than 5 nM. When the researchers redocked the 490 million molecules to the σ2 AF2 model, the previously high-ranking 138 molecules dropped in rank to such an extent that they no longer appeared within the top 30,000. This likely reflects a slight contraction in the AF2 positive site compared to the crystal structure, with the computed volume reducing from 268 ų to 213 ų. Correspondingly, when using the crystal structure (logAUC: 39) versus the AF2 model (logAUC: 16) to dock known σ2 ligands from the ChEMBL database along with a property-matched decoy library, the crystal structure yielded higher enrichment. Similar results have been observed in retrospectives involving cryo-EM and AF2 structures of the 5-HT2A receptor: experimental structures outperformed AF2 structures in the retrospective enrichment of known ligands. Unlike the situation with the σ2 receptor, the AF2 model of the 5-HT2A receptor shows significant rotameric variations at several key residues compared to the cryo-EM structure. Although the RMSD (Root-Mean-Square Deviation) of most key residues is less than 1.5 Å, critical residues such as Phe-2345.38 on TM5 and Leu-22945.52 in the ECL2 loop show deviations of 2.5 Å and 3.1 Å, respectively, and adopt different rotamers in the two structures. This difference likely arises because the AF2 model leverages the overall sequence similarity within the 5-HT receptor family, whereas the cryo-EM structure represents the active state of the receptor in complex with Gαq, while the AF2 model is closer to the inactive state. Researchers expect that the experimentally determined structure is more likely to select agonists with 5-HT2A selectivity and Gαq bias. Subsequently, researchers docked over 1.6 billion molecules into both the cryo-EM and AF2 receptor structures. They considered the top 3 million molecules (top 0.2%) for each structure based on their docking scores. Consistent filtering, clustering, and hit selection criteria were applied to these molecules to narrow down to a set of priority compounds for synthesis and testing. Ultimately, 223 high-ranking molecules resulting from docking to the cryo-EM structure and 161 high-ranking molecules from docking to the AF2 structure were synthesized. In preliminary radioligand binding screening assays, out of 223 molecules docked against cryo-EM structures, 51 substituted more than 50% of [3H]-LSD at a docking ligand concentration of 10 μM, with a hit rate of 23%. Additionally, out of 161 molecules docked against AF2 models, 42 met this threshold, resulting in a hit rate of 26%. Unexpectedly, the three highest affinity compounds (15 to 24 nM) all came from AF2 docking, whereas the three best compounds from cryo-EM docking had affinities approximately five times weaker (ranging from 71 to 114 nM). Despite the higher hit rate from AF2 docking, there was no significant difference in hit rates between AF2 and experimental docking based on the z-test. Moreover, among the 93 active molecules common to both tests, no two shared the same scaffold, with an average pairwise similarity (Tc) of 0.27, akin to randomness. Given that the 5-HT receptor has multiple subtypes in the human body, to evaluate the subtype selectivity of the molecules, researchers assessed the functional activity (including agonist or antagonist activity) of 338 priority molecules from AF2 and experimental docking. This included subtype selectivity across 5-HT2A, 5-HT2B, and 5-HT2C receptors, as well as ligand bias between Gαq activation or β-arrestin2 recruitment. Upon treatment with 3 μM of each compound, 10 compounds from the cryo-EM docking group and 6 compounds from the AF2 docking group exhibited activity as 5-HT2A agonists, defined as ≥10% of the 5-HT response; additionally, another 10 compounds (5 cryo-EM and 5 AF2) and 6 compounds (3 cryo-EM and 3 AF2) were identified as 5-HT2B and 5-HT2C agonists, respectively. In antagonist screening mode at 3 μM, 5 compounds (1 cryo-EM and 4 AF2) antagonized 5-HT2A receptor activity, defined as ≥20% of Clozapine activity. Furthermore, 17 compounds (10 cryo-EM and 7 AF2) and 1 compound (cryo-EM) were discovered to have antagonist activity on 5-HT2B and 5-HT2C receptors, respectively. Further activity studies revealed that the potency (pEC50 values) of top agonists from the cryo-EM docking group ranged from 246 nM to 3 µM, while those from the AF2 docking group ranged from 42 nM to 1.6 µM. Among the top five AF2 agonists, three (Q2118, Z7757, and Z2504) exhibited subtype selectivity for the 5-HT2A receptor over the 5-HT2B and 5-HT2C receptors. In contrast, none of the top cryo-EM agonists demonstrated selectivity for the 5-HT2A receptor. During comprehensive dose-response studies, the AF2-derived agonists emerged as the most effective and selective molecules. Research SummaryContrary to expectations, the study results indicate that large library docking using the AF2 model is equally effective as docking using experimental structures. For the σ2 and 5-HT2A receptors, hundreds of molecules were experimentally tested on both structures, yielding a high hit rate with no significant differences between the AF2 model and the experimental structures. Comparisons of the affinity of these molecules led to similar conclusions. Specifically, for the 5-HT2A receptor, compounds generated using the AF2 structure might even be more potent and selective, with docking hits showing sub-nanometer EC50 values that rank among the most potent and selective molecules in extensive structure-based docking for this target. Cryo-EM structures validated the model predictions for the novel agonist Z7757: the docking predictions overlapped well with the new cryo-EM structure positions, and some key structural differences between the original AF2 model and the lisuride/5-HT2A cryo-EM structure were reproduced in the new Z7757/5-HT2A complex's cryo-EM structure. These observations are consistent with the view that the AF2 model captures low-energy, accessible states of the 5-HT2A receptor. In conclusion, the authors propose that alternative conformations sampled by the AF2 model capture low-energy conformations, which are useful for identifying ligands different from those identified by the experimental structures. In docking tests against the 5-HT2A receptor, the functional properties of hits from AF2 docking were not inferior to those from experimental structure docking. Notably, three 5-HT2A receptor hits derived from AF2 model docking were subtype-selective, a feature not observed in docking against the cryo-EM structures. The AF2 model can expand the range of proteins targeted by structure-based drug discovery and broaden the spectrum of effective chemical types discovered. How to obtain the latest research advancements in the field of biopharmaceuticals? In the Synapse database, you can keep abreast of the latest research and development advances in drugs, targets, indications, organizations, etc., anywhere and anytime, on a daily or weekly basis. Click on the image below to embark on a brand new journey of drug discovery!