Competitive Inhibitor Research Articles

Crucial role of a takeout protein in white-backed planthopper Sogatella furcifera (Horváth) orientation towards its host rice plants.

The takeout (TO) gene family impacts diverse physiological and behavioral functions in insects, yet specific olfactory-associated roles for the family have yet to be fully elucidated. To provide insights into TO function in rice planthoppers, the genomes of three rice planthoppers (white-backed planthopper, brown planthopper and small brown planthopper) were searched for TO homologs and their degree of conservation assessed via chromosomal localization, exon-intron boundaries, phylogenetic relationships and protein domains/motifs. We performed a tissue-specific expression analysis of the 20 TO genes in the white-backed planthopper (WBPH, Sogatella furcifera) and found that SfTO17 is enriched in adult antennae. RNAi-mediated knockdown of SfTO17 impaired WBPH olfaction and reduced host-seeking responses following exposure to rice plants. The binding profile of β-ionone, hexyl benzoate and benzyl benzoate with recombinant SfTO17 was evaluated via competitive fluorescence binding assays. Conformational prediction of SfTO17 coupled with molecular docking analyses revealed several amino acid residues potentially critical for odorant binding. This study demonstrates the olfactory function of SfTO17 in WBPH and highlights its potential as a target for controlling this rice pest. © 2024 Society of Chemical Industry.

Identification of Novel PPARγ Partial Agonists Based on Virtual Screening Strategy: In Silico and In Vitro Experimental Validation.

Thiazolidinediones (TZDs) including rosiglitazone and pioglitazone function as peroxisome proliferator-activated receptor gamma (PPARγ) full agonists, which have been known as a class to be among the most effective drugs for the treatment of type 2 diabetes mellitus (T2DM). However, side effects of TZDs such as fluid retention and weight gain are associated with their full agonistic activities toward PPARγ induced by the AF-2 helix-involved "locked" mechanism. Thereby, this study aimed to obtain novel PPARγ partial agonists without direct interaction with the AF-2 helix. Through performing virtual screening of the Targetmol L6000 Natural Product Library and utilizing molecular dynamics (MD) simulation, as well as molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) analysis, four compounds including tubuloside b, podophyllotoxone, endomorphin 1 and paliperidone were identified as potential PPARγ partial agonists. An in vitro TR-FRET competitive binding assay showed podophyllotoxone displayed the optimal binding affinity toward PPARγ among the screened compounds, exhibiting IC50 and ki values of 27.43 µM and 9.86 µM, respectively. Further cell-based transcription assays were conducted and demonstrated podophyllotoxone's weak agonistic activity against PPARγ compared to that of the PPARγ full agonist rosiglitazone. These results collectively demonstrated that podophyllotoxone could serve as a PPARγ partial agonist and might provide a novel candidate for the treatment of various diseases such as T2DM.

Hyaluronic acid/lactoferrin–coated polydatin/PLGA nanoparticles for active targeting of CD44 receptors in lung cancer

Traditional chemotherapeutic drugs lack optimal efficacy and invoke severe adverse effects in cancer patients. Polydatin (PD), a phytomedicine, has gradually gained attention due to its antitumor activity. However, its low solubility and poor bioavailability are still cornerstone issues. The present study aimed to fabricate and develop hyaluronic acid/lactoferrin–double coated PD/PLGA nanoparticles via a layer-by-layer self-assembly technique for active targeting of CD44 receptors in lung cancer. Different molecular weights (M.wt.) of HA (32 and 110 kDa) were exploited to study the relationship between the HA M.wt. and the NPs targeting efficacy. The optimized formulations were fully characterized. Their cytotoxicity and cellular uptake were investigated against A549 cell line by CCK-8 kit and fluorescence imaging, respectively. Finally, HA110/Lf-coated PD/PLGA NPs (F9) were subjected to a competitive inhibition study to prove internalization through CD44 overexpressed receptors. The results verified the fabrication of F9 with a particle size of 174.87 ± 3.97 nm and a zeta potential of −24.37 ± 1.19 mV as well as spherical NPs architecture. Importantly, it provoked enhanced cytotoxicity (IC50 = 0.57 ± 0.02 µg/mL) and superior cellular uptake efficacy. To conclude, the current investigation lays the foundation for the prospective therapeutic avenue of F9 for active targeting of CD44 receptors in lung cancer.

Mechanism-Based Allylic Carbasugar Chlorides That Form Covalent Intermediates with α- and β-Galactosidases.

Glycoside hydrolases have been implicated in a wide range of human conditions including lysosomal storage diseases. Consequently, many researchers have directed their efforts towards identifying new classes of glycoside hydrolase inhibitors, both synthetic and from natural sources. A large percentage of such inhibitors are reversible competitive inhibitors that bind in the active site often due to them possessing structural features, often a protonatable basic nitrogen atom, that mimic the enzymatic transition state. We report that mechanism-based small molecule galacto-like configured cyclohexenyl carbasugars form reversible covalent complexes with both α-galactosidase and β-galactosidase. In addition, we show that the β-galactosidase from Aspergillus oryzae reacts with three different carbasugar inhibitors, with three different second-order rate constants (kinact/Ki), to give the same enzyme-carbasugar covalent intermediate. The surprising observation that the α-galacto-configured inhibitor covalently labels the A. oryzae β-galactosidase highlights the catalytic versatility of glycoside hydrolases. We expect that cyclohexenyl covalent inhibitors will become an important class of compounds in the chemical biologist's tool box.

Extracellular Vesicle Mobility in Collagen I Hydrogels Is Influenced by Matrix-Binding Integrins.

Extracellular vesicles (EVs) are a diverse population of membrane structures produced and released by cells into the extracellular space for the intercellular trafficking of cargo molecules. They are implicated in various biological processes, including angiogenesis, immunomodulation, and cancer cell signaling. While much research has focused on their biogenesis or their effects on recipient cells, less is understood about how EVs are capable of traversing diverse tissue environments and crossing biological barriers. Their interactions with extracellular matrix components are of particular interest, as such interactions govern diffusivity and mobility, providing a potential basis for organotropism. To start to untangle how EV-matrix interactions affect diffusivity, we use high speed epifluorescence microscopy, single particle tracking, and confocal reflectance microscopy to analyze particle mobility and localization in extracellular matrix-mimicking hydrogels composed of collagen I. EVs are compared with synthetic liposomes and extruded plasma membrane vesicles to better understand the importance of membrane composition on these interactions. By treating EVs with trypsin to digest surface proteins, we determine that proteins are primarily responsible for EV immobilization in collagen I hydrogels. We next use a synthetic peptide competitive inhibitor to narrow down the identity of the proteins involved to argynylglycylaspartic acid (RGD) motif-binding integrins, which interact with unincorporated or denatured nonfibrillar collagen. Moreover, the effect of integrin inhibition with RGD peptides has strong implications for the use of RGD-peptide-based drugs to treat certain cancers, as integrin inhibition appears to increase EV mobility, improving their ability to infiltrate tissue-like environments.

Synthesis of 2,4-Bis(trifluoromethyl)benzaldehyde Hybrid Thiosemicarbazones as Prolyl Oligopeptidase Inhibitors for Neurodegenerative Disorders and their In-silico Analysis.

Prolyl-specific oligopeptidase (POP), one of the brain's highly expressed enzymes, is an important target for the therapy of central nervous system disorders, notably autism spectrum disorder, schizophrenia, Parkinson's, Alzheimer's disease, and dementia. The current study was designed to investigate 2,4-bis(trifluoromethyl) benzaldehyde- based thiosemicarbazones as POP inhibitors to treat the above-mentioned disorders. A variety of techniques, such as nuclear magnetic resonance (NMR), mass spectrometry (MS), and Fourier-transform infrared spectroscopy (FTIR), were used for the structural confirmation of synthesized compounds. After in-vitro evaluation, all of these compounds were found to be prominent inhibitors of the POP enzyme (IC50= 10.14 - 41.73 μM). Compound 3a emerged as the most active compound (IC50 10.14 ± 0.72 μM) of the series. The kinetic study of the most active 3a (Ki =13.66 0.0012 μM) indicated competitive inhibition of the aforementioned enzyme. Moreover, molecular docking depicted a noticeable role of thiosemicarbazide moiety in the binding of these molecules within the active site of the POP enzyme.

Potent α-glucosidase inhibitors with benzimidazole-propionitrile hybridization; synthesis, bioassay and docking study.

Background: Diabetes is characterized by a lack of insulin and insensitivity to insulin. In 2013, the global diabetes population was 382million, with 90% of them having type 2 diabetes (non-insulin-dependent). It is predicted that this number will increase to 592million by 2035.Aim: Here, we aimed to synthesize a series of benzimidazole-based derivatives B1-B32 with α-glucosidase inhibition potential as antidiabetic agents.Methods: Compounds B1-B32 were prepared in three three-step reactions, and the structures were elucidated using spectroscopic methods, namely 1H NMR, 13C NMR, MS and IR. Enzyme inhibition and kinetic study were done using commercial assay kits, and molecular docking study using autodock4.Results: Bioassay data showed that twenty-four out of the thirty-two tested compounds exhibited IC50 values ranging from 44 to 745μM, surpassing the standard molecule, acarbose (IC50: 750μM). it was determined that the best compound, B10, functions as a competitive inhibitor. Additionally, a molecular docking study provided insights into the interactions between the four most promising compounds (B5, B6, B10 and B28) and the active site residues within the enzyme.Conclusion: The tested compounds are interesting α-glucosidase inhibitors, which indicates the benefit of more bioassay studies, especially in vivo studies.

VGLL3 modulates chemosensitivity through promoting DNA double-strand break repair.

Transcription cofactor vestigial-like 3 (VGLL3), as a master regulator of female-biased autoimmunity, also functions in tumor development, while the underlying mechanisms remain largely elusive. Here, we report that VGLL3 plays an important role in DNA damage response (DDR). VGLL3 can be recruited to damage sites in a PARylation-dependent manner. VGLL3 depletion impairs the accumulation of RNF8 and RAD51 at sites of DNA damage, leading to reduced homologous recombination efficiency and increased cellular sensitivity to chemotherapeutic drugs. Mechanistically, VGLL3 can prevent CtIP from KLHL15-mediated ubiquitination and degradation through competitive binding with KLHL15 and, meanwhile, stabilize MDC1 by limiting TRIP12-MDC1 but promoting USP7-MDC1 associations for optimal RNF8 signaling initiation. Consistently, VGLL3 depletion delays tumor development and sensitizes the xenografts to etoposide treatment. Overall, our results reveal an unexpected role of VGLL3 in DDR, which is distinct from its transcriptional cofactor function and not conserved among VGLL family members.

A novel molecularly imprinted solid phase based on SiO2-Ag2O double-nucleation doped with graphene oxide for extraction and determination of flavonoids from Acanthaceae ebracteatus via HPLC-MS/MS

(Objectives) A novel method was developed for extracting and determining flavonoids from marine mangrove medicinal plants. This approach utilizes surface molecular imprinting to increase selectivity. Graphene oxide doped with a SiO2–Ag2O dual core structure served as a scaffold for synthesizing multilayer surface-imprinted polymers. The surface of the SiO2-Ag2O@GO composite was modified through self-polymerization. (Methods) Acacetin molecularly imprinted polymers (AMIPs) were created via surface-initiated precipitation polymerization with acacetin as the template. These AMIPs were synthesized and characterized via ultraviolet (UV) spectroscopy, Fourier transform infrared (FT-IR) spectroscopy, scanning electron microscopy (SEM), X-ray diffraction (XRD), and transmission electron microscopy (TEM). Recycling experiments demonstrated the stability of the AMIPs. Analysis of the adsorption mechanism via the Langmuir and Freundlich models indicated that the Langmuir model was more suitable for predicting AMIPs. (Results) Scatchard plots showing two different binding sites of AMIPs for acacetin adsorption at various concentrations. The class selectivity of AMIPs for flavonoids was found in Acanthaceae ebracteatus. Compared with other analogs, competitive binding experiments revealed that the polymer had greater selectivity for acacetin. After AMIP solid-phase extraction (AMISPE), the linearity, LOD, LOQ, intraday and interday variabilities, recovery, and repeatability of AMIPs combined with HPLC–MS/MS were validated, confirming the method’s selectivity, precision, accuracy, and reproducibility. The contents of acacetin, apigenin, kaempferol, luteolin, quercetin, and genistein in Acanthaceae ebracteatus were determined. (Conclusions) These results indicate that AMISPE coupled with HPLC–MS/MS is a new, simple, and practical method for extracting, purifying, and enriching flavonoids from complex samples, such as those of herbs and other natural marine plants.

Investigation of ponatinib metabolism and drug-drug interactions with lycopene and shikonin in vitro and invivo

Ponatinib is approved for use in patients with chronic myeloid leukemia (CML) who are resistant to or intolerant to prior tyrosine kinase inhibitor (TKI) therapy. Given that ponatinib can induce significant cardiotoxicity when taken, and that most Chinese medicines have cardioprotective effects, it is possible to administer them in combination in clinic to alleviate adverse effects. The quantitative determination of ponatinib and its metabolite N-desmethyl ponatinib was optimized and fully verified by ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). And the drug-drug interactions (DDI) of ponatinib with lycopene and shikonin, both in vivo and in vitro, were studied. The results of bioanalytical methodology showed that ponatinib and N-desmethyl ponatinib had good linearity in plasma samples, and their selectivity, accuracy, precision, stability, matrix effect and recovery were all satisfied with the need of quantitative analysis of samples. In animal experiments, compared with the control group, lycopene and shikonin significantly changed the pharmacokinetic parameters of ponatinib, including AUC(0-t), AUC(0-∞) and CLz/F, while having no effect on the pharmacokinetic parameters of N-desmethyl ponatinib. In vitro interaction studies indicated that lycopene showed mixed inhibition mechanism on ponatinib metabolism in both rat liver microsomes (RLM) and human liver microsomes (HLM). And, shikonin displayed mixed inhibition mechanism in RLM and competitive inhibition mechanism in HLM, respectively. In summary, the UPLC-MS/MS method can accurately and sensitively quantify ponatinib and N-desmethyl ponatinib, and provide further reference for clinical drug combination between ponatinib and lycopene or shikonin.

Visnagin alleviates rheumatoid arthritis via its potential inhibitory impact on malate dehydrogenase enzyme: in silico, in vitro, and in vivo studies

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disorder. The present study aimed to evaluate the in silico, in vitro, and in vivo inhibitory effect of visnagin on malate dehydrogenase activity and elucidate its inflammatory efficacy when combined with methotrexate in the RA rat model. The molecular docking, ADMET simulations, MDH activity, expression, and X-ray imaging were detected. Moreover, CRP, RF, (anti-CCP) antibody, (TNF-α), (IL-6), (IL-17), and (IL-10) were evaluated. The expression levels of MMP3 and FOXP3 genes and CD4, CD25, and CD127 protein levels were assessed. Histological assessment of ankle joints was evaluated. The results revealed that visnagin showed reversible competitive inhibition on MDH with inhibitory constant (Ki) equal to 141 mM with theoretical IC50 equal to 1202.7 mM, LD50 equal to 155.39 mg/kg, and LD25 equal to 77.69 mg/kg. In vivo studies indicated that visnagin exhibited anti-inflammatory effects through decreasing MDH1 activity and expression and induced proliferation of anti-inflammatory CD4+CD25+FOXP3 regulatory T cells with increasing the anti-inflammatory cytokine IL-10 levels. Moreover, visnagin reduced the levels of inflammatory cytokines and the immuno-markers. Our findings elucidate that visnagin exhibits an anti-inflammatory impact against RA through its ability to inhibit the MDH1 enzyme, improve methotrexate efficacy, and reduce oxidative stress.Graphical

Selenium polysaccharide form sweet corn cob mediated hypoglycemic effects in vitro and untargeted metabolomics study on type 2 diabetes

Type 2 diabetes mellitus (T2D) causes complications due to metabolic disorders besides increasing blood glucose. Sweet corn cob selenium polysaccharide (SeSCP) is a complex of Se with Sweet corn cob polysaccharide that has good hypoglycemic efficacy, but its effect on T2D metabolism has not been determined. In this study, the hypoglycemic effect of SeSCP was investigated by in vitro and in vivo experiments, and the levels of metabolites in feces were analyzed in a high-fat diet and STZ-induced T2D mouse model by Liquid chromatography-mass spectrometry (LC-MS). The results indicated that SeSCP regulates α-amylase and α-glucosidase through competitive reversible inhibition, and the reaction is spontaneous, driven by van der Waals forces and hydrogen bonding. In vivo, SeSCP modulates glucose transport decreasing glucose entry into the bloodstream. The metabolites mainly affected by SeSCP-MC were adenine, LysoPA (0:0/18:2(9Z, 12Z)), cysteine-S-sulfate, and demeclocycline (hydrochloride) metabolites. SeSCP interfered with β-alanine metabolism, starch and sucrose metabolism, ether lipid metabolism, glycerophospholipid metabolism, glyoxylate and dicarboxylate metabolism, pantothenate and CoA biosynthesis, etc. Additionally, SeSCP exhibited more effective metabolic interventions than metformin and SCP. Therefore, SeSCP can reduce complications while improving T2D blood glucose.

Hinokiflavone from Platycladi cacumen as a potent broad-spectrum inhibitor of gut microbial Loop-1 β-glucuronidases: Inhibition kinetics and molecular simulation

Gut microbial Loop-1 β-glucuronidases (gmGUS) played an important role in irinotecan-induced gastrointestinal toxicity by regulating the level of its active metabolite SN38 through enterohepatic recirculation. gmGUS inhibition has emerged as a promising approach to relieve its dose-limiting intestinal toxicity and improve its medication efficacy. This study aims to investigate the inhibitory effects and mechanisms of Platycladi cacumen and its main constituent hinokiflavone against four different types of Loop-1 gmGUS (EeGUS, SaGUS, CpGUS and EcGUS). Our results showed that the ethanol extract of Platycladi cacumen displayed strong broad-spectrum inhibition against four gmGUS, and hinokiflavone could potently inhibit EeGUS, SaGUS, CpGUS and EcGUS with IC50 values of 0.09 ± 0.01 μM, 0.44 ± 0.01 μM, 0.20 ± 0.01 μM and 0.69 ± 0.10 μM, respectively. Inhibition kinetic analyses demonstrated that hinokiflavone acted as a strong competitive inhibitor of EeGUS with Ki value of 0.13 μM, while it displayed non-competitive inhibition against SaGUS, CpGUS and EcGUS, with the Ki values of 0.43 μM, 0.33 μM and 0.76 μM, respectively. Docking simulations revealed that hinokiflavone could tightly bind with Tyr-485 and Glu-516 in catalytic sites of EeGUS, as well it created strong interactions with amino acids in loop structures of SaGUS (Asn-362), CpGUS (Phe-363, Met-364, Ala-365 and Arg-375) and EcGUS (Leu-361) to interfere the substrate entry into the catalytic pocket. Collectively, these results confirmed that hinokiflavone from Platycladi cacumen is a potent naturally occurring inhibitor of gmGUS with broad efficiency, suggesting hinokiflavone will be helpful for alleviating intestinal toxicity in irinotecan therapy.

BRCA1 levels and DNA-damage response are controlled by the competitive binding of circHIPK3 or FMRP to the BRCA1 mRNA

Circular RNAs (circRNAs) are covalently closed RNA molecules widely expressed in eukaryotes and deregulated in several pathologies, including cancer. Many studies point to their activity as microRNAs (miRNAs) and protein sponges; however, we propose a function based on circRNA-mRNA interaction to regulate mRNA fate. We show that the widely tumor-associated circHIPK3 directly interacts invivo with the BRCA1 mRNA through the back-splicing region in human cancer cells. This interaction increases BRCA1 translation by competing for the binding of the fragile-X mental retardation 1 protein (FMRP) protein, which we identified as a BRCA1 translational repressor. CircHIPK3 depletion or disruption of the circRNA-mRNA interaction decreases BRCA1 protein levels and increases DNA damage, sensitizing several cancer cells to DNA-damage-inducing agents and rendering them susceptible to synthetic lethality. Additionally, blocking FMRP interaction with BRCA1 mRNA with locked nucleic acid (LNA) restores physiological protein levels in BRCA1 hemizygous breast cancer cells, underscoring the importance of this circRNA-mRNA interaction in regulating DNA-damage response.

Exploration of thiazine Schiff bases as promising urease inhibitors: Design, synthesis, enzyme inhibition, kinetic analysis, ADME/T evaluation, and molecular docking studies

Urease catalyzes the hydrolysis of urea, leading to an increase in stomach pH and supporting Helicobacter pylori survival, which is linked to several gastrointestinal disorders. In this study, thiazine-based Schiff bases were explored as promising urease inhibitors. Various spectroscopic techniques characterized the synthetic library of thiazine Schiff bases 2–36 and also evaluated for their inhibitory activities against urease. The derivatives demonstrated significant inhibitory potential with IC50 values ranging from 0.14 ± 0.08 to 3.66 ± 0.21 μM, outperforming the standard inhibitor thiourea (IC50 = 19.43 ± 0.18 μM). Structure-activity relationship (SAR) studies revealed that specific substitutions (type and positions) on the aryl ring significantly affect the inhibition potential. The most potent derivative, compound 7, possessed 2-methoxy-5-trifluoromethyl substitutions and exhibited an IC50 of 0.14 ± 0.08 μM. Enzyme kinetics studies revealed that the most potent derivatives function as competitive inhibitors. Additionally, molecular docking studies provided insights into the binding interactions between the molecule and the urease active site, highlighting key residues involved in inhibitor binding. These findings highlight the therapeutic potential of thiazine-based Schiff bases as urease inhibitors and provide insights for the development of new anti-ulcer agents.

Virtual and in Vitro Screening Employing a Repurposing Approach Reveal 13-cis-Retinoic Acid is a PTP1B Inhibitor.

Current treatments for type 2 diabetes (T2D) mainly rely on exercise, dietary control, and anti-diabetic drugs to enhance insulin secretion and improve insulin sensitivity. However, there is a need for more therapeutic options, as approved drugs targeting different pharmacological objectives are still unavailable. One potential target that has attracted attention is the protein tyrosine phosphatase 1B (PTP1B), which negatively regulates the insulin signaling pathway. In this work, a comprehensive computational screening was carried out using cheminformatics and molecular docking on PTP1B, employing a rigorous repurposing approach. The screening involved approved drugs and compounds under research as anti-diabetics that bind to targets such as peroxisome proliferator-activated receptor gamma (PPAR-γ) and α-glucosidase. Several computational hits were then meticulously tested in vitro against PTP1B, with 13-cis-retinoic acid (3a) showing an IC50 of 0.044 mM and competitive inhibition. Molecular dynamics studies further confirmed that 3a can bind to the catalytic binding site of PTP1B. Finally, 3a is the first time it has been reported as an inhibitor of PTP1B, making it a potentially valuable candidate for further studies in D2T treatment.

In Silico Approach for Assessment of the Anti-Tumor Potential of Cannabinoid Compounds by Targeting Glucose-6-Phosphate Dehydrogenase Enzyme.

Glucose-6-phosphate dehydrogenase (G6PD) is a pentose phosphate pathway (PPP) enzyme that generates NADPH, which is required for cellular redox equilibrium and reductive biosynthesis. It has been demonstrated that abnormal G6PD activation promotes cancer cell proliferation and metastasis. To date, no G6PD inhibitor has passed clinical testing successfully enough to be launched as a medicine. As a result, in this investigation, cannabinoids were chosen to evaluate their anticancer potential by targeting G6PD. Molecular docking indicated that three molecules, Tetrahydrocannabinolic acid (THCA), Cannabichromenic acid (CBCA), and tetrahydrocannabivarin (THCV), have the highest binding affinities for G6PD of -8.61, -8.39, and 8.01 Kcal mol. ADMET analysis found that all of them were safe prospective drug candidates. Molecular dynamics (MD) simulation and MM-PBSA analysis confirm the structural compactness and lower conformational variation of protein-ligand complexes, thereby maintaining structural stability and rigidity. Thus, our in silico investigation exhibited all three cannabinoids as potential competitive inhibitors of G6PD.

Biochemical and kinetic properties of three indoleamine 2,3-dioxygenases of Aspergillus fumigatus: mechanism of increase in the apparent Km by ascorbate.

Indoleamine 2,3-dioxygenase (IDO) is a monomeric heme enzyme that catalyzes the oxidative cleavage of tryptophan (L-Trp) to form N-formyl-kynurenine. Similar to other heme proteins, IDO only binds to O2 when the heme iron is ferrous (FeII), thereby rendering the enzyme active. Thus, ascorbate (Asc, a reducing agent) and methylene blue (MB, an electron carrier) are commonly added to in vitro IDO assay systems. However, Asc and MB have been recently reported to significantly impact the measurement of the enzymatic parameters of vertebrate IDO. Aspergillus fumigatus is a filamentous fungus and the most common cause of invasive aspergillosis; it has three IDO genes (IDOα, IDOβ, and IDOγ). The FeII-O2 IDOs of A. fumigatus, particularly FeII-O2 IDOγ, have relatively long half-lives in their autoxidation; however, the autoxidation was accelerated by Asc. Similar to vertebrate IDOs, Asc acted as a competitive (or mixed-competitive) inhibitor of the IDOs of A. fumigatus. A positive correlation (in the order of IDOγ > IDOβ > IDOα) was observed between the inhibitory sensitivity of the IDOs to Asc and the facilitation of their autoxidation by Asc. The FeII-O2 IDO can repeat the dioxygenase reaction as long as it reacts with L-Trp; however, substrate-free FeII-O2 IDO is converted into inactive FeIII-IDO by autoxidation. Thus, L-Trp (which keeps the IDO active) competes with Asc (which inactivates IDO by accelerating autoxidation). This is probably why Asc, which is structurally quite different from L-Trp, appears to function as a competitive (or mixed-competitive) inhibitor of IDOs.

Substrate recognition principles for the PP2A-B55 protein phosphatase.

The PP2A-B55 phosphatase regulates a plethora of signaling pathways throughout eukaryotes. How PP2A-B55 selects its substrates presents a severe knowledge gap. By integrating AlphaFold modeling with comprehensive high-resolution mutational scanning, we show that α helices in substrates bind B55 through an evolutionary conserved mechanism. Despite a large diversity in sequence and composition, these α helices share key amino acid determinants that engage discrete hydrophobic and electrostatic patches. Using deep learning protein design, we generate a specific and potent competitive peptide inhibitor of PP2A-B55 substrate interactions. With this inhibitor, we uncover that PP2A-B55 regulates the nuclear exosome targeting (NEXT) complex by binding to an α-helical recruitment module in the RNA binding protein 7 (RBM7), a component of the NEXT complex. Collectively, our findings provide a framework for the understanding and interrogation of PP2A-B55 function in health and disease.

Development of Tunable Mechanism-Based Carbasugar Ligands that Stabilize Glycoside Hydrolases through the Formation of Transient Covalent Intermediates.

Mutations in many members of the set of human lysosomal glycoside hydrolases cause a wide range of lysosomal storage diseases. As a result, much effort has been directed toward identifying pharmacological chaperones of these lysosomal enzymes. The majority of the candidate chaperones are active site-directed competitive iminosugar inhibitors but these have met with limited success. As a first step toward an alternative class of pharmacological chaperones we explored the potential of small molecule mechanism-based reversible covalent inhibitors to form transient enzyme-inhibitor adducts. By serial synthesis and kinetic analysis of candidate molecules, we show that rational tuning of the chemical reactivity of glucose-configured carbasugars delivers cyclohexenyl-based allylic carbasugar that react with the lysosomal enzyme β-glucocerebrosidase (GCase) to form covalent enzyme-adducts with different half-lives. X-ray structural analysis of these compounds bound noncovalently to GCase, along with the structures of the covalent adducts of compounds that reacted with the catalytic nucleophile of GCase, reveal unexpected reactivities of these compounds. Using differential scanning fluorimetry, we show that formation of a transient covalent intermediate stabilizes the folded enzyme against thermal denaturation. In addition, these covalent adducts break down to liberate the active enzyme and a product that is no longer inhibitory. We further show that the one compound, which reacts through an unprecedented SN1'-like mechanism, exhibits exceptional reactivity-illustrated by this compound also covalently labeling an α-glucosidase. We anticipate that such carbasugar-based single turnover covalent ligands may serve as pharmacological chaperones for lysosomal glycoside hydrolases and other disease-associated retaining glycosidases. The unusual reactivity of these molecules should also open the door to creation of new chemical biology probes to explore the biology of this important superfamily of glycoside hydrolases.