Understanding Allosteric Mechanisms in Drug Development

Happy to share our collaborative study, led by the Jana Shen Lab, which unravels the mechanism of dimer selectivity and binding cooperativity of BRAF inhibitors using a combination of molecular dynamics simulations and experimental insights. Notably, we highlight the PHI1 inhibitor, developed by our group, which exhibits positive cooperativity, priming the BRAF dimer for enhanced inhibitor binding. https://lnkd.in/egAbiSBv Key findings include: Dimerization reshapes BRAF conformation, stabilizing the αC-helix and increasing DFG flexibility. Hydrogen bond interactions drive dimer selectivity by favoring the αC-in state. PHI1 shows positive cooperativity, preorganizing the opposite protomer for enhanced binding. The study provides a new empirical framework to assess dimer-selective BRAF inhibitors. These insights deepen our understanding of kinase allostery and aid in the design of next-generation RAF inhibitors against mutant BRAF-driven cancers. Excited to see where this leads! Jana Shen Poulikos Poulikakos Evangelia Matenoglou #CancerResearch #BRAF #KinaseInhibitors #ComputationalBiology #DrugDiscovery

Want to understand how protein dynamics influence cellular signaling in #BRAF? The interaction between BRAF and MEK1 is a critical step in the RAF/MEK/ERK pathway, which regulates cell growth and survival. Dysregulation of this pathway is linked to many cancers. Mutations like BRAF V600E cause continuous activation of MEK1 and downstream signaling, driving uncontrolled cell proliferation. Targeting this interaction is a key strategy in #cancer research to disrupt aberrant signaling in BRAF-mutant cancers. So, how does a mutation in the activation loop of BRAF modulate the BRAF-MEK1 interaction? Wild-type BRAF, remains in a dynamic equilibrium between active and inactive states, with the inactive state being predominant. The inactive form interacts with MEK1 and forms an auto-inhibited complex with the 14-3-3 protein, which is key in the signaling pathway. The V600E mutation "allosterically induces a population shift" from the inactive state to the active state, leading to activation of MEK1 and downstream signaling, thereby driving uncontrolled cell proliferation. Imagine having this much prior information before launching a drug discovery campaign. One could design small molecules that shift the population from the active to the inactive state, leading to the development of selective therapeutics against the BRAF V600E mutation, which occurs in over 90% of all #melanoma. Indeed, small molecules like Vemurafenib and Dabrafenib have been designed to precisely shift the population, restoring the autoinhibited BRAF-MEK1 complex. This highlights the power of #protein dynamics and its role in cellular signaling. We are building a generalizable foundation model of protein dynamics at scale, which will elucidate how population shifts between multiple metastable states govern protein-protein interactions. This foundation model will provide unprecedented predictive power to design #precision #therapeutics that can be rapidly advanced to the clinic, impacting millions of lives. #AI #DrugDiscovery #StructuralBiology #ProteinDynamics #cancer #biotech

𝐀𝐭𝐭𝐞𝐧𝐭𝐢𝐨𝐧 𝐓𝐘𝐊2 𝐚𝐧𝐝 𝐉𝐀𝐊 𝐈𝐧𝐡𝐢𝐛𝐢𝐭𝐨𝐫 𝐀𝐟𝐢𝐜𝐢𝐨𝐧𝐚𝐝𝐨𝐬! Our recent study published in the 𝐉𝐨𝐮𝐫𝐧𝐚𝐥 𝐨𝐟 𝐈𝐧𝐯𝐞𝐬𝐭𝐢𝐠𝐚𝐭𝐢𝐯𝐞 𝐃𝐞𝐫𝐦𝐚𝐭𝐨𝐥𝐨𝐠𝐲 explores how allosteric TYK2 inhibitors block immune signaling at the molecular level—revealing a triple-action mechanism: • Stabilizing TYK2 in its inactive (autoinhibited) conformation • Blocking ATP binding at the pseudokinase domain • Preventing formation of the active signaling complex through steric interference In addition, we developed structural models of the TYK2/JAK1 signaling complex, which allowed us to explore how this heterodimer becomes activated and how it phosphorylates STAT proteins. These models provide insight into the structural choreography of cytokine signaling and how it might be disrupted by targeted therapies. These structural insights deepen our understanding of TYK2 regulation and may inform future efforts to develop more selective, pathway-specific therapies targeting IL-12, IL-23, and type I interferon signaling. Grateful to my Yale colleagues Jimin Wang, Ivan Lomakin, and Victor Batista for their contributions to this work. Please read the full article: https://lnkd.in/eigW4TMm #Dermatology #Immunology #Psoriasis #TYK2 #JAKinhibitors #Mechanism #JID #DrugDiscovery

#compchem #computationalchemistry #drugdesign #drugdiscovery #medicinalchemistry Large library docking identifies positive allosteric modulators of the calcium-sensing receptor Liu et al. (Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA 94143, USA) demonstrate the value of large library screens in identifying allosteric ligands for the calcium-sensing receptor. Optimization of several hits yielded leads with nanomolar efficacy, two of which were captured bound to the receptor in cryo–electron microscopy structures. "Four key observations emerged from this study. First, from a structure-based screen of a 1.2-billion-molecule tangible library emerged a spectrum of diverse chemotypes that potently enhanced CaSR activation. These molecules are among the first PAMs discovered through large library docking and are among the first structure-based ligands discovered for family C GPCRs. The potency of the initial docking hits was relatively high, with EC50 values down to 273 nM, and all were structurally dissimilar to known CaSR PAMs. Structure-based optimization improved affinity between 40- and 600-fold, leading to molecules that were up to 50-fold more potent than cinacalcet in vitro and 10- to 100-fold more potent at suppressing PTH secretion from organs ex vivo and in vivo. Second, the docking predictions were largely confirmed by the subsequent cryo-EM structures, with an important exception (see below), including selecting for and correctly predicting extended and bent conformations in the 7TMA and 7TMB sites of the CaSR dimer. Third, docking a library of 1.2 billion molecules led to 2.7-fold higher experimental hit rates and PAMs that were up to 37 times more potent than docking a smaller (2.7 million) molecule library. This offers among the first experimental support for simulations suggesting that docking results improve with library size increases. Fourth, the chemotypes reported here make interactions with the receptor that have not been seen in prior PAMs, promoting new active-state dimer interfaces that are closer to the G protein–coupled state and which were not observed with the established drugs. The more extensive dimer interfaces adopted in complex with the new PAMs, closer to that of the G protein–coupled state, may be behind the improved efficacy and reduced hypocalcemia of ‘54149 versus the established drugs" Science https://lnkd.in/evsyexK5

Cube Biotech's Membrane Marvels #108 🔬 Protein: GLP-1R-Gs complex with GLP-1 peptide and a positive allosteric modulator 🏷️ Family: Class B1 G Protein-Coupled Receptors 👤 Species: Homo sapiens 📏 Resolution: 3.30 Å 🗂️ PDB ID: 6VCB 📅 Year: 2020 📖 Publication: https://lnkd.in/gbdm25h4 Overview: 1. GLP-1R, a critical receptor for metabolic homeostasis, mediates insulin release and appetite regulation. The 3.30 Å cryo-EM structure captures GLP-1R bound to its natural peptide GLP-1 and the positive allosteric modulator (PAM) LSN3160440, providing a detailed look at their cooperative activation mechanism. 2. The structure highlights how LSN3160440 binds at a distinct allosteric pocket formed by TM1 and TM2, stabilizing the receptor’s active conformation. This modulation enables GLP-1(9-36), a less active GLP-1 fragment, to robustly activate GLP-1R and enhance glucose-dependent insulin secretion. 3. Key findings include: • LSN3160440 interacts directly with Y145 and L142 in TM1 through π-π stacking and hydrophobic contacts. These interactions strengthen the receptor’s affinity for GLP-1(9-36), enhancing its potency over 1500-fold. • The modulator bridges critical residues of GLP-1(9-36), such as F12, V16, and L20, with TM1 and TM2, reinforcing peptide-receptor contacts and stabilizing the active receptor state. • Functional studies show that this allosteric mechanism amplifies glucose-stimulated insulin release, while structural insights reveal how subtle changes in ligand and receptor interactions drive this potent activation. 4. This study highlights a new paradigm for GLP-1R modulation, where cooperative binding between the peptide and a PAM amplifies receptor activation. These findings provide a roadmap for developing next-generation therapies targeting metabolic disorders, such as type 2 diabetes and obesity.   ---   Check out our NativeMP™ Platform:   👉 Over 70 stabilized full-length, functional membrane protein targets with retained native lipids in our copolymer nanodiscs, including hot targets like GLP1R, P2X4, and ABCA4.   👉 Off-the-shelf membrane proteins ideal for drug binding assays at 37°C, supporting drug discovery, target exploration, compound optimization, and structural studies.   🖥 https://lnkd.in/gtmXkTme   #cubebiotech #MembraneProteins #StructuralBiology #Proteomics

Allostery represents a fundamental mechanism in protein regulation, enabling modulation of protein function from sites distal to the active site. While traditionally explored in the context of small molecules, allosteric modulation is gaining traction as a main mode of action in the realm of antibodies, which offer enhanced specificity and reduced toxicity. Successful discoveries of allosteric antibodies against previously antibody-undruggable targets, such as G protein-coupled receptors (GPCRs) or ligand-gated ion channels, are shedding light on potential new druggability avenues with antibodies. Allosteric antibodies are also of interest for small molecules discovery, opening up a new era by integrating the two technologies. Additionally, recent efforts in the fields of computational biology and artificial intelligence (AI) hold promise for integrating allosteric site detection with de novo antibody design, paving the way for efficient allosteric antibody discovery. This review delves into the rapidly growing field of allosteric antibodies, highlighting recent therapeutic advancements and novel druggability avenues. We also explore the potential of these antibodies as innovative tools in drug discovery and discuss contemporary strategies for designing novel allosteric antibodies, leveraging state-of-the-art computational approaches. Interesting review written by Léxane F., Enrico Guarnera, Harald Kolmar, and @Stefan Beckeron on the development of therapeutic antibodies which utilize allosteric modulation to create new biologics with innovative mechanisms of action. The text above is from the author's abstract, the full paper can be found here: https://lnkd.in/eYHHcPgs

As our Drug Hunter team was compiling the November Molecule Roundup, one molecule that really caught our attention was Compound 4—a mutant-selective, salicylaldehyde-based covalent inhibitor designed to target the AKT1 (E17K) oncogenic mutation. This discovery, reported in collaboration between Jack Taunton’s group at University of California, San Francisco and Terremoto Biosciences in Nature Magazine, addresses a mutation frequently observed in solid tumors, where it drives persistent oncogenic signaling via constitutive membrane localization. What makes Compound 4 stand out to me is its design strategy. Unlike traditional pan-AKT inhibitors, which are often plagued by dose-limiting hyperglycemia, Compound 4 leverages a unique allosteric, lysine-targeted mechanism to achieve exceptional selectivity for AKT1 (E17K) over wild-type AKT isoforms. Structural analysis uncovered an unexpected twist: the salicylaldimine adduct, formed with the mutant lysine, recruits endogenous Zn²⁺. This zinc ion coordinates with two proximal cysteines in the kinase activation loop, resulting in sustained inhibition of AKT1 (E17K) while leaving wild-type AKT isoforms largely unaffected. The outcome? Robust anti-tumor efficacy in AKT1 (E17K) xenograft models, without inducing the hyperglycemia often seen with traditional inhibitors. What excites me most is the broader potential of this approach. The chelation-enhanced binding mechanism suggests that this strategy could be extended to other proteins with lysines near metal-binding regions, such as metalloenzymes and zinc fingers. This could open entirely new avenues for targeting previously elusive protein classes. What other targets do you think could benefit from this chelation-enhanced strategy? Explore our full November Molecule Roundup here: https://lnkd.in/e6Jsny6A And stay tuned—we’re finalizing our December molecules now!

Experimental drug supercharges medicine that reverse opioid overdose. Researchers from Washington University School of Medicine in St. Louis, Stanford University and University of Florida have identified potential drugs that make naloxone more potent and longer lasting, capable of reversing effects of opioids in mice at low doses, without worsening withdrawal symptoms. July 03, 2024. Excerpt: Opioids such as oxycodone and fentanyl work by slipping inside a pocket on the opioid receptor, found primarily on neurons in the brain. The presence of opioids activates the receptor, setting off a cascade of molecular events that temporarily alters how the brain functions: reducing perception of pain, inducing euphoria and slowing down breathing. It is suppression of breathing that makes opioids deadly. The molecular compound is a negative allosteric modulator (NAM) of the opioid receptor. Allosteric modulators are an exciting area of pharmacology research. NAM influence how the body responds to drugs by fine-tuning activity of drug receptors rather than the drugs themselves. Co-author Vipin Rangari, PhD, a postdoc fellow in Majumdar lab, performed experiments to chemically characterize the compound. The research team led by co-senior authors Majumdar; Brian K. Kobilka, PhD, a professor of molecular and cellular physiology at Stanford University; and Jay P. McLaughlin, PhD, a professor of pharmacodynamics at University of Florida, set out to find NAMs that strengthen naloxone and suppress activation of the opioid receptor more effectively. Note: A library of 4.5 billion molecules was screened in the lab in search of molecules that bound to the opioid receptor with naloxone already tucked into the receptor’s pocket. Compounds representing several molecular families passed initial screening, with one of the most promising named compound 368. Further experiments in cells revealed, in the presence of compound 368, naloxone was 7.6 times more effective at inhibiting activation of the opioid receptor, partly because naloxone stayed in the binding pocket at least 10 times longer. Compound 368 improved naloxone’s ability to counteract opioid overdoses in mice and enabled naloxone to reverse effects of fentanyl and morphine at 1/10th the usual doses. People who overdose on opioids and are revived with naloxone can experience withdrawal symptoms: pain, chills, vomiting and irritability. In this study, while addition of compound 368 boosted naloxone’s potency, it did not worsen the mice’s withdrawal symptoms. Compound 368 is one of several molecules that show potential as NAMs of the opioid receptor. The researchers have filed a patent on the NAMs, and are working on narrowing and characterizing the most promising candidates. Majumdar estimates it will be 10 to 15 years before a naloxone-enhancing NAM is brought to market. Nature |  03 July 2024 A µ-opioid receptor modulator that works cooperatively with naloxone

They write: Here, we present a large dataset of molecular dynamics simulations covering 60% of currently available GPCR structures. Our analysis reveals extensive local “breathing” motions of the receptor on a nano- to microsecond timescale and provides access to numerous previously unexplored receptor conformational states. Furthermore, we reveal that receptor flexibility impacts the shape of allosteric drug binding sites, which frequently adopt partially or completely closed states in the absence of a molecular modulator. We demonstrate that exploring membrane lipid dynamics and their interaction with GPCRs is an efficient approach to expose such hidden allosteric sites and even lateral ligand entrance gateways. The obtained insights and generated dataset on conformations, allosteric sites and lateral entrance gates in GPCRs allows us to better understand the functionality of these receptors and opens new therapeutic avenues for drug-targeting strategies.