Hinge Region Research Articles

Structural and dynamic characterization of the hexa-coordinated globin from Spisula solidissima

High energy consumption in the nervous system requires a continuous supply of O2. This role is assisted by proteins from the globin super-family in the nerve cells of invertebrates, where ‘nerve hemoglobins’ (nHbs) are mainly present at mM concentrations and exhibit oxygen affinities comparable to those of vertebrate myoglobins. To gain insight into the structural bases of this function, we report the crystal structure of nHb from the Atlantic surf clam Spisula solidissima (SsHb), previously suggested to display a bis-histidyl hexa-coordinated heme in the deoxy state, high O2 affinity, and ligand binding cooperativity when assayed in situ. The crystallized protein forms a dimer through packing of a 4-helix bundle involving helices E and F of each subunit. The SsHb ‘classic’ globin fold displays bis-histidyl (His71(E7) and His103(F8)) hexa-coordination of the heme-Fe atom, with structural and dynamics variations found in the inter-helix hinge regions. Molecular Dynamics simulations of both monomeric and dimeric species in the bis-histidyl hexa-coordinated, deoxy penta-coordinated, and O2-bound hexa-coordinated states reveal distinct structural rearrangements at the interface between subunits in the dimer; these would affect the magnitude of the conformational fluctuations observed between monomer and dimer, and the topology of cavities within the protein matrix and at the interface. These results point to a distal site opening mechanism allowing access of the exogenous ligand to the heme and cast hypotheses on the dimer interface structural and dynamic properties that may support ligand binding cooperativity in dimeric SsHb.-

Ala307Thr variation modulates FSHR structure and impairs its binding affinity for FSH: Implications in polycystic ovarian syndrome.

Follicle-stimulating hormone receptor (FSHR) belongs to the family of G-protein coupled receptors and acts as a cognate receptor for follicle-stimulating hormone (FSH). Among the various polymorphic changes reported in FSHR, rs6165 polymorphism leading to Ala307Thr variation in the extracellular domain of the FSHR (FSHRED ) is widely reported. Therefore we attempted to evaluate the functional implications of this variation by studying its effects on FSHRED structure as well as FSH binding. Our atomic-scale investigations reveal that the hinge region, a key hormone interaction site in the extracellular domain of Wt FSHR, exhibits significantly more flexibility compared with the variant structure. Moreover, the Wt receptor in complex with FSH was observed to form a pocket-like structure in its hinge region whereas such a structure was not detected in the variant. The study further reveals that the key residue, sTyr335, required for FSH recognition and FSHR activation, exhibits lower binding free energy in the variant structure as compared to the Wt. In conclusion, our results point out that Ala307Thr variation leads to structural and conformational anomalies in FSHRED which may alter its FSH binding and affect its activation.

Influence of different combinations of impact mass and velocity with identical kinetic energy or momentum on the impact response of RC piles

Marine engineering structures may be subjected to various levels of vessel collisions, causing different degrees of damage to the RC piles. However, the influence of different combinations of impact mass and velocity under the same initial kinetic energy or momentum on the impact behavior of RC piles has not been explored. A numerical investigation of the impact responses of RC pile structures (including plumb piles and inclined pile groups) considering the material strain rate effect is carried out using the software ABAQUS/Explicit in this paper. Parametric studies are conducted based on the validated finite element models of RC piles subjected to horizontal impact. The internal force distributions, as well as the effect of the impact kinetic energy and the momentum (including different combinations of impact mass and initial velocity) on the impact response of RC piles, are discussed. The results indicate that the development path of the bending moment‒overall structural displacement curves in the plastic hinge regions of RC piles are almost identical under both impact and static loading conditions. Compared to momentum, the impact response of RC piles is insensitive to the variation of impact kinetic energy with different combinations of impact mass and velocity. Therefore, the impact kinetic energy is more suitable for evaluating the structural deformation of RC piles after impact. Finally, a performance-based impact-resistance design framework for RC piles is proposed.

Elucidating the potential effects of point mutations on FGFR3 inhibitor resistance via combined molecular dynamics simulation and community network analysis.

FGFR3 kinase mutations are associated with a variety of malignancies, but FGFR3 mutant inhibitors have rarely been studied. Furthermore, the mechanism of pan-FGFR inhibitors resistance caused by kinase domain mutations is still unclear. In this study, we try to explain the mechanism of drug resistance to FGFR3 mutation through global analysis and local analysis based on molecular dynamics simulation, binding free energy analysis, umbrella sampling and community network analysis. The results showed that FGFR3 mutations caused a decrease in the affinity between drugs and FGFR3 kinase, which was consistent with the reported experimental results. Possible mechanisms are that mutations affect drug-protein affinity by altering the environment of residues near the hinge region where the protein binds to the drug, or by affecting the A-loop and interfering with the allosteric communication networks. In conclusion, we systematically elucidated the underlying mechanism of pan-FGFR inhibitor resistance caused by FGFR3 mutation based on molecular dynamics simulation strategy, which provided theoretical guidance for the development of FGFR3 mutant kinase inhibitors.

Behavior of CFRP confined circular CFST columns reinforced with internal latticed steel angles under seismic loading

This paper investigated the hysteretic behavior of CFRP confined concrete-filled steel tube (CFST) reinforced with internal latticed steel angles. The parameters studied were the diameter-to-thickness ratio, the CFRP wrapping layers and the axial compression ratio. The failure modes, hysteretic curves, skeleton curves, stiffness degradation, ductility and energy dissipation capacity were presented and discussed. Experimental results revealed that the ultimate load and drift capacity increased by 18.8% and 33.5% when the plastic hinge region was confined by CFRP, all specimens failed due to the compression-bending failure in the plastic hinge region. Moreover, the CFRP can significantly improve the energy dissipation capacity under higher axial compression ratios. After testing, the developed finite element model was also used to perform parameter analysis, with a total of 53 specimens analyzed, and based on the parameter analysis results, the design equation for the drift capacity was proposed.

Crystal structures of the human IgD Fab reveal insights into CH1 domain diversity

Antibodies of the IgD isotype remain the least well characterized of the mammalian immunoglobulin isotypes. Here we report three-dimensional structures for the Fab region of IgD, based on four different crystal structures, at resolutions of 1.45–2.75 Å. These IgD Fab crystals provide the first high-resolution views of the unique Cδ1 domain. Structural comparisons identify regions of conformational diversity within the Cδ1 domain, as well as among the homologous domains of Cα1, Cγ1 and Cμ1. The IgD Fab structure also possesses a unique conformation of the upper hinge region, which may contribute to the overall disposition of the very long linker sequence between the Fab and Fc regions found in human IgD. Structural similarities observed between IgD and IgG, and differences with IgA and IgM, are consistent with predicted evolutionary relationships for the mammalian antibody isotypes.

AD‐Associated Variants Directly Impair Surface Interactions Between the Hydrophobic Site of TREM2 and the Hinge Region of ApoE

AbstractBackgroundTriggering Receptor Expressed on Myeloid Cells 2 (TREM2) and Apolipoprotein E (ApoE) are two of the strongest genetic risk factors for late‐onset Alzheimer’s disease (AD), but while their interplay in the microglial response to AD pathology is hotly studied, surprisingly little is known about the mechanism by which their variants impair this role.MethodTo define the structural mechanism by which AD‐associated variants impair the TREM2‐ApoE interaction, we used sequence‐based in silico methods to predict the binding site between TREM2 and ApoE, validated the predicted binding site with biolayer interferometry (BLI), then constructed initial models of TREM2‐ApoE complex structures using the identified binding site between TREM2 and APOE as restraints for unbiased molecular docking methods. The top four initial models were re‐ranked by comparing the effect of key residue mutations in the TREM2‐ApoE complex on binding affinities calculated from molecular dynamics‐molecular mechanics Poisson‐Boltzmann surface area (MD/MM‐PBSA) binding free energy analyses with binding affinities measured by BLI. The final validated TREM2‐ApoE complex structure model provides for further investigating the molecular and structural effects of AD‐associated TREM2 variants on interactions between hydrophobic site of TREM2 and hinge region of ApoE.ResultOur iterative MD/MM‐PBSA–BLI procedure generated an all‐atom model of the TREM2‐ApoE complex that replicates the binding effects of both engineered mutations in TREM2 and AD‐associated variants in both TREM2 and ApoE. The validated model shows the interaction centered on the apical hydrophobic site of TREM2 and the hinge region of ApoE. The interaction sites in the TREM2‐ApoE complex model are consistent with regions affected by AD‐associated variants in simulations of the individual proteins, with biophysical studies using truncated constructs of the two proteins, and with a recently reported minimal active peptide of TREM2 that is sufficient to stimulate its cytoprotective and cytokine regulating roles in primary microglia.ConclusionIterative molecular modeling and biophysical binding affinity assays show the hydrophobic site of TREM2 binding the hinge region of ApoE, an interaction which is impaired by AD‐associated variants and which can be targeted to generate novel therapeutics for AD.

Experimental investigation on plastic hinge length of RC round-ended hollow columns with a variable section

In the ductile design, it is an essential task to accurately estimate the equivalent plastic hinge length of a reinforced concrete (RC) column. The plastic behavior of a RC hollow column is influenced significantly by tension shift and bond-slip according to the test results, which hasn’t yet received adequate attention in the existing models of equivalent plastic hinge in the literatures. Few publications and even no explicit provision in the current codes provide the method to compute the plastic hinge of hollow RC columns, especially those with a variable section. To bridge this knowledge gap, in this study, based on the equivalent principle of plastic displacement, an innovative formula of equivalent plastic hinge length is derived for the hollow columns, which considers both the effects of plastic tension shift and bond-slip based upon the authors’ previous research on solid columns. The quasi-static tests on two rectangular and two round-ended hollow columns were conducted to calibrate the undetermined parameters of the proposed formula. The accuracy and applicability of the proposed formula were then investigated based on the available test results of 30 rectangular and 3 round-ended hollow columns in literature. The results show that different slip behavior leads to distinct plastic deformation between these two types of hollow columns. Compared to the rectangular columns with a uniform section, the plastic hinge length of round-ended columns is longer due to the presence of the bottom solid segment, chamfer, and variable section. For both types of hollow columns, the equivalent plastic hinge region increases with the specimen length, section height, and strain penetration. The comparison suggests that the proposed formula is more appropriate to calculate the plastic hinge length of hollow columns with a uniform or variable section than the existing models.

Thermodynamic and kinetic analysis of the LAO binding protein and its isolated domains reveal non-additivity in stability, folding and function.

Substrate-binding proteins (SBPs) are used by organisms from the three domains of life for transport and signalling. SBPs are composed of two domains that collectively trap ligands with high affinity and selectivity. To explore the role of the domains and the integrity of the hinge region between them in the function and conformation of SBPs, here, we describe the ligand binding, conformational stability and folding kinetics of the Lysine Arginine Ornithine (LAO) binding protein from Salmonella thiphimurium and constructs corresponding to its two independent domains. LAO is a class II SBP formed by a continuous and a discontinuous domain. Contrary to the expected behaviour based on their connectivity, the discontinuous domain shows a stable native-like structure that binds l-arginine with moderate affinity, whereas the continuous domain is barely stable and shows no detectable ligand binding. Regarding folding kinetics, studies of the entire protein revealed the presence of at least two intermediates. While the unfolding and refolding of the continuous domain exhibited only a single intermediate and simpler and faster kinetics than LAO, the folding mechanism of the discontinuous domain was complex and involved multiple intermediates. These findings suggest that in the complete protein the continuous domain nucleates folding and that its presence funnels the folding of the discontinuous domain avoiding nonproductive interactions. The strong dependence of the function, stability and folding pathway of the lobes on their covalent association is most likely the result of the coevolution of both domains as a single unit.

Design, Synthesis, and Evaluation of a New Series of Hydrazones as Small-Molecule Akt Inhibitors for NSCLC Therapy.

In an endeavor to identify small molecules for the management of non-small-cell lung carcinoma, 10 new hydrazone derivatives (3a-j) were synthesized. MTT test was conducted to examine their cytotoxic activities against human lung adenocarcinoma (A549) and mouse embryonic fibroblast (L929) cells. Compounds 3a, 3e, 3g, and 3i were determined as selective antitumor agents on A549 cell line. Further studies were conducted to figure out their mode of action. Compounds 3a and 3g markedly induced apoptosis in A549 cells. However, both compounds did not show any significant inhibitory effect on Akt. On the other hand, in vitro experiments suggest that compounds 3e and 3i are potential anti-NSCLC agents acting through Akt inhibition. Furthermore, molecular docking studies revealed a unique binding mode for compound 3i (the strongest Akt inhibitor in this series), which interacts with both hinge region and acidic pocket of Akt2. However, it is understood that compounds 3a and 3g exert their cytotoxic and apoptotic effects on A549 cells via different pathway(s).

Decoupling the dynamic mechanism revealed by FGFR2 mutation-induced population shift

The fibroblast growth factor receptor 2 (FGFR2) is a key component in cellular signaling networks, and its dysfunctional activation has been implicated in various diseases including cancer and developmental disorders. Mutations at the activation loop (A-loop) have been suggested to trigger an increased basal kinase activity. However, the molecular mechanism underlying this highly dynamic process has not been fully understood due to the limitation of static structural information. Here, we conducted multiple, large-scale Gaussian accelerated molecular dynamics simulations of five (K659E, K659N, K659M, K659Q, and K659T) FGFR2 mutants at the A-loop, and comprehensively analyzed the dynamic molecular basis of FGFR2 activation. The results quantified the population shift of each system, revealing that all mutants had a higher proportion of active-like states. Using Markov state models, we extracted the representative structure of different conformational states and identified key residues related to the increased kinase activity. Furthermore, community network analysis showed enhanced information connections in the mutants, highlighting the long-range allosteric communication between the A-loop and the hinge region. Our findings may provide insights into the dynamic mechanism for FGFR2 dysfunctional activation and allosteric drug discovery. Communicated by Ramaswamy H. Sarma

Synthesis and structure-activity relationship studies of naphthoquinones as STAT3 inhibitors

Based on previous studies, we synthesized a novel class of ortho- and para-naphthoquinones derivatives bearing a phenolic hydroxy or sulfonamide moiety and evaluated their in vitro antiproliferative and signal transducer and activator of transcription-3 (STAT3) phosphorylation inhibitory activities. The biological evaluations of these naphthoquinones revealed that ortho-naphthoquinones containing a phenolic hydroxyl group exhibited greater antiproliferative activity compared to compounds without a phenolic hydroxyl group. Among the synthesized para-naphthoquinones, 21, which has a condensed sulfonamide structure, showed substantially higher antiproliferative activity than that of the parent compound, and was also found to inhibit the phosphorylation of STAT3(Y705) in a dose-dependent manner. A docking simulation using AutoDock Vina suggested that 21 could directly bind to the hinge region of STAT3.

Torsional behavior of hybrid fiber reinforced shear walls an experimental point of view

AbstractThe shear wall system is one of the most commonly used lateral load‐resisting systems in reinforced concrete (RC) high‐rise buildings. The present work is designed to investigate the behavior of reinforced shear walls upon their hybrid fiber ratio, the horizontal reinforcement ratio, and for the reinforcement aspect ratio. For this purpose, nine‐shear walls were manufactured. The same longitudinal reinforcement ratio and the steel and carbon fibers were the main design concept. Withal, the self‐compacting concrete with fiber hybridization was the general perspective for the RC. All the shear wall models were inspected not only for torsion, but also for the energy dissipation capacity, crack propagation, etc. Torsional moment capacity increased, when the hybrid fiber ratio rose up from 0% to 1.0% and then 1.5%. However, this increase was larger in case of increasing the hybrid fiber ratio from 0% to 1.0%. Increasing the hybrid fiber ratio from 1.0% to 1.5% did not result in a performance increase as in the first case. Increasing the fiber ratio from 0% to 1% and from 1% to 1.5% increased the maximum torsional moment between 7.39% and 33.41%. Torsional moment and twist angle capacities increase with the increase in hybrid fiber and horizontal reinforcement ratio and the decrease in the aspect ratio in RC shear walls. Withal, the energy absorption capacity, ductility, and stiffness are also increased as the hybrid fiber ratio. Increasing the hybrid fiber ratio decreased the crack density in the plastic hinge region. Decreasing the aspect ratio from 1.5 to 1.25 increased the dissipation energy capacity between 68.75% and 100%. The addition of 1.0% fiber, a significant increase in the value of ~30% and 53% was achieved stiffness after the crack.

High-throughput Western Blot for in vitro and in vivo assessment of human Ig specific cleaving enzymes

Abstract Antibodies play a vital role in the body’s immune defense mechanism. However, pathogenic Ig antibodies are thought to contribute to several autoimmune conditions and transplant rejection. Proteases derived from human pathogens can specifically cleave IgG into F(ab’)2 and Fc fragments, thus allowing for rapid clearance of IgG. This unique trait suggests a novel opportunity to use these molecules to treat autoantibody mediated diseases. Using our IMPACT platform leveraging machine learning, we have engineered Ig proteases that specifically cleave IgG, and treatment with these proteases causes cleavage below the hinge region, which can result in the generation of the following degradation products: 1) Fab’2 and Fc fragments lacking effector function; or 2) single chain cleaved fragments with reduced effector functions. To detect these species, we developed a method using JESS, an automated high throughput western blot instrument. Using this system, we can detect intact, single cleaved and fully cleaved IgG in a time and dose dependent manner in both in vitro and in vivo assays. In conclusion, we successfully optimized a high throughput assay format that is highly specific and sensitive in evaluating the cleavage activity of different Ig proteases and could be a powerful tool to assess efficacy of IgG cleaving enzymes in the clinic.

Engineering Antibody Fusion Proteins for Targeted Intracellular Therapeutic Delivery

Abstract Intracellular (IC) proteins make up a significant portion of the proteome, leading to an interest in achieving immune modulation via IC targeted therapeutics. Most IC protein-targeted drugs are small molecules, which can easily pass through the cell membrane but are hindered by a lack of specificity and short half-life. Antibodies, thanks to their greater contact surface area, can have superior target specificity, and persist longer in the blood. Fusing a therapeutic to an antibody presents a promising solution, but antibodies lack a good path into the cell. We recently discovered a multiparatopic antibody that binds multiple epitopes on the immune checkpoint protein programed death-ligand 1 (PD-L1) to induce its downregulation. This platform promotes endocytosis through clustering receptors together on the surface and enhancing internalization. We sought to exploit this mechanism for IC delivery by introducing endosomalytic peptides to induce escape of the multiparatopic antibody into the cytosol. We inserted two different endosomalytic peptides (EPs) into the downregulating antibody, one before and after the hinge region. We found that these EPs do not hinder downregulation and observed decreased lysosomal accumulation of the antibodies. To determine the efficacy of this modified antibody as a vehicle for an IC therapeutic, we fused Cas9 to the C-terminus of the heavy chain (HC) of our anti-PD-L1 multiparatopic antibody. We further demonstrated robust PD-L1 downregulation and endosomal escape for these fusion proteins. These data generate proof-of-concept results, establishing a promising new vehicle for IC delivery, with wide-ranging applications including gene therapy, signaling modulation, and genetic engineering.

START domain-mediated nuclear translocation of Them1 regulates the expression of metabolic genes relative to thermogenesis in brown adipocytes

Thioesterase superfamily member 1 (Them1) is a long-chain fatty acyl-CoA thioesterase that is highly expressed in brown adipose tissue (BAT), where it limits energy expenditure. Them1 is comprised of tandem enzymatic domains and a C-terminal lipid-binding steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain. Fatty acid binding to the START domain stabilizes the enzymatic domains and increases activity, whereas lysophosphatidylcholine (LPC) acts in the opposite manner. In brown adipocytes, Them1 localizes to puncta within the cytoplasm, but diffuses in response to β-adrenergic stimulation after which it is also found in the nucleus. The aim of this study was to determine the mechanism of Them1 nuclear trafficking and the metabolic consequences. METHODS: Identification of a putative nuclear localization signal (NLS) was determined by computational analysis. Full-length, NLS-deleted, or truncated Them1 cDNA constructs were cloned and linked to EGFP. Plasmid or adenoviral vectors were transfected into immortalized brown adipose cells (iBAs) that do not express Them1. Them1 was visualized by live cell confocal imaging via EGFP and with NucSpot nuclear staining. Signal quantification was performed with Imaris software. Them1 nuclear localization was evaluated in the presence or absence of norepinephrine (NE), leptomycin B, cycloheximide, or lipids that bind the START domain. Them1 nuclear localization was also quantified in BAT from mice treated with a β3-adrenergic receptor agonist, CL316,243. mRNA expression in iBAs expressing full-length or NLS-mutant Them1 in response to NE was determined by RNA-Seq. RESULTS: In the diffuse state, Them1 localized to the nucleus in a time-dependent manner. The NLS resides in a hinge region between the thioesterase domains and the START domain. Deletion of either the NLS or the START domain blocked nuclear targeting. Conversely, truncated Them1 containing only the START domain was targeted to the nucleus. Entry into the nucleus was balanced by trafficking out of the nucleus and new protein synthesis was not required, as evidenced by using leptomycin B to block nuclear efflux and cycloheximide to block protein synthesis, respectively. Incubation of NE-stimulated iBAs with LPC promoted nuclear Them1 translocation. Them1 protein was also detected in the nucleus of BAT from wild-type mice in response to CL administration. Nuclear Them1 localization in iBAs was associated with down-regulated mRNA expression of genes that control lipid and glucose metabolism during cold adaptation. CONCLUSIONS: Our data suggest that LPC generated during thermogenesis binds and destabilizes the START domain, leading to exposure of the NLS and nuclear trafficking of Them1 in response to β-adrenergic stimulation. Once inside the nucleus, Them1 regulates the transcription of lipid and glucose metabolic enzymes that are required for chronic cold adaptation. This work was supported by NIH DK103046 to DEC, SJH, and EAO This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

In Silico-Motivated Discovery of Novel Potent Glycogen Synthase-3 Inhibitors: 1-(Alkyl/arylamino)-3H-naphtho[1,2,3-de]quinoline-2,7-dione Identified as a Scaffold for Kinase Inhibitor Development.

Glycogen synthase kinase-3 (GSK-3) isoforms α and β have diverse roles within cell biology, and have been linked with multiple diseases that include prominent CNS conditions such as Alzheimer's disease and several psychiatric disorders. In this study, motivated by computation, we aimed to identify novel ATP-binding site inhibitors of GSK-3 with CNS-active potential. A ligand screening (docking) protocol against GSK-3β was first optimized, employing an active/decoy benchmarking set, with the final protocol selected based on statistical performance analysis. The optimized protocol involved pre-filtering of ligands using a three-point 3D-pharmacophore, followed by Glide-SP docking applying hinge region hydrogen bonding constraints. Using this approach, the Biogenic subset of the ZINC15 compound database was screened, focused on compounds with potential for CNS-activity. Twelve compounds (generation I) were selected for experimental validation using in vitro GSK-3β binding assays. Two hit compounds, 1 and 2, with 6-amino-7H-benzo[e]perimidin-7-one and 1-(phenylamino)-3H-naphtho[1,2,3-de]quinoline-2,7-dione type scaffolds were identified with IC50 values of 1.63 µM and 20.55 µM, respectively. Ten analogues of 2 (generation II) were selected for structure activity relationship (SAR) analysis and revealed four low micromolar inhibitors (<10 µM), with 19 (IC50 = 4.1 µM)~five times more potent than initial hit compound 2. Selectivity screening of low micromolar inhibitors 14 and 19 (comparing aryl- and alkyl-substituents) against 10 homologous kinases revealed unique selectivity profiles, with both compounds more potent against the GSK-3α isoform (IC50s~2 µM) and, additionally, inhibitors of PKBβ (IC50s < 25 µM). Compound 14 also inhibited ERK2 and 19, PKCγ, but generally good selectivity for GSK-3 isoforms over the other kinases was observed. The compounds had excellent predicted oral bioavailability and CNS-activity profiles, presenting promising candidates for future testing in cellular models of disease.

Mutation of the TGN1412 anti-CD28 monoclonal antibody lower hinge confers specific FcγRIIb binding and retention of super-agonist activity.

The agonistic action of several immunomodulatory monoclonal antibodies (mAbs) requires both target antigen binding and clustering of this mAb:target complex by the Fcs interacting with Fcγ receptors (FcγRs), in particular FcγRIIb, on neighboring bystander cells. Fc mutations were made in the immunoglobulin G4 (IgG4)-based TGN1412 anti-CD28 mAb to define the role of FcγR interactions in its "super-agonist" activity. The dual mutation, IgG4-ED269,270 AA, ablated interaction with all human FcγRs and agonistic action was consequentially lost, confirming the FcγR dependence on the action of TGN1412. The IgG4 lower hinge region (F234 L235 G236 G237 ) was modified by L235 E mutation (F234 E235 G236 G237 ), a mutation commonly used to ablate FcγR binding, including in approved therapeutic mAbs. However, rather than ablating all FcγR binding, IgG4-L235 E conferred specific binding to FcγRIIb, the inhibitory Fc receptor. Furthermore, in combination with the core hinge-stabilizing mutation (IgG4-S228 P, L235 E), this mutation increased affinity for FcγRIIb compared with wild-type IgG4. In addition to having FcγRIIb specificity, these engineered TGN1412 antibodies retained their super-agonistic ability, demonstrating that CD28- and FcγRIIb-specific binding are together sufficient for agonistic function. The FcγRIIb-specific nature of IgG4-L235 E has utility for mAb-mediated immune agonism therapies that are dependent on FcγRIIb interaction and of anti-inflammatory mAbs in allergy and autoimmunity that harness FcγRIIb inhibitory signaling.

A PD-L1/EGFR bispecific antibody combines immune checkpoint blockade and direct anti-cancer action for an enhanced anti-tumor response

ABSTRACT Immune checkpoint blockade (ICB) with antibodies has shown durable clinical responses in a wide range of cancer types, but the overall response rate is still limited. Other effective therapeutic modalities to increase the ICB response rates are urgently needed. New bispecific antibody (bsAb) formats combining the ICB effect and a direct action on cancer cells could improve the efficacy of current immunotherapies. Here, we report the development of a PD-L1/EGFR symmetric bsAb by fusing a dual-targeting tandem trimmer body with the human IgG1 hinge and Fc regions. The bsAb was characterized in vitro and the antitumor efficacy was evaluated in humanized mice bearing xenografts of aggressive triple-negative breast cancer and lung cancer. The IgG-like hexavalent bsAb, designated IgTT-1E, was able to simultaneously bind both EGFR and PD-L1 antigens, inhibit EGF-mediated proliferation, effectively block PD-1/PD-L1 interaction, and induce strong antigen-specific antibody-dependent cellular cytotoxicity activity in vitro. Potent therapeutic efficacies of IgTT-1E in two different humanized mouse models were observed, where tumor growth control was associated with a significantly increased proportion of CD8+ T cells. These results support the development of IgTT-1E for the treatment of EGFR+ cancers.

Discovery of a cryptic pocket in the AI-predicted structure of PPM1D phosphatase explains the binding site and potency of its allosteric inhibitors.

Virtual screening is a widely used tool for drug discovery, but its predictive power can vary dramatically depending on how much structural data is available. In the best case, crystal structures of a ligand-bound protein can help find more potent ligands. However, virtual screens tend to be less predictive when only ligand-free crystal structures are available, and even less predictive if a homology model or other predicted structure must be used. Here, we explore the possibility that this situation can be improved by better accounting for protein dynamics, as simulations started from a single structure have a reasonable chance of sampling nearby structures that are more compatible with ligand binding. As a specific example, we consider the cancer drug target PPM1D/Wip1 phosphatase, a protein that lacks crystal structures. High-throughput screens have led to the discovery of several allosteric inhibitors of PPM1D, but their binding mode remains unknown. To enable further drug discovery efforts, we assessed the predictive power of an AlphaFold-predicted structure of PPM1D and a Markov state model (MSM) built from molecular dynamics simulations initiated from that structure. Our simulations reveal a cryptic pocket at the interface between two important structural elements, the flap and hinge regions. Using deep learning to predict the pose quality of each docked compound for the active site and cryptic pocket suggests that the inhibitors strongly prefer binding to the cryptic pocket, consistent with their allosteric effect. The predicted affinities for the dynamically uncovered cryptic pocket also recapitulate the relative potencies of the compounds (τb = 0.70) better than the predicted affinities for the static AlphaFold-predicted structure (τb = 0.42). Taken together, these results suggest that targeting the cryptic pocket is a good strategy for drugging PPM1D and, more generally, that conformations selected from simulation can improve virtual screening when limited structural data is available.