Discovery of Potent, Selective, and Orally Available IRE1α Inhibitors Demonstrating Comparable PD Modulation to IRE1 Knockdown in a Multiple Myeloma Model.

The lack of selective and safe in vivo IRE1α tool molecules has limited the evaluation of IRE1α as a viable target to treat multiple myeloma. Focus on improving the physicochemical properties of a literature compound by decreasing lipophilicity, molecular weight, and basicity allowed the discovery of a novel series with a favorable in vitro safety profile and good oral exposure. These efforts culminated in the identification of a potent and selective in vivo tool compound, G-5758, that was well tolerated following multiday oral administration of doses up to 500 mg/kg. G-5758 demonstrated comparable pharmacodynamic effects to induced IRE1 knockdown as measured by XBP1s levels in a multiple myeloma model (KMS-11).

HPK1 citron homology domain regulates phosphorylation of SLP76 and modulates kinase domain interaction dynamics.

Hematopoietic progenitor kinase 1 (HPK1) is a negative regulator of T-cell receptor signaling and as such is an attractive target for cancer immunotherapy. Although the role of the HPK1 kinase domain (KD) has been extensively characterized, the function of its citron homology domain (CHD) remains elusive. Through a combination of structural, biochemical, and mechanistic studies, we characterize the structure-function of CHD in relationship to KD. Crystallography and hydrogen-deuterium exchange mass spectrometry reveal that CHD adopts a seven-bladed ß-propellor fold that binds to KD. Mutagenesis associated with binding and functional studies show a direct correlation between domain-domain interaction and negative regulation of kinase activity. We further demonstrate that the CHD provides stability to HPK1 protein in cells as well as contributes to the docking of its substrate SLP76. Altogether, this study highlights the importance of the CHD in the direct and indirect regulation of HPK1 function.

Injectable ChitHCl-DDA tissue adhesive with high adhesive strength and biocompatibility for torn meniscus repair and regeneration.

Suture pull-through is a clinical problem in meniscus repair surgery due to the sharp leading edge of sutures. Several tissue adhesives have been developed as an alternative to traditional suturing; however, there is still no suitable tissue adhesive specific for meniscus repair treatment due to unsatisfactory biosafety, biodegradable, sterilizable, and tissue-bonding characteristics. In this study, we used a tissue adhesive composed of chitosan hydrochloride reacted with oxidative periodate-oxidized dextran (ChitHCl-DDA) combined with a chitosan-based hydrogel and oxidative dextran to attach to the meniscus. We conducted viscoelastic tests, viscosity tests, lap shear stress tests, Fourier transform infrared (FTIR) spectroscopy, swelling ratio tests, and degradation behavior tests to characterize these materials. An MTT assay, alcian blue staining, migration assay, cell behavior observations, and protein expression tests were used to understand cell viability and responses. Moreover, ex vivo and in vivo tests were used to analyze tissue regeneration and biocompatibility of the ChitHCl-DDA tissue adhesive. Our results revealed that the ChitHCl-DDA tissue adhesive provided excellent tissue adhesive strength, cell viability, and cell responses. This tissue adhesive has great potential for torn meniscus tissue repair and regeneration.

Plasma-Enabled Graphene Quantum Dot Hydrogel-Magnesium Composites as Bioactive Scaffolds for In Vivo Bone Defect Repair.

Bioactive and mechanically stable metal-based scaffolds are commonly used for bone defect repair. However, conventional metal-based scaffolds induce nonuniform cell growth, limiting damaged tissue restoration. Here, we develop a plasma nanotechnology-enhanced graphene quantum dot (GQD) hydrogel-magnesium (Mg) composite scaffold for functional bone defect repair by integrating a bioresource-derived nitrogen-doped GQD (NGQD) hydrogel into the Mg ZK60 alloy. Each scaffold component brings major synergistic advantages over the current alloy-based state of the art, including (1) mechanical support of the cortical bone and calcium deposition by the released Mg2+ during degradation; (2) enhanced uptake, migration, and distribution of osteoblasts by the porous hydrogel; and (3) improved osteoblast adhesion and proliferation, osteogenesis, and mineralization by the NGQDs in the hydrogel. Through an in vivo study, the hybrid scaffold with the much enhanced osteogenic ability induced by the above synergy promotes a more rapid, uniform, and directional bone growth across the hydrogel channel, compared with the control Mg-based scaffold. This work provides insights into the design of multifunctional hybrid scaffolds, which can be applied in other areas well beyond the demonstrated bone defect repair.

Publisher Erratum to: The rationale and potential for using Lactobacillus in the management of periodontitis.

Due to a technical defect the following articles have been mistakenly published Open Access:The rationale and potential for using Lactobacillus in the management of periodontitis. J. Microbiol. 60, 355-363 (doi: https://doi.org/10.1007/s12275-022-1514-4 ).The copyright of these article changes to 'Copyright © 2022, Author(s) under the exclusive license with the Microbiological Society of Kore' with all rights reserved.

A saturation mutagenesis screen uncovers resistant and sensitizing secondary KRAS mutations to clinical KRASG12C inhibitors.

Mutant-specific inhibitors of KRASG12C, such as AMG510 (sotorasib) and MRTX849 (adagrasib), offer the unprecedented opportunity to inhibit KRAS, the most frequently mutated and heretofore undruggable oncoprotein. While clinical data are still limited, on-target mutations in KRASG12C at position 12 and other sites are emerging as major drivers of clinical relapse. We identified additional mutations in KRASG12C that impact inhibitor sensitivity through a saturation mutagenesis screen in the KRASG12C NCI-H358 non­small-cell lung cancer (NSCLC) cell line. We also identified individuals in population genetic databases harboring these resistance mutations in their germline and in tumors, including a subset that co-occur with KRASG12C, indicating that these mutations may preexist in patients treated with KRASG12C inhibitors. Notably, through structural modeling, we found that one such mutation (R68L) interferes with the critical protein­drug interface, conferring resistance to both inhibitors. Finally, we uncovered a mutant (S17E) that demonstrated a strong sensitizing phenotype to both inhibitors. Functional studies suggest that S17E sensitizes KRASG12C cells to KRASG12C inhibition by impacting signaling through PI3K/AKT/mTOR but not the MAPK signaling pathway. Our studies highlight the utility of unbiased mutation profiling to understand the functional consequences of all variants of a disease-causing genetic mutant and predict acquired resistant mutations in the targeted therapeutics.

The rationale and potential for using Lactobacillus in the management of periodontitis.

Periodontitis refers to a wide range of the inflammatory conditions of supporting dental structures. For some patients with periodontitis, antibacterial agents are needed as an adjuvant to mechanical debridement treatments and oral hygiene maintenance. However, the widespread use of broad-spectrum antibiotics for the prophylaxis and treatment of periodontal infections results in the emergence of resistant pathogens. Therefore, probiotics have become markedly interesting to researchers as a potentially safe alternative to periodontal treatment and maintenance. Probiotics have been used in medicine for decades and extensively applied to the treatment of inflammatory diseases through the modulation of microbial synergy and other mechanisms. A growing amount of evidence has shown that using Lactobacillus strains for oral cavity maintenance could improve periodontal health. In this study, we reviewed studies showing proof of the inhibitory effects of Lactobacillus species on periodontal inflammation. We also explored the rationale and potential for using Lactobacillus species in the management of periodontitis.

Using Cu as a Spacer to Fabricate and Control the Porosity of Titanium Zirconium Based Bulk Metallic Glass Foams for Orthopedic Implant Applications.

In this study, a porous titanium zirconium (TiZr)-based bulk metallic foam was successfully fabricated using the Cu spacer by employing the hot press method. TiZr-based bulk metallic foams with porosities ranging from 0% to 50% were fabricated and analyzed. The results indicate that thermal conductivity increased with the addition of Cu spacer; the increased thermal conductivity reduced the holding time in the hot press method. Moreover, the compressive strength decreased from 1261 to 76 MPa when the porosity of the TiZr-based bulk metallic foam increased to 50%, and the compressive strength was predictable. In addition, the foam demonstrated favorable biocompatibility in cell viability, cell migration capacity, and calcium deposition tests. Moreover, the pore size of the porous TiZr-based bulk metallic foam was around 120 µm. In conclusion, TiZr-based bulk metallic foam has favorable biocompatibility, mechanical property controllability, and porous structure for bone ingrowth and subsequent enhanced osteointegration. This porous TiZr-based bulk metallic foam has great potential as an orthopedic implant to enhance bone healing and decrease healing time.

Discovery of Spiro-azaindoline Inhibitors of Hematopoietic Progenitor Kinase 1 (HPK1).

Hematopoietic progenitor kinase 1 (HPK1) is implicated as a negative regulator of T-cell receptor-induced T-cell activation. Studies using HPK1 kinase-dead knock-in animals have demonstrated the loss of HPK1 kinase activity resulted in an increase in T-cell function and tumor growth inhibition in glioma models. Herein, we describe the discovery of a series of small molecule inhibitors of HPK1. Using a structure-based drug design approach, the kinase selectivity of the molecules was significantly improved by inducing and stabilizing an unusual P-loop folded binding mode. The metabolic liabilities of the initial 7-azaindole high-throughput screening hit were mitigated by addressing a key metabolic soft spot along with physicochemical property-based optimization. The resulting spiro-azaindoline HPK1 inhibitors demonstrated improved in vitro ADME properties and the ability to induce cytokine production in primary human T-cells.

Conformation-locking antibodies for the discovery and characterization of KRAS inhibitors.

Small molecules that stabilize inactive protein conformations are an underutilized strategy for drugging dynamic or otherwise intractable proteins. To facilitate the discovery and characterization of such inhibitors, we created a screening platform to identify conformation-locking antibodies for molecular probes (CLAMPs) that distinguish and induce rare protein conformational states. Applying the approach to KRAS, we discovered CLAMPs that recognize the open conformation of KRASG12C stabilized by covalent inhibitors. One CLAMP enables the visualization of KRASG12C covalent modification in vivo and can be used to investigate response heterogeneity to KRASG12C inhibitors in patient tumors. A second CLAMP enhances the affinity of weak ligands binding to the KRASG12C switch II region (SWII) by stabilizing a specific conformation of KRASG12C, thereby enabling the discovery of such ligands that could serve as leads for the development of drugs in a high-throughput screen. We show that combining the complementary properties of antibodies and small molecules facilitates the study and drugging of dynamic proteins.

Temporal-spatial variations, source apportionment, and ecological risk of trace elements in sediments of water-level-fluctuation zone in the Three Gorges Reservoir, China.

The Three Gorges Reservoir (TGR) plays a crucial role in providing electricity for mega-cities across China. However, since the impoundment was completed in 2006, attention to environmental concerns has also been intensive. In order to determine the distribution, sources, and pollution status of trace elements in the water fluctuation zone of the TGR following ten years of repeated "submergence" and "exposure", we systematically collected 16 paired surface sediment samples (n = 32) covering the entire main body of the TGR in March 2018 (following 6 months of submergence) and September 2018 (after 6 months of exposure), and quantitatively analyzed 13 elements (e.g., Mn, Fe, V, Cr, Ni, Cu, Zn, As, Sr, Y, Zr, Ba, and Pb) using X-ray fluorescence spectrophotometry (XRF). The results showed that, except for Sr, concentrations of trace metals following submergence were generally higher than those after exposure due to the less settling of suspended solids at the faster flow velocity during the drawdown period. Assessment using enrichment factors (EFs) and a geo-accumulation index (Igeo) both characterized a relatively serious anthropogenic pollution status of metals in the upper reaches of the TGR with respect to the middle-lower reaches. Source apportionment by positive matrix factorization (PMF) analysis indicated that agricultural activities (24.8 and 24.3%, respectively) and industrial emissions (24.5 and 22.9%, respectively) were the two major sources in these two periods, followed by natural sources, domestic sewage, and ore mining. Ecological risk assessment showed that metalloid arsenic (As) could be the main potential issue of risk to aquatic organisms and human health. A new source-specific risk assessment method (pRI) combined with PMF revealed that agricultural activities could be the major source of potential ecological risk and should be prioritized as the focus of metal/metalloid risk management in the TGR.

Decoding non-canonical mRNA decay by the endoplasmic-reticulum stress sensor IRE1α.

Inositol requiring enzyme 1 (IRE1) mitigates endoplasmic-reticulum (ER) stress by orchestrating the unfolded-protein response (UPR). IRE1 spans the ER membrane, and signals through a cytosolic kinase-endoribonuclease module. The endoribonuclease generates the transcription factor XBP1s by intron excision between similar RNA stem-loop endomotifs, and depletes select cellular mRNAs through regulated IRE1-dependent decay (RIDD). Paradoxically, in mammals RIDD seems to target only mRNAs with XBP1-like endomotifs, while in flies RIDD exhibits little sequence restriction. By comparing nascent and total IRE1α-controlled mRNAs in human cells, we identify not only canonical endomotif-containing RIDD substrates, but also targets without such motifs-degraded by a process we coin RIDDLE, for RIDD lacking endomotif. IRE1α displays two basic endoribonuclease modalities: highly specific, endomotif-directed cleavage, minimally requiring dimers; and more promiscuous, endomotif-independent processing, requiring phospho-oligomers. An oligomer-deficient IRE1α mutant fails to support RIDDLE in vitro and in cells. Our results advance current mechanistic understanding of the UPR.

Two-Step Approach Using Degradable Magnesium to Inhibit Surface Biofilm and Subsequently Kill Planktonic Bacteria.

Bacterial infection remains a great risk in medical implantation surgery. In this paper, we found that degradable metals may be a feasible alternative option of antibacterial implantation materials. It is known that the spalling mechanism of magnesium (Mg) during degradation leads to Mg ions-induced alkaline environment, which is harmful to planktonic bacteria. In this study, we showed that alkaline pH environment is almost harmless to those adhesive bacteria protected in well-formed biofilms. Moreover, experimental results demonstrated that the biofilm formed in the place where Mg spalls are destroyed, releasing the covered bacteria to be planktonic in the alkaline environment. As a result, the colonization of biofilms continues to shrink during the degradation of Mg. It implies that if degradable metal is employed as implantation material, even if bacterial infection occurs, it may be possibly cured without second surgery.

Targeting KRAS Mutant Cancers via Combination Treatment: Discovery of a Pyridopyridazinone pan-RAF Kinase Inhibitor.

Structure-based optimization of a set of aryl urea RAF inhibitors has led to the identification of Type II pan-RAF inhibitor GNE-9815 (7), which features a unique pyrido[2,3-d]pyridazin-8(7H)-one hinge-binding motif. With minimal polar hinge contacts, the pyridopyridazinone hinge binder moiety affords exquisite kinase selectivity in a lipophilic efficient manner. The improved physicochemical properties of GNE-9815 provided a path for oral dosing without enabling formulations. In vivo evaluation of GNE-9815 in combination with the MEK inhibitor cobimetinib demonstrated synergistic MAPK pathway modulation in an HCT116 xenograft mouse model. To the best of our knowledge, GNE-9815 is among the most highly kinase-selective RAF inhibitors reported to date.

Targeting KRAS Mutant Cancers via Combination Treatment: Discovery of a 5-Fluoro-4-(3H)-quinazolinone Aryl Urea pan-RAF Kinase Inhibitor.

Optimization of a series of aryl urea RAF inhibitors led to the identification of type II pan-RAF inhibitor GNE-0749 (7), which features a fluoroquinazolinone hinge-binding motif. By minimizing reliance on common polar hinge contacts, this hinge binder allows for a greater contribution of RAF-specific residue interactions, resulting in exquisite kinase selectivity. Strategic substitution of fluorine at the C5 position efficiently masked the adjacent polar NH functionality and increased solubility by impeding a solid-state conformation associated with stronger crystal packing of the molecule. The resulting improvements in permeability and solubility enabled oral dosing of 7. In vivo evaluation of 7 in combination with the MEK inhibitor cobimetinib demonstrated synergistic pathway inhibition and significant tumor growth inhibition in a KRAS mutant xenograft mouse model.

An Approach to Bioactivity Assessment for Critical Quality Attribute Identification Based on Antibody-Antigen Complex Structure.

Identification of critical quality attributes (CQAs) is an important step for development of biopharmaceuticals with intended performance. An accurate CQA assessment is needed to ensure product quality and focusing on development efforts where control is needed. The assignment of criticality is based on safety and efficacy. Efficacy is related to PK and bioactivity. Here, we developed a novel approach based on antibody-antigen complex structure and modeling as a complementary method for bioactivity assessment. To validate this approach, common product related quality attributes and mutagenesis data from several IgGs were assessed using available antibody-antigen complex structures, and results were compared with experimental data from bioactivity or binding affinity measurements. A stepwise evaluation scheme for structural based analysis is proposed; based on systematic assessment following the scheme, good correlation has been observed between structural analysis and experimental data. This demonstrates that such an approach can be applied as a complementary tool for bioactivity assessment. Main applications are 1) To decouple multiple attributes to achieve amino acid resolution for bioactivity assessment, 2) To assess bioactivity of attributes that cannot be experimentally generated, 3) To provide molecular mechanism for experimental observation and understand structure function relationship. Examples are provided to illustrate these applications.

Identification of BRaf-Sparing Amino-Thienopyrimidines with Potent IRE1α Inhibitory Activity.

Amino-quinazoline BRaf kinase inhibitor 2 was identified from a library screen as a modest inhibitor of the unfolded protein response (UPR) regulating potential anticancer target IRE1α. A combination of crystallographic and conformational considerations were used to guide structure-based attenuation of BRaf activity and optimization of IRE1α potency. Quinazoline 6-position modifications were found to provide up to 100-fold improvement in IRE1α cellular potency but were ineffective at reducing BRaf activity. A salt bridge contact with Glu651 in IRE1α was then targeted to build in selectivity over BRaf which instead possesses a histidine in this position (His539). Torsional angle analysis revealed that the quinazoline hinge binder core was ill-suited to accommodate the required conformation to effectively reach Glu651, prompting a change to the thienopyrimidine hinge binder. Resulting analogues such as 25 demonstrated good IRE1α cellular potency and imparted more than 1000-fold decrease in BRaf activity.

Activation of the IRE1 RNase through remodeling of the kinase front pocket by ATP-competitive ligands.

Inositol-Requiring Enzyme 1 (IRE1) is an essential component of the Unfolded Protein Response. IRE1 spans the endoplasmic reticulum membrane, comprising a sensory lumenal domain, and tandem kinase and endoribonuclease (RNase) cytoplasmic domains. Excess unfolded proteins in the ER lumen induce dimerization and oligomerization of IRE1, triggering kinase trans-autophosphorylation and RNase activation. Known ATP-competitive small-molecule IRE1 kinase inhibitors either allosterically disrupt or stabilize the active dimeric unit, accordingly inhibiting or stimulating RNase activity. Previous allosteric RNase activators display poor selectivity and/or weak cellular activity. In this study, we describe a class of ATP-competitive RNase activators possessing high selectivity and strong cellular activity. This class of activators binds IRE1 in the kinase front pocket, leading to a distinct conformation of the activation loop. Our findings reveal exquisitely precise interdomain regulation within IRE1, advancing the mechanistic understanding of this important enzyme and its investigation as a potential small-molecule therapeutic target.

Disruption of IRE1α through its kinase domain attenuates multiple myeloma.

Multiple myeloma (MM) arises from malignant immunoglobulin (Ig)-secreting plasma cells and remains an incurable, often lethal disease despite therapeutic advances. The unfolded-protein response sensor IRE1α supports protein secretion by deploying a kinase-endoribonuclease module to activate the transcription factor XBP1s. MM cells may co-opt the IRE1α-XBP1s pathway; however, the validity of IRE1α as a potential MM therapeutic target is controversial. Genetic disruption of IRE1α or XBP1s, or pharmacologic IRE1α kinase inhibition, attenuated subcutaneous or orthometastatic growth of MM tumors in mice and augmented efficacy of two established frontline antimyeloma agents, bortezomib and lenalidomide. Mechanistically, IRE1α perturbation inhibited expression of key components of the endoplasmic reticulum-associated degradation machinery, as well as secretion of Ig light chains and of cytokines and chemokines known to promote MM growth. Selective IRE1α kinase inhibition reduced viability of CD138+ plasma cells while sparing CD138- cells derived from bone marrows of newly diagnosed or posttreatment-relapsed MM patients, in both US- and European Union-based cohorts. Effective IRE1α inhibition preserved glucose-induced insulin secretion by pancreatic microislets and viability of primary hepatocytes in vitro, as well as normal tissue homeostasis in mice. These results establish a strong rationale for developing kinase-directed inhibitors of IRE1α for MM therapy.

Activation loop dynamics are controlled by conformation-selective inhibitors of ERK2.

Conformational selection by small molecules expands inhibitory possibilities for protein kinases. Nuclear magnetic resonance (NMR) measurements of the mitogen-activated protein (MAP) kinase ERK2 have shown that activation by dual phosphorylation induces global motions involving exchange between two states, L and R. We show that ERK inhibitors Vertex-11e and SCH772984 exploit the small energetic difference between L and R to shift the equilibrium in opposing directions. An X-ray structure of active 2P-ERK2 complexed with AMP-PNP reveals a shift in the Gly-rich loop along with domain closure to position the nucleotide in a more catalytically productive conformation relative to inactive 0P-ERK2:ATP. X-ray structures of 2P-ERK2 complexed with Vertex-11e or GDC-0994 recapitulate this closure, which is blocked in a complex with a SCH772984 analog. Thus, the L→R shift in 2P-ERK2 is associated with movements needed to form a competent active site. Solution measurements by hydrogen-exchange mass spectrometry (HX-MS) reveal distinct binding interactions for Vertex-11e, GDC-0994, and AMP-PNP with active vs. inactive ERK2, where the extent of HX protection correlates with R state formation. Furthermore, Vertex-11e and SCH772984 show opposite effects on HX near the activation loop. Consequently, these inhibitors differentially affect MAP kinase phosphatase activity toward 2P-ERK2. We conclude that global motions in ERK2 reflect conformational changes at the active site that promote productive nucleotide binding and couple with changes at the activation loop to allow control of dephosphorylation by conformationally selective inhibitors.