Biophysical Techniques Research Articles

Structure-Guided Bacteria Specificity and Wide Activity Spectrum of Endotoxin-Responsive Peptide Nanonets.

Peptide nanonets offer a promising avenue for constructing anti-infective biomaterials. Our group recently reported innovative designs of synthetic BTT nanonets that fibrillate selectively in response to bacterial endotoxins. Herein, we delved deeper into the molecular interactions between our peptides and these bacteria-specific biomolecules, which is an aspect critically missing from major works in the field. Using microscopic and biophysical techniques, we identified phosphate moieties in endotoxins as being the most essential to the initiation of peptide fibrillation. This was strongly supported by molecular dynamics simulations in an outer membrane environment with variable states of phosphorylation. To support the claim over bacterial specificity, we demonstrated a lack of nanonet formation in the presence of various phosphate-containing biomolecules native to human biology. The structural importance of phosphate moieties among pathogenic strains strongly indicates a wide clinical spectrum of our peptides, which was experimentally verified.

Into the Groove: A Multitechnique Insight into the DNA–Vemurafenib Interaction

This study explores the interaction between Vemurafenib (VEM), a potent BRAF inhibitor, and calf thymus DNA (ctDNA) using a comprehensive array of biophysical and computational techniques. The primary objective is to understand the potential off-target effects of VEM on DNA, given its established role in melanoma therapy targeting the BRAF V600E mutation. The investigation employed methods such as ultraviolet–visible absorption spectroscopy, steady-state fluorescence, circular dichroism, isothermal titration calorimetry, and advanced molecular dynamics simulations. The results indicate that VEM interacts with DNA primarily through a minor groove-binding mechanism, causing minimal structural disruption to the DNA double helix. Viscosity measurements and melting temperature analyses further confirmed this non-intercalative mode of binding. Calorimetry data revealed an exothermic, thermodynamically favorable interaction between VEM and ctDNA, driven by both enthalpic and entropic factors. Finally, computer simulations identified the most probable binding site and mode of VEM within the minor groove of the nucleic acid, providing a molecular basis for the experimental findings.

Design of a Cereblon construct for crystallographic and biophysical studies of protein degraders

The ubiquitin E3 ligase cereblon (CRBN) is the target of therapeutic drugs thalidomide and lenalidomide and is recruited by most targeted protein degraders (PROTACs and molecular glues) in clinical development. Biophysical and structural investigation of CRBN has been limited by current constructs that either require co-expression with the adaptor DDB1 or inadequately represent full-length protein, with high-resolution structures of degrader ternary complexes remaining rare. We present the design of CRBNmidi, a construct that readily expresses from E. coli with high yields as soluble, stable protein without DDB1. We benchmark CRBNmidi for wild-type functionality through a suite of biophysical techniques and solve high-resolution co-crystal structures of its binary and ternary complexes with degraders. We qualify CRBNmidi as an enabling tool to accelerate structure-based discovery of the next generation of CRBN based therapeutics.

Molecular Interactions between Tau Protein and TIA1: Distinguishing Physiological Condensates from Pathological Fibrils.

The interaction of tau protein with other key proteins essential for stress granule formation determines their functional and pathological impact. In a biological framework, the synergy between Alzheimer's associated tau protein and the stress granule core protein TIA1 is widely recognized. However, the molecular details of this association remain unclear. In this study, we throw light on the importance of the state in which the TIA1 exists in mediating its association with the tau protein. Investigations were carried out on the three repeat constructs of tau (K19) and different structures formed by TIA1. Specifically, the condensate formed by TIA1 full-length (TIA1-FL) protein as well as fibril formed by low complexity domain of TIA1 (TIA1-LCD). The dynamics of K19 inside TIA1-FL condensates and the aggregation kinetics of K19 in the presence of TIA1-LCD fibrils were examined using various biophysical techniques. Relaxation-based solution NMR spectroscopic investigations suggest a weak interaction with TIA1 condensates and indicated a reduction in the dynamics of K19 within these TIA1 condensates. In contrast, a significant interaction was observed between K19, and TIA1-LCD fibrils primarily mediated through 321KCGS324 and 306VQIVYKPVDLSKV317. Our findings emphasize that the interaction between Tau and TIA1 varies depending on whether TIA1 is in its physiological condensate form or its pathological fibril state.

Functional role of myosin-binding protein H in thick filaments of developing vertebrate fast-twitch skeletal muscle.

Myosin-binding protein H (MyBP-H) is a component of the vertebrate skeletal muscle sarcomere with sequence and domain homology to myosin-binding protein C (MyBP-C). Whereas skeletal muscle isoforms of MyBP-C (fMyBP-C, sMyBP-C) modulate muscle contractility via interactions with actin thin filaments and myosin motors within the muscle sarcomere "C-zone," MyBP-H has no known function. This is in part due to MyBP-H having limited expression in adult fast-twitch muscle and no known involvement in muscle disease. Quantitative proteomics reported here reveal that MyBP-H is highly expressed in prenatal rat fast-twitch muscles and larval zebrafish, suggesting a conserved role in muscle development and prompting studies to define its function. We take advantage of the genetic control of the zebrafish model and a combination of structural, functional, and biophysical techniques to interrogate the role of MyBP-H. Transgenic, FLAG-tagged MyBP-H or fMyBP-C both localize to the C-zones in larval myofibers, whereas genetic depletion of endogenous MyBP-H or fMyBP-C leads to increased accumulation of the other, suggesting competition for C-zone binding sites. Does MyBP-H modulate contractility in the C-zone? Globular domains critical to MyBP-C's modulatory functions are absent from MyBP-H, suggesting that MyBP-H may be functionally silent. However, our results suggest an active role. In vitro motility experiments indicate MyBP-H shares MyBP-C's capacity as a molecular "brake." These results provide new insights and raise questions about the role of the C-zone during muscle development.

Force-mediated recruitment and reprogramming of healthy endothelial cells drive vascular lesion growth

Force-driven cellular interactions are crucial for cancer cell invasion but remain underexplored in vascular abnormalities. Cerebral cavernous malformations (CCM), a vascular abnormality characterized by leaky vessels, involves CCM mutant cells recruiting wild-type endothelial cells to form and expand mosaic lesions. The mechanisms behind this recruitment remain poorly understood. Here, we use an in-vitro model of angiogenic invasion with traction force microscopy to reveal that hyper-angiogenic Ccm2-silenced endothelial cells enhance angiogenic invasion of neighboring wild-type cells through force and extracellular matrix-guided mechanisms. We demonstrate that mechanically hyperactive CCM2-silenced tips guide wild-type cells by transmitting pulling forces and by creating paths in the matrix, in a ROCKs-dependent manner. This is associated with reinforcement of β1 integrin and actin cytoskeleton in wild-type cells. Further, wild-type cells are reprogrammed into stalk cells and activate matrisome and DNA replication programs, thereby initiating proliferation. Our findings reveal how CCM2 mutants hijack wild-type cell functions to fuel lesion growth, providing insight into the etiology of vascular malformations. By integrating biophysical and molecular techniques, we offer tools for studying cell mechanics in tissue heterogeneity and disease progression.

Rational design, synthesis, and biophysical characterization of a peptidic MDM2-MDM4 interaction inhibitor

In recent years, the restoration of p53 physiological functions has become an attractive therapeutic approach to develop novel and efficacious cancer therapies. Among other mechanisms, the oncosuppressor protein p53 is functionally regulated by MDM2 through its E3 ligase function. MDM2 promotes p53 ubiquitination and degradation following homodimerization or heterodimerization with MDM4. Recently, we discovered Pep3 (1, Pellegrino et al., 2015), a novel peptidic inhibitor of MDM2 dimerization able to restore p53 oncosuppressive functions both in vitro and in vivo. In this work, we were able to identify the key interactions between peptide 1 and MDM2 RING domain and to design peptide 2, a truncated version of 1 that is still able to bind MDM2. Integrating both computational and biophysical techniques, we show that peptide 2 maintains the conserved peptide 1-MDM2 interactions and is still able to bind to full-length MDM2.

B-064 Bovine Serum Albumin Coats Silicon Dioxide More Uniformly Near its Isoelectric Point Based on Atomic Force Microscopy Analysis

Abstract Background Bovine serum albumin (BSA) serves as a blocking agent in in-vitro diagnostic (IVD) applications, with fatty acid stabilized (FAS) and fatty acid free (FAF) BSA as common options. The presence of fatty acids and pH influence the conformation of BSA. While previous studies have explored the individual effects of fatty acids or pH on BSA blocking, limited data exists on their combined impact. This study employs atomic force microscopy (AFM) to investigate how pH and fatty acids jointly affect BSA adsorption onto silica, a surface relevant within diagnostics. Methods AFM, a sensitive microscopic technique, provides topographic images of surfaces with superior resolution compared to optical microscopy. We conducted experiments to assess the adsorption of 1% FAS and FAF BSA on a silica surface at pH 5.2 and pH 7. Evaluation of the coatings involved scratching to assess overall thickness, and roughness, indicating thickness variability. Results Results revealed that both fatty acids and pH play significant roles in shaping the BSA coating characteristics on silica surfaces. In three out of four experimental conditions, the average thickness of the BSA coating ranged between 3-4 nm, except for FAS BSA at pH 7, which exhibited a thicker average coating measuring 5.6 nm. The pH impacted the roughness of the BSA coating. Coatings prepared at pH 5.2 displayed smoother surfaces compared to those at pH 7. Utilizing the root mean square average height variability analysis, there is 95% confidence that the FAF and FAS BSA surfaces at pH 5.2 had a height range between 2.4-4.3 nm and 2.0-4.9 nm, respectively, indicating complete coverage of the silica surface without much height variation. In contrast, the FAF and FAS BSA coatings at pH 7 exhibited a wider range of heights, with 95% confidence intervals spanning from -1.5-8.6 nm and 0.0-11.2 nm, respectively. This variability suggests potential issues including incomplete BSA coverage at the lower range, and steric hindrance at the higher end, highlighting the importance of pH in optimizing BSA coatings for various applications. Conclusions Silica is commonly used in IVD assays, with BSA frequently employed to coat and passivate the surface. Our AFM experiments highlight the impact of pH and fatty acid presence on coating thickness and uniformity. Notably, FAS BSA at pH 7 exhibited the thickest coating; however, its increased roughness may pose challenges in IVD applications due to steric hindrance blocking antibody-antigen interactions. Alternatively, FAF BSA at pH 7 displayed an adequate thickness but also considerable roughness, suggesting potential insufficiencies in surface coverage and passivation. FAS and FAF BSA coatings at pH 5.2 showed relatively consistent thickness, indicating a more uniform surface which may be ideal for IVD coating applications. These AFM experiments were conducted on pure silica surfaces, which may differ from surfaces engineered for IVD. Additionally, both FAF and FAS BSA grades are proven to be effective in IVD applications. Further research employing AFM and other biophysical techniques is warranted to deepen our understanding of pH and fatty acid effects on BSA-surface interactions, aiming to enhance blocking strategies in IVD.

Phytochemical Characterization and Synergistic Antibacterial Effects of Colebrookea Oppositifolia Essential Oil as Adjuvants to Modern Antibiotics in Combating Drug Resistance.

The global threat of multi-drug-resistant bacteria has severely limited the options available for effective antibiotics. This study focuses on the antimicrobial activity and phytochemical characterization of C. oppositifolia extracts, aiming to identify novel plant-based therapeutic agents. C. oppositifolia specimens-leaves and inflorescence. Specimens were cleaned, sterilized, dried, and ground into a fine powder. Extracts were obtained using methanol and petroleum ether via a Soxhlet apparatus, followed by fractionation with chloroform, n-butanol, and ethyl acetate. Volatile oil was extracted through hydro distillation using a Clevenger apparatus. Phytochemical analysis was conducted to identify bioactive compounds. Biophysical techniques, including UV-visible spectrophotometry, TLC, HPLC, GC-MS, FTIR, and NMR, were employed for characterization. Antimicrobial activity was tested against S. aureus ATCC25922 and E. coli ATCC25922 using agar well and disc diffusion methods, and synergistic effects were assessed with erythromycin and amoxicillin. Methanol extract exhibited bacteriostatic activity with inhibition zones of 13.0 ± 0.2 mm for both S. aureus and E. coli. Petroleum ether, chloroform, n-butanol, and ethyl acetate fractions showed varying inhibition zones. Erythromycin demonstrated bactericidal activity, which was enhanced synergistically when combined with methanol extract and volatile oil, increasing inhibition zones against S. aureus. Phytochemical analysis identified phenols, flavonoids, tannins, coumarins, alkaloids, terpenoids, saponins, and glycosides. FTIR analysis revealed functional groups such as amines, aldehydes, nitriles, alkenes, and sulfones. GC-MS identified 24 compounds, with α-pinene, caryophyllene, and carene as major components. NMR spectra indicated no complex formation between oils and antibiotics, suggesting the compounds act as synergists. The C. oppositifolia extracts possess significant antimicrobial activity and synergistic potential, particularly against S. aureus. The presence of various bioactive compounds suggests a promising role in developing new plant-based therapeutics.

The molecular dissection of TRIM25’s RNA-binding mechanism provides key insights into its antiviral activity

TRIM25 is an RNA-binding ubiquitin E3 ligase with central but poorly understood roles in the innate immune response to RNA viruses. The link between TRIM25’s RNA binding and its role in innate immunity has not been established. Thus, we utilized a multitude of biophysical techniques to identify key RNA-binding residues of TRIM25 and developed an RNA-binding deficient mutant (TRIM25-m9). Using iCLIP2 in virus-infected and uninfected cells, we identified TRIM25’s RNA sequence and structure specificity, that it binds specifically to viral RNA, and that the interaction with RNA is critical for its antiviral activity.

The Effect of Reactive Oxygen Species on Respiratory Complex I Activity in Liposomes.

Respiratory complex I (R-CI) is an essential enzyme in the mitochondrial electron transport chain but also a major source of reactive oxygen species (ROS), which are implicated in neurodegenerative diseases and ageing. While the mechanism of ROS production by R-CI is well-established, the feedback of ROS on R-CI activity is poorly understood. Here, we perform EPR spectroscopy on R-CI incorporated in artificial membrane vesicles to reveal that ROS (particularly hydroxyl radicals) reduce R-CI activity by making the membrane more polar and by increasing its hydrogen bonding capability. Moreover, the mechanism that we have uncovered reveals that the feedback of ROS on R-CI activity via the membrane is transient and not permanent; lipid peroxidation is negligible for the levels of ROS generated under these conditions. Our successful use of modular proteoliposome systems in conjunction with EPR spectroscopy and other biophysical techniques is a powerful approach for investigating ROS effects on other membrane proteins.

Harnessing Light for G-Quadruplex Modulation: Dual Isomeric Effects of an Ortho-Fluoroazobenzene Derivative.

G-quadruplexes (G4s) are important therapeutic and photopharmacological targets in cancer research. Small-molecule ligands targeting G4s offer a promising strategy to block DNA transactions and induce genetic instability in cancer cells. While numerous G4-ligands have been reported, relatively few examples exist of compounds whose G4-interactive binding properties can be modulated using light. Herein, we report the photophysical characterization of a novel ortho-fluoroazobenzene derivative, Py-Azo4F-3N, that undergoes reversible two-way isomerization upon visible light exposure. Using a combination of biophysical techniques, including affinity and selectivity assays, structural and computational analysis, and cytotoxicity experiments in cancer cell lines, we carefully characterized the G4-interactive binding properties of both isomers. We identify the trans isomer as the most promising form of interacting and stabilizing G4s, enhancing their ablation capability in cancer cells. Our research highlights the importance of light-responsive molecules in achieving precise control over G4 structures, demonstrating their potential in innovative anticancer strategies.

A single diiron enzyme catalyses the oxidative rearrangement of tryptophan to indole nitrile.

Nitriles are uncommon in nature and are typically constructed from oximes through the oxidative decarboxylation of amino acid substrates or from the derivatization of carboxylic acids. Here we report a third nitrile biosynthesis strategy featuring the cyanobacterial nitrile synthase AetD. During the biosynthesis of the eagle-killing neurotoxin, aetokthonotoxin, AetD transforms the 2-aminopropionate portion of 5,7-dibromo-L-tryptophan to a nitrile. Employing a combination of structural, biochemical and biophysical techniques, we characterized AetD as a non-haem diiron enzyme that belongs to the emerging haem-oxygenase-like dimetal oxidase superfamily. High-resolution crystal structures of AetD together with the identification of catalytically relevant products provide mechanistic insights into how AetD affords this unique transformation, which we propose proceeds via an aziridine intermediate. Our work presents a unique template for nitrile biogenesis and portrays a substrate binding and metallocofactor assembly mechanism that may be shared among other haem-oxygenase-like dimetal oxidase enzymes.

Peptides as modulators of FPPS enzyme: A multifaceted evaluation from the design to the mechanism of action

Bone diseases are medical conditions caused by the loss of bone homeostasis consecutive to increased osteoclast activity and diminished osteoblast activity. The mevalonate pathway (MVA) is crucial for maintaining this balance since it drives the post-translational prenylation of small guanosine triphosphatases (GTPases) proteins. Farnesyl pyrophosphate synthase (FPPS) plays a crucial role in the MVA pathway. Consequently, in the treatment of bone-related diseases, FPPS is the target of FDA-approved nitrogen-containing bisphosphonates (N-BPs), which have tropism mainly for bone tissue due to their poor penetration in soft tissues. The development of inhibitors targeting the FPPS enzyme has garnered significant interest in recent decades due to FPPS's role in the biosynthesis of cholesterol and other isoprenoids, which are implicated in cancer, bone diseases, and other conditions. In this study, we describe a multidisciplinary approach to designing novel FPPS inhibitors, combining computational modeling, biochemical assays, and biophysical techniques. A series of peptides and phosphopeptides were designed, synthesized, and evaluated for their ability to inhibit FPPS activity. Molecular docking was employed to predict the binding modes of these compounds to FPPS, while Surface Plasmon Resonance (SPR) and Nuclear Magnetic Resonance (NMR) spectroscopy experiments – based on Saturation Transfer Difference (STD) and an enzymatic NMR assay – were used to measure their binding affinities and kinetics. The biological activity of the most promising compounds was further assessed in cellular assays using murine colorectal cancer (CRC) cells. Additionally, genomics and metabolomics profiling allowed to unravel the possible mechanisms underlying the activity of the peptides, confirming their involvement in the modulation of the MVA pathway. Our findings demonstrate that the designed peptides and phosphopeptides exhibit significant inhibitory activity against FPPS and possess antiproliferative effects on CRC cells, suggesting their potential as therapeutic agents for cancer.

The Efficacy and Safety of CollaSel Pro® Hydrolyzed Collagen Peptide Supplementation without Addons in Improving Skin Health in Adult Females: A Double Blind, Randomized, Placebo-Controlled Clinical Study Using Biophysical and Skin Imaging Techniques.

Background: This study aimed to evaluate the efficacy and safety of oral hydrolyzed collagen peptide (HCP) in healthy females by assessing the skin parameters via biophysical and skin imaging techniques. Methods: 112 females were randomly assigned to receive either HCP (n = 57; 10 g CollaSel Pro®) or placebo (n = 55; 10 g maltodextrin) daily for eight weeks. The contribution of HCP to skin elasticity, hydration, and roughness was investigated against a placebo, while the facial soft tissue sagging (RMS) and safety data were also recorded. Results: HCP was associated with significant improvements in skin elasticity (p = 0.009), skin hydration (p ranged from 0.003 to <0.001), and skin roughness (p ranged from 0.002 to <0.001). In the HCP vs. the placebo group, week eight values for skin elasticity (43.0 (7.4) vs. 40.3 (3.3) mPa, p = 0.017), skin hydration (65.8 (18.9) vs. 53.1 (14.9) g/m3, p < 0.001) and skin roughness (40.2 (20.4) vs. 24.9 (20.9) g/m3, p < 0.001) were significantly higher. In the HCP group, week 8 RMS values were significantly lower than baseline values (1.02 (0.21) vs. 1.10 (0.21) mm, p = 0.012). Conclusions: CollaSel Pro® HCP can be considered a well-tolerated, safe product that effectively improves dermal health and the appearance of sagging and ameliorates the signs of the aging process.

Multivalency drives interactions of alpha-synuclein fibrils with tau.

Age-related neurodegenerative disorders like Alzheimer's disease (AD) and Parkinson's disease (PD) are characterized by deposits of protein aggregates, or amyloid, in various regions of the brain. Historically, aggregation of a single protein was observed to be correlated with these different pathologies: tau in AD and α-synuclein (αS) in PD. However, there is increasing evidence that the pathologies of these two diseases overlap, and the individual proteins may even promote each other's aggregation. Both tau and αS are intrinsically disordered proteins (IDPs), lacking stable secondary and tertiary structure under physiological conditions. In this study we used a combination of biochemical and biophysical techniques to interrogate the interaction of tau with both soluble and fibrillar αS. Fluorescence correlation spectroscopy (FCS) was used to assess the interactions of specific domains of fluorescently labeled tau with full length and C-terminally truncated αS in both monomer and fibrillar forms. We found that full-length tau as well as individual tau domains interact with monomer αS weakly, but this interaction is much more pronounced with αS aggregates. αS aggregates also mildly slow the rate of tau aggregation, although not the final degree of aggregation. Our findings suggest that co-occurrence of tau and αS in disease are more likely to occur through monomer-fiber binding interactions, rather than monomer-monomer or co-aggregation.

A Few Charged Residues in Galectin-3's Folded and Disordered Regions Regulate Phase Separation.

Proteins with intrinsically disordered regions (IDRs) often undergo phase separation to control their functions spatiotemporally. Changing the pH alters the protonation levels of charged sidechains, which in turn affects the attractive or repulsive force for phase separation. In a cell, the rupture of membrane-bound compartments, such as lysosomes, creates an abrupt change in pH. However, how proteins' phase separation reacts to different pH environments remains largely unexplored. Here, using extensive mutagenesis, NMR spectroscopy, and biophysical techniques, it is shown that the assembly of galectin-3, a widely studied lysosomal damage marker, is driven by cation-π interactions between positively charged residues in its folded domain with aromatic residues in the IDR in addition to π-π interaction between IDRs. It is also found that the sole two negatively charged residues in its IDR sense pH changes for tuning the condensation tendency. Also, these two residues may prevent this prion-like IDR domain from forming rapid and extensive aggregates. These results demonstrate how cation-π, π-π, and electrostatic interactions can regulate protein condensation between disordered and structured domains and highlight the importance of sparse negatively charged residues in prion-like IDRs.

Allosteric modulation of the CXCR4:CXCL12 axis by targeting receptor nanoclustering via the TMV-TMVI domain

CXCR4 is a ubiquitously expressed chemokine receptor that regulates leukocyte trafficking and arrest in both homeostatic and pathological states. It also participates in organogenesis, HIV-1 infection, and tumor development. Despite the potential therapeutic benefit of CXCR4 antagonists, only one, plerixafor (AMD3100), which blocks the ligand-binding site, has reached the clinic. Recent advances in imaging and biophysical techniques have provided a richer understanding of the membrane organization and dynamics of this receptor. Activation of CXCR4 by CXCL12 reduces the number of CXCR4 monomers/dimers at the cell membrane and increases the formation of large nanoclusters, which are largely immobile and are required for correct cell orientation to chemoattractant gradients. Mechanistically, CXCR4 activation involves a structural motif defined by residues in TMV and TMVI. Using this structural motif as a template, we performed in silico molecular modeling followed by in vitro screening of a small compound library to identify negative allosteric modulators of CXCR4 that do not affect CXCL12 binding. We identified AGR1.137, a small molecule that abolishes CXCL12-mediated receptor nanoclustering and dynamics and blocks the ability of cells to sense CXCL12 gradients both in vitro and in vivo while preserving ligand binding and receptor internalization.

Allosteric modulation of the CXCR4:CXCL12 axis by targeting receptor nanoclustering via the TMV-TMVI domain

CXCR4 is a ubiquitously expressed chemokine receptor that regulates leukocyte trafficking and arrest in both homeostatic and pathological states. It also participates in organogenesis, HIV-1 infection, and tumor development. Despite the potential therapeutic benefit of CXCR4 antagonists, only one, plerixafor (AMD3100), which blocks the ligand-binding site, has reached the clinic. Recent advances in imaging and biophysical techniques have provided a richer understanding of the membrane organization and dynamics of this receptor. Activation of CXCR4 by CXCL12 reduces the number of CXCR4 monomers/dimers at the cell membrane and increases the formation of large nanoclusters, which are largely immobile and are required for correct cell orientation to chemoattractant gradients. Mechanistically, CXCR4 activation involves a structural motif defined by residues in TMV and TMVI. Using this structural motif as a template, we performed in silico molecular modeling followed by in vitro screening of a small compound library to identify negative allosteric modulators of CXCR4 that do not affect CXCL12 binding. We identified AGR1.137, a small molecule that abolishes CXCL12-mediated receptor nanoclustering and dynamics and blocks the ability of cells to sense CXCL12 gradients both in vitro and in vivo while preserving ligand binding and receptor internalization.

Biostructural, biochemical and biophysical studies of mutant IDH1

We report bio-structural, bio-chemical and bio-physical evidence demonstrating how small molecules can bind to both wild-type and mutant IDH1, but only inhibit the enzymatic activity of the mutant isoform. Enabled through x-ray crystallography, we characterized a series of small molecule inhibitors that bound to mutant IDH1 differently than the marketed inhibitor Ivosidenib, for which we have determined the x-ray crystal structure. Across the industry several mutant IDH1 inhibitor chemotypes bind to this allosteric IDH1 pocket and selectively inhibit the mutant enzyme. Detailed characterization by a variety of biophysical techniques and NMR studies led us to propose how compounds binding in the allosteric IDH1 R132H pocket inhibit the production of 2-Hydroxy glutarate.