Inhibitor Affinity Research Articles

An exploration of human d-crystallin affinity for potential aggregation inhibitors: a molecular docking investigation

An exploration of human d-crystallin affinity for potential aggregation inhibitors: a molecular docking investigation

Indazole to 2‐Cyanoindole Scaffold Progression for Mycobacterial Lipoamide Dehydrogenase Inhibitors Achieves Extended Target Residence Time and Improved Antibacterial Activity

AbstractTuberculosis remains a leading cause of death from a single infection worldwide. Drug resistance to existing and even new antimycobacterials calls for research into novel targets and unexplored mechanisms of action. Recently we reported on the development of tight‐binding inhibitors of Mycobacterium tuberculosis (Mtb) lipoamide dehydrogenase (Lpd), which selectively inhibit the bacterial but not the human enzyme based on a differential modality of inhibitor interaction with these targets. Here we report on the striking improvement in inhibitor residence time on the Mtb enzyme associated with scaffold progression from an indazole to 2‐cyanoindole. Cryo‐EM of Lpd with the bound 2‐cyanoindole inhibitor 19 confirmed displacement of the buried water molecule deep in the binding channel with a cyano group. The ensuing hours‐long improvement in on‐target residence time is associated with enhanced antibacterial activity in axenic culture and in primary mouse macrophages. Resistance to 2‐cyanoindole inhibitors involves mutations within the inhibitor binding site that have little effect on inhibitor affinity but change the modality of inhibitor‐target interaction, resulting in fast dissociation from Lpd. These findings underscore that on‐target residence time is a major determinant of antibacterial activity and in vivo efficacy.

Indazole to 2-Cyanoindole Scaffold Progression for Mycobacterial Lipoamide Dehydrogenase Inhibitors Achieves Extended Target Residence Time and Improved Antibacterial Activity.

Tuberculosis remains a leading cause of death from a single infection worldwide. Drug resistance to existing and even new antimycobacterials calls for research into novel targets and unexplored mechanisms of action. Recently we reported on the development of tight-binding inhibitors of Mycobacterium tuberculosis (Mtb) lipoamide dehydrogenase (Lpd), which selectively inhibit the bacterial but not the human enzyme based on a differential modality of inhibitor interaction with these targets. Here we report on the striking improvement in inhibitor residence time on the Mtb enzyme associated with scaffold progression from an indazole to 2-cyanoindole. Cryo-EM of Lpd with the bound 2-cyanoindole inhibitor 19 confirmed displacement of the buried water molecule deep in the binding channel with a cyano group. The ensuing hours-long improvement in on-target residence time is associated with enhanced antibacterial activity in axenic culture and in primary mouse macrophages. Resistance to 2-cyanoindole inhibitors involves mutations within the inhibitor binding site that have little effect on inhibitor affinity but change the modality of inhibitor-target interaction, resulting in fast dissociation from Lpd. These findings underscore that on-target residence time is a major determinant of antibacterial activity and in vivo efficacy.

Synergistic effect of pectin and the flavanols mixture on in vitro starch digestion and the corresponding mechanism

In this study, the effect and mechanism of pectin on the inhibition of in vitro starch digestion by a mixture of flavanols (rutin, quercetin, and kaempferol [R–Q–K]) were investigated, mainly from an enzymatic point of view. Results showed that the association of pectin and R–Q–K decreased the hydrolysis rate, rapidly digestible starch content, estimated glycemic index (eGI) values and hydrolysis index (HI) values, and increased the resistant starch content of different crystalline starches. Among them, cassava starch exhibited a significant decrease in terms of HI and eGI values (32.81% ± 0.65% and 10.57% ± 0.37%, respectively). Meanwhile, the lowest rate of decrease in terms of HI and eGI values was found for breadfruit starch (22.97% ± 0.39% and 7.23% ± 0.62%, respectively). The inhibitory effect of R–Q–K on α-glucosidase was attenuated by low concentrations of pectin, which was correlated to the non-covalent binding among pectin, α-glucosidase, quercetin, and kaempferol. The hydrogen bonding between pectin–R–Q–K and starch may be responsible for the ability of low concentrations of pectin and R–Q–K to synergistically delay starch digestion. Higher concentrations of pectin were noted to have the opposite trend, which was attributed to forming the binary complex of pectin–α-glucosidase. The inhibitory affinity and quenching affinity of R–Q–K for α-glucosidase were enhanced by low concentrations of pectin and attenuated by high concentrations of pectin. This study aims to provide theoretical support for the development of pectin and flavonoids in starch-based healthy foods.

Determining the Affinity and Kinetics of Small Molecule Inhibitors of Galectin-1 Using Surface Plasmon Resonance.

The beta-galactoside-binding mammalian lectin galectin-1 can bind, via its carbohydrate recognition domain (CRD), to various cell surface glycoproteins and has been implicated in a range of cancers. As a consequence of binding to sugar residues on cell surface receptors, it has been shown to have a pleiotropic effect across many cell types and mechanisms, resulting in immune system modulation and cancer progression. As a result, it has started to become a therapeutic target for both small and large molecules. In previous studies, we used fluorescence polarization (FP) assays to determine KD values to screen and triage small molecule glycomimetics that bind to the galectin-1 CRD. In this study, surface plasmon resonance (SPR) was used to compare human and mouse galectin-1 affinity measures with FP, as SPR has not been applied for compound screening against this galectin. Binding affinities for a selection of mono- and di-saccharides covering a 1000-fold range correlated well between FP and SPR assay formats for both human and mouse galectin-1. It was shown that slower dissociation drove the increased affinity at human galectin-1, whilst faster association was responsible for the effects in mouse galectin-1. This study demonstrates that SPR is a sound alternative to FP for early drug discovery screening and determining affinity estimates. Consequently, it also allows association and dissociation constants to be measured in a high-throughput manner for small molecule galectin-1 inhibitors.

In silico design of peptide inhibitors for Dengue virus to treat Dengue virus-associated infections

Dengue virus is a single positive-strand RNA virus that is composed of three structural proteins including capsid, envelope, and precursor membrane while seven non-structural proteins (NS1, NS2A, NS2B, NS3A, NS3B, NS4, and NS5). Dengue is a viral infection caused by the dengue virus (DENV). DENV infections are asymptomatic or produce only mild illness. However, DENV can occasionally cause more severe cases and even death. There is no specific treatment for dengue virus infections. Therapeutic peptides have several important advantages over proteins or antibodies: they are small in size, easy to synthesize, and have the ability to penetrate the cell membranes. They also have high activity, specificity, affinity, and less toxicity. Based on the known peptide inhibitor, the current study designs peptide inhibitors for dengue virus envelope protein using an alanine and residue scanning technique. By replacing I21 with Q21, L14 with H14, and V28 with K28, the binding affinity of the peptide inhibitors was increased. The newly designed peptide inhibitors with single residue mutation improved the binding affinity of the peptide inhibitors. The inhibitory capability of the new promising peptide inhibitors was further confirmed by the utilization of MD simulation and free binding energy calculations. The molecular dynamics simulation demonstrated that the newly engineered peptide inhibitors exhibited greater stability compared to the wild-type peptide inhibitors. According to the binding free energies MM(GB)SA of these developed peptides, the first peptide inhibitor was the most effective against the dengue virus envelope protein. All peptide derivatives had higher binding affinities for the envelope protein and have the potential to treat dengue virus-associated infections. In this study, new peptide inhibitors were developed for the dengue virus envelope protein based on the already reported peptide inhibitor.

AI-based prediction of protein–ligand binding affinity and discovery of potential natural product inhibitors against ERK2

Determination of protein–ligand binding affinity (PLA) is a key technological tool in hit discovery and lead optimization, which is critical to the drug development process. PLA can be determined directly by experimental methods, but it is time-consuming and costly. In recent years, deep learning has been widely applied to PLA prediction, the key of which lies in the comprehensive and accurate representation of proteins and ligands. In this study, we proposed a multi-modal deep learning model based on the early fusion strategy, called DeepLIP, to improve PLA prediction by integrating multi-level information, and further used it for virtual screening of extracellular signal-regulated protein kinase 2 (ERK2), an ideal target for cancer treatment. Experimental results from model evaluation showed that DeepLIP achieved superior performance compared to state-of-the-art methods on the widely used benchmark dataset. In addition, by combining previously developed machine learning models and molecular dynamics simulation, we screened three novel hits from a drug-like natural product library. These compounds not only had favorable physicochemical properties, but also bound stably to the target protein. We believe they have the potential to serve as starting molecules for the development of ERK2 inhibitors.

Exploring the conformational landscapes of protein kinases: perspectives from FRET and DEER.

Conformational changes of catalytically-important structural elements are a key feature of the regulation mechanisms of protein kinases and are important for dictating inhibitor binding modes and affinities. The lack of widely applicable methods for tracking kinase conformational changes in solution has hindered our understanding of kinase regulation and our ability to design conformationally selective inhibitors. Here we provide an overview of two recently developed methods that detect conformational changes of the regulatory activation loop and αC-helix of kinases and that yield complementary information about allosteric mechanisms. An intramolecular Förster resonance energy transfer-based approach provides a scalable platform for detecting and classifying structural changes in high-throughput, as well as quantifying ligand binding cooperativity, shedding light on the energetics governing allostery. The pulsed electron paramagnetic resonance technique double electron-electron resonance provides lower throughput but higher resolution information on structural changes that allows for unambiguous assignment of conformational states and quantification of population shifts. Together, these methods are shedding new light on kinase regulation and drug interactions and providing new routes for the identification of novel kinase inhibitors and allosteric modulators.

Identification of highly potent and selective HTRA1 inhibitors

High temperature requirement A serine peptidase 1 (HTRA1) is a serine protease involved in an array of signaling pathways. It is also responsible for the regulation of protein aggregates via refolding, translocation, and degradation. It has subsequently been found that runaway proteolytic HTRA1 activity plays a role in a variety of diseases, including Age-Related Macular Degeneration (AMD), osteoarthritis, and Rheumatoid Arthritis. Selective inhibition of serine protease HTRA1 therefore offers a promising new strategy for the treatment of these diseases. Herein we disclose structure–activity-relationship (SAR) studies which identify key interactions responsible for binding affinity of small molecule inhibitors to HTRA1. The study results in highly potent molecules with IC50′s less than 15 nM and excellent selectivity following a screen of 35 proteases.

Purification and characterization of glutamate dehydrogenase from rainbow trout (Oncorhynchus mykiss) liver and molecular docking studies.

Glutamate dehydrogenase (GDH) participates in the energy metabolism of proteins and the synthesis of metabolites important for the organism. In this study, GDH enzyme was purified from the liver of rainbow trout (Oncorhynchus mykiss) by 2',5'-ADP Sepharose 4B affinity chromatography in one step. As a result of this purification process, GDH enzyme was purified 171-fold with 5.83U/mg protein-specific activity. The characterization experiments presented that the storage stability of the purified GDH enzyme was determined as -80°C; optimum temperature 40°C; it was determined that the optimum ionic strength was 100mM phosphate buffer and the optimum pH was 8.00. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and PAGE studies showed that the natural molar mass of the purified GDH enzyme was 346.74kDa, and the molar mass of its subunits was 53.71kDa. Km and Vmax values for substrates and coenzymes of GDH enzyme purified from rainbow trout liver were calculated, and the lowest Km value was found in NAD+ (1.86mM) and the highest Vmax value in NH4 + (1.79U/mL). The effects of some metal ions, vitamins, and solvents on the activity of the purified GDH enzyme were investigated and also IC50 values and inhibition types. The metal ion with the lowest IC50 value is Ag+ (8.65 ± 1.68μM), and the vitamin is B6 (0.77 ± 0.04mM). The binding affinities of inhibitors were investigated with molecular docking, based on the conformational state of GDH.

GS-441524-Diphosphate-Ribose Derivatives as Nanomolar Binders and Fluorescence Polarization Tracers for SARS-CoV-2 and Other Viral Macrodomains.

Viral macrodomains that can bind to or hydrolyze protein adenosine diphosphate ribosylation (ADP-ribosylation) have emerged as promising targets for antiviral drug development. Many inhibitor development efforts have been directed against the severe acute respiratory syndrome coronavirus 2 macrodomain 1 (SARS-CoV-2 Mac1). However, potent inhibitors for viral macrodomains are still lacking, with the best inhibitors still in the micromolar range. Based on GS-441524, a remdesivir precursor, and our previous studies, we have designed and synthesized potent binders of SARS-CoV-2 Mac1 and other viral macrodomains including those of Middle East respiratory syndrome coronavirus (MERS-CoV), Venezuelan equine encephalitis virus (VEEV), and Chikungunya virus (CHIKV). We show that the 1'-CN group of GS-441524 promotes binding to all four viral macrodomains tested while capping the 1″-OH of GS-441524-diphosphate-ribose with a simple phenyl ring further contributes to binding. Incorporating these two structural features, the best binders show 20- to 6000-fold increases in binding affinity over ADP-ribose for SARS-CoV-2, MERS-CoV, VEEV, and CHIKV macrodomains. Moreover, building on these potent binders, we have developed two highly sensitive fluorescence polarization tracers that only require nanomolar proteins and can effectively resolve the binding affinities of nanomolar inhibitors. Our findings and probes described here will facilitate future development of more potent viral macrodomain inhibitors.

SARS-CoV-2 and Angiotensin-Converting Enzyme-2 Receptor Interaction Blocker– an In-Silico Approach

The global COVID-19 pandemic, instigated by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has led to substantial morbidity and mortality on a worldwide scale. While COVID-19 vaccines offer hope, the emergence of mutated viral strains necessitates the development of FDA-approved drugs to address future outbreaks. Objective: To examine prospective antiviral medications through an analysis of the interaction between the spike protein of SARS-CoV-2 and Angiotensin-converting enzyme-2 (ACE-2) receptors, which play a pivotal role in facilitating viral entry into host cells. Methods: Molecular docking was employed to assess the binding affinities of various protease inhibitors with ACE-2 receptors. Natural proteases, including Furin and Transmembrane serine protease 2 (TMPRSS2), cleave viral spike proteins into S1 and S2 subunits, facilitating fusion with ACE-2 receptors. We assessed the binding energies of Indinavir, Nafamostat, Fosamprenavir, Lopinavir, and Boceprevir to inhibit this interaction with a sense of optimism for their potential therapeutic applications. Results: Our findings suggest potential treatments for COVID-19, with Indinavir, Nafamostat, Fosamprenavir, Lopinavir, and Boceprevir displaying promising binding energies of -9.6 kcal/mol, -8.4 kcal/mol, -7.7 kcal/mol, and -7.5 kcal/mol, respectively. Conclusions: While promising, further clinical trials are important to potentially evaluate the efficacy and safety of these proposed drugs in combating COVID-19 and its variants.

Abstract 5740: Peptide-functionalized dendrimers for enhanced binding to immune checkpoint proteins

Abstract The human immune system has complex and dynamic mechanisms to distinguish and eliminate malignant cells and defend the body against cancer. Various therapeutic approaches to stimulate the immune system have thus been investigated, demonstrating strong anti-tumor immune responses capable of controlling metastasis and preventing recurrence. Nevertheless, existing cancer immunotherapies suffer from several limitations, including inconsistent benefits across cancer types and patients. In recent years, the utilization of peptides for immunotherapy has attracted a great deal of interest due to their ability to mimic protein functionalities while maintaining a high degree of modularity and synthetic reproducibility in their molecular design. However, the clinical translation of otherwise promising peptide-based immunotherapeutics has been hindered due to their significantly weaker binding to target proteins and short half-life in plasma circulation in comparison to their monoclonal antibody counterparts. Nevertheless, these issues can potentially be addressed by conjugation to nanoparticles, which enhances cell binding/uptake and prolongs circulation. To this end, our group has extensively investigated the use of poly(amidoamine) (PAMAM) dendrimers to improve the therapeutic utility of peptides. These dendrimers can facilitate the simultaneous binding of conjugated peptides to multiple cell receptors, resulting in a significantly enhanced binding interaction through the multivalent binding effect. Here, we report an efficient de novo nanoparticle design strategy that combines organic synthesis, phage display, and molecular dynamics to identify a novel immuno-oncology therapeutic modality. We first employed phage display to identify several highly selective sequences that bind to the mouse ortholog of TIGIT—an inhibitory receptor expressed on lymphocytes that has been identified as a potential therapeutic target to enhance anti-tumor immune responses. Subsequent computational modeling (NAMD with CHARMM force field) revealed several conserved regions of interaction that we hypothesize drive favorable binding interactions and enhance the binding affinity of the peptide inhibitors. We next incorporated experimental kinetic measurements of the identified sequences with adaptive evolution computational modeling to identify optimized mutant analogs for dendrimer conjugation. It was found that the TIGIT targeting DPCs (dendrimer-peptide conjugates) demonstrated increased binding avidity in vitro and anti-tumor efficacy in vivo, most notably when administered in combination with PD-L1 targeting DPCs to a syngeneic mouse model bearing mouse oral carcinoma (MOC1) tumors. Overall, this work demonstrates the potential of rational peptide design and our multi-functional DPC platform for the development of both effective and novel immuno-oncology therapeutics. Citation Format: Piper Anne Rawding, Francesco Coppola, DaWon Kim, Michael Poellmann, Deric Wheeler, Petr Král, Seungpyo Hong. Peptide-functionalized dendrimers for enhanced binding to immune checkpoint proteins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5740.

Abstract 7114: Exploring the landscape of PARP and PARG inhibitor selectivity in live cells using NanoBRET™ target engagement assays

Abstract The 17-member poly(ADP-ribose) polymerase (PARP) family of proteins mediate a number of cellular processes including gene transcription, cell-division and DNA-repair. PARPs catalyze the addition of poly- or mono- ADP-ribose chains on target proteins, modifying their function and/or recruitment in various pathways. Poly(ADP-ribose) Glycohydrolase (PARG) catabolizes PAR units and facilitates the recycling of PARP and other modified effector proteins. A number of PARPS are implicated in human disease, and PARP inhibitors (PARPi’s) have seen major clinical success, however, many PARPs (especially mono PARPs) are understudied and chemical tools for understanding cellular PARPi selectivity and potency across the entire family are lacking. Similarly, several PARG inhibitors have been developed to date, but tools for directly assessing target engagement in a cellular context are sorely needed. Here we describe the development and application of target engagement assays for characterizing PARP and PARG inhibitor selectivity and affinity in live cells. This method is based on the NanoBRET™ assay platform, which is a proximity-based probe displacement assay that operates in live cells. Using NanoBRET™ technology, we have developed a single probe that covers 12 out of 17 PARP family members. We explored the family-wide selectivity of a diverse panel of clinical and preclinical PARP inhibitors in live cells, representing the first family-wide, cell-based method for profiling PARP selectivity. We also describe the development of a second probe that reports on PARG target engagement. Target engagement of PARG was observed for three out of five tested inhibitors, suggesting a potential indirect mode of action for certain inhibitors on PARG glycohydrolase activity. These assays offer a complementary approach for studying target engagement within the PARylation cycle. Citation Format: Ani Michaud, Kelly Teske, Jennifer Wilkinson, Chad Zimprich, James Vasta, Cesear Corona, Min Zhou, Elizabeth Dominguez, Kaitlin Dunn-Hoffman, Matthew Robers. Exploring the landscape of PARP and PARG inhibitor selectivity in live cells using NanoBRET™ target engagement assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7114.

Inhibitor Trapping in Kinases.

Recently, we identified a novel mechanism of enzyme inhibition in N-myristoyltransferases (NMTs), which we have named 'inhibitor trapping'. Inhibitor trapping occurs when the protein captures the small molecule within its structural confines, thereby preventing its free dissociation and resulting in a dramatic increase in inhibitor affinity and potency. Here, we demonstrate that inhibitor trapping also occurs in the kinases. Remarkably, the drug imatinib, which has revolutionized targeted cancer therapy, is entrapped in the structure of the Abl kinase. This effect is also observed in p38α kinase, where inhibitor trapping was found to depend on a 'magic' methyl group, which stabilizes the protein conformation and increases the affinity of the compound dramatically. Altogether, these results suggest that inhibitor trapping is not exclusive to N-myristoyltransferases, as it also occurs in the kinase family. Inhibitor trapping could enhance the binding affinity of an inhibitor by thousands of times and is as a key mechanism that plays a critical role in determining drug affinity and potency.

Integrated Virtual Screening and Validation toward Potential HPPD Inhibition Herbicide.

4-Hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27, HPPD) is one of the most widely studied herbicide targets and has gained significant attention. To identify potential effective HPPD inhibitors, a rational multistep virtual screening workflow was built, which included CBP models (based on the receptor-ligand interactions in the crystal complex), Hypogen models with activity prediction ability (according to the derivation of structure-activity relationships from a set of molecules with reported activity values), and a consensus docking procedure (consisting of LibDock, Glide, and CDOCKER). About 1 million molecules containing diketone or β-keto-enol substructures were filtered by Lipinski's rules, CBP model, and Hypogen model. A total of 12 compounds with similar docking postures were generated by consensus docking. Eventually, four molecules were screened based on the specific binding pattern and affinity of the HPPD inhibitor. The biological evaluation in vivo displayed that compounds III-1 and III-2 exhibited comparable herbicidal activity to isoxaflutole and possessed superior safety on various crops (wheat, rice, sorghum, and maize). The ADMET prediction (absorption, distribution, metabolism, excretion, and toxicity) showed that compound III possessed relatively good toxicological results. This work provides a theoretical basis and valuable reference for the virtual screening and molecular design of novel HPPD inhibition herbicides.

Intracellular Ellagic Acid Derived from Goat Urine DMSO Fraction (GUDF) Predicted as an Inhibitor of c-Raf Kinase.

Dietary chemicals and their gut-metabolized products are explored for their anti-proliferative and pro-cell death effects. Dietary and metabolized chemicals are different from ruminants such as goats over humans. Loss of cell viability and induction of death due to goat urine DMSO fraction (GUDF) derived chemicals were assessed by routine in vitro assays upon MCF-7 breast cancer cells. Intracellular metabolite profiling of MCF-7 cells treated with goat urine DMSO fraction (GUDF) was performed using an in-house designed vertical tube gel electrophoresis (VTGE) assisted methodology, followed by LC-HRMS. Next, identified intracellular dietary chemicals such as ellagic acid were evaluated for their inhibitory effects against transducers of the c-Raf signaling pathway employing molecular docking and molecular dynamics (MD) simulation. GUDF treatment upon MCF-7 cells displayed significant loss of cell viability and induction of cell death. A set of dietary and metabolized chemicals in the intracellular compartment of MCF-7 cells, such as ellagic acid, 2-hydroxymyristic acid, artelinic acid, 10-amino-decanoic acid, nervonic acid, 2,4-dimethyl-2-eicosenoic acid, 2,3,4'- Trihydroxy,4-Methoxybenzophenone and 9-amino-nonanoic acid were identified. Among intracellular dietary chemicals, ellagic acid displayed a strong inhibitory affinity (-8.7 kcal/mol) against c-Raf kinase. The inhibitory potential of ellagic acid was found to be significantly comparable with a known c-Raf kinase inhibitor sorafenib with overlapping inhibitory site residues (ARG450, GLU425, TRP423, VA403). Intracellular dietary-derived chemicals such as ellagic acid are suggested for the induction of cell death in MCF-7 cells. Ellagic acid is predicted as an inhibitor of c-Raf kinase and could be explored as an anti-cancer drug.

Investigation of how gate residues in the main channel affect the catalytic activity of Scytalidium thermophilum catalase.

Catalase is an antioxidant enzyme that breaks down hydrogen peroxide (H2O2) into molecular oxygen and water. In all monofunctional catalases the pathway that H2O2 takes to the catalytic centre is via the `main channel'. However, the structure of this channel differs in large-subunit and small-subunit catalases. In large-subunit catalases the channel is 15 Å longer and consists of two distinct parts, including a hydrophobic lower region near the heme and a hydrophilic upper region where multiple H2O2 routes are possible. Conserved glutamic acid and threonine residues are located near the intersection of these two regions. Mutations of these two residues in the Scytalidium thermophilum catalase had no significant effect on catalase activity. However, the secondary phenol oxidase activity was markedly altered, with kcat and kcat/Km values that were significantly increased in the five variants E484A, E484I, T188D, T188I and T188F. These variants also showed a lower affinity for inhibitors of oxidase activity than the wild-type enzyme and a higher affinity for phenolic substrates. Oxidation of heme b to heme d did not occur in most of the studied variants. Structural changes in solvent-chain integrity and channel architecture were also observed. In summary, modification of the main-channel gate glutamic acid and threonine residues has a greater influence on the secondary activity of the catalase enzyme, and the oxidation of heme b to heme d is predominantly inhibited by their conversion to aliphatic and aromatic residues.

Functional dissection of the PME and PMEI super families in the protection of Populus pectin during Fusarium wilt

Integration of the pectin homogalacturonan (HG) integrity and remodeling into defense immunity facilitates building blocks of stress resilience within the host and pathogen interactions in the forests. Recent advances in apoplastic pectin methylesterases (PMEs) and coordinated PME inhibitors (PMEIs) deployed immune signaling, highlighting their critical roles in regulating biotic stress adaptation. Nevertheless, the PME and PMEI multiple isoenzyme families involved in defense priming in the model tree species are largely unknown. The in silico demonstration of the molecular profile identified 70 PME- (50 type-I proPME and 20 type-II PME) and 49 PMEI-encoding genes in the Populus genome. Evaluation of the transcript abundance verified 20 PtproPMEs and 18 PtPMEIs that showed predominant expression in the root and genetic redundancy for type-II PtPMEs. Dynamic changes in the transcriptome and experimental evaluation revealed a minuscule number of type-I proPMEs and PMEIs that showed marked promotions during a time-course Fusarium solani (Fs) infection. Using GC/LC-mass-based non-targeted metabolomics to inspect structure-related carbohydrates and PME activities suggested a correlation between proPME-PMEI co-expression and deformed pectin HG biosynthesis. Further functional examination of the enzyme inhibitory affinities of recombinant PtPMEI1 and 39 in vitro and apoplastic patterns suggested a clustering of the type-I proPME in the regulation of the innate immunity by maintaining homeostasis of the pectin HG de-methylesterification (DME) in the cell wall. The coherent omics-wide survey of the fungi pathogen-induced PME-PMEIs and disturbed carbohydrate accumulation provide theoretical cues for in-depth mining of the biological significance of the underlying immune signaling networks within an apoplastic niche in trees.

Adrenodoxin allosterically alters human cytochrome P450 11B enzymes to accelerate substrate binding and decelerate release.

Two human mitochondrial membrane CYP11B enzymes play a pivotal role in steroidogenesis. CYP11B1 generates the major glucocorticoid cortisol, while CYP11B2 catalysis yields the primary mineralocorticoid aldosterone. Catalysis by both requires electron delivery by a soluble iron-sulfur adrenodoxin redox partner. However recent studies have shown that adrenodoxin/CYP11B interaction alone allosterically increases substrate and inhibitor affinity as exhibited by decreased dissociation constant (K d) values. The current study moves beyond such equilibrium studies, by defining adrenodoxin effects on the rates of P450 ligand binding and release separately. Stopped-flow data clearly demonstrate that adrenodoxin interaction with the P450 proximal surfaces increases ligand binding in both P450 CYP11B active sites by increasing the on rate constant and decreasing the off rate constant. As substrate entry and exit from the sequestered P450 active site requires conformational changes on the distal side of the P450 enzyme, a likely explanation is that adrenodoxin binding allosterically modulates CYP11B conformational changes. The 93% identical CYP11B enzymes can bind and hydroxylate each other's native substrates differing only by a hydroxyl. However, CYP11B1 exhibits monophasic substrate binding and CYP11B2 biphasic substrate binding, even when the substrates are swapped. This indicates that small differences in amino acid sequence between human CYP11B1 and CYP11B2 enzymes are more functionally important in ligand binding and could suggest avenues for more selective inhibition of these drug targets. Both protein/protein interactions and protein/substrate interactions are most likely to act by modulating CYP11B conformational dynamics.