Reduced Binding Affinity Research Articles

Comprehensive structure-function analysis reveals gain- and loss-of-function mechanisms impacting oncogenic KRAS activity.

To dissect variant-function relationships in the KRAS oncoprotein, we performed deep mutational scanning (DMS) screens for both wild-type and KRAS G12D mutant alleles. We defined the spectrum of oncogenic potential for nearly all possible KRAS variants, identifying several novel transforming alleles and elucidating a model to describe the frequency of KRAS mutations in human cancer as a function of transforming potential, mutational probability, and tissue-specific mutational signatures. Biochemical and structural analyses of variants identified in a KRAS G12D second-site suppressor DMS screen revealed that attenuation of oncogenic KRAS can be mediated by protein instability and conformational rigidity, resulting in reduced binding affinity to effector proteins, such as RAF and PI3-kinases, or reduced SOS-mediated nucleotide exchange activity. These studies define the landscape of single amino acid alterations that modulate the function of KRAS, providing a resource for the clinical interpretation of KRAS variants and elucidating mechanisms of oncogenic KRAS inactivation for therapeutic exploitation.

Molecular Modeling and In Vitro Functional Analysis of the RGS12 PDZ Domain Variant Associated with High-Penetrance Familial Bipolar Disorder.

Bipolar disorder's etiology involves genetics, environmental factors, and gene-environment interactions, underlying its heterogeneous nature and treatment complexity. In 2020, Forstner and colleagues catalogued 378 sequence variants co-segregating with familial bipolar disorder. A notable candidate was an R59Q missense mutation in the PDZ (PSD-95/Dlg1/ZO-1) domain of RGS12. We previously demonstrated that RGS12 loss removes negative regulation on the kappa opioid receptor, disrupting basal ganglia dopamine homeostasis and dampening responses to dopamine-eliciting psychostimulants. Here, we investigated the R59Q variation in the context of potential PDZ domain functional alterations. We first validated a new target for the wildtype RGS12 PDZ domain-the SAPAP3 C-terminus-by molecular docking, surface plasmon resonance (SPR), and co-immunoprecipitation. While initial molecular dynamics (MD) studies predicted negligible effects of the R59Q variation on ligand binding, SPR showed a significant reduction in binding affinity for the three peptide targets tested. AlphaFold2-generated models predicted a modest reduction in protein-peptide interactions, which is consistent with the reduced binding affinity observed by SPR, suggesting that the substituted glutamine side chain may weaken the affinity of RGS12 for its in vivo binding targets, likely through allosteric changes. This difference may adversely affect the CNS signaling related to dynorphin and dopamine in individuals with this R59Q variation, potentially impacting bipolar disorder pathophysiology.

Stereochemical influence of 4ʹ-methyl substitutions on truncated 4ʹ-thioadenosine derivatives: Impact on A3 adenosine receptor binding and antagonism

Herein, we investigated the stereochemical effects of 4ʹ-methyl substitution on A3 adenosine receptor (A3AR) ligands by synthesizing and evaluating a series of truncated 4ʹ-thioadenosine derivatives featuring 4ʹ-α-methyl, 4ʹ-β-methyl, and 4ʹ,4ʹ-dimethyl substitutions. We successfully synthesized these derivatives, using the stereoselective addition of an organometallic reagent, KSAc-mediated sulfur cyclization, and Vorbrüggen condensation. Binding assays demonstrated that the 4ʹ-β-methyl substitution conferred the highest affinity for A3AR, with compound 1 h exhibiting a Ki = 3.5 nM, followed by the 4ʹ,4ʹ-dimethyl and 4ʹ-α-methyl substitutions. Notably, despite the absence of the 5ʹ-OH group, compound 1 h unexpectedly displayed partial agonism. Computational docking studies indicated that compound 1 h, the β-methyl derivative, adopted a South conformation and maintained strong interactions within the receptor, including a critical interaction with Thr94, a residue known to be notable for agonistic effects. Conversely, compound 2 h, the α-methyl derivative, also adopted a South conformation but resulted in a flattened structure that hindered interactions with Thr94 and Asn250. The dimethyl derivative 3 h exhibited steric clashes with Thr94, contributing to a reduction in binding affinity. However, the docking results for 3 h indicated a North conformation, suggesting that the change in sugar conformation due to the additional 4ʹ-methyl group altered the angle between the α-methyl group and the sugar plane, enabling binding despite the increased steric bulk. These findings suggest that not only do the substituents and their stereochemistry influence receptor–ligand interactions, but the conformation and the resulting spatial orientation of the substituents also play a crucial role in modulating receptor–ligand interaction. This stereochemical insight offers a valuable framework for the design of new, selective, and potent A3AR ligands, potentially facilitating the development of novel therapeutics for A3AR-related diseases such as glaucoma, inflammation, and cancer.

Molecular Mechanisms for the Selective Transport of Dichlorofluorescein by Human Organic Anion Transporting Polypeptide 1B1.

Human organic anion transporting polypeptide (OATP) 1B1 and 1B3 are two highly homologous liver-specific uptake transporters. However, 2',7'-dichlorofluorescein (DCF) is preferably transported by OATP1B1. In the present study, the molecular mechanisms for the selective transport of DCF by OATP1B1 were investigated by constructing and characterizing an array of OATP1B1/1B3 chimeras and site-directed mutagenesis. Our results show that transmembrane domain (TM) 10 is crucial for the surface expression and function of OATP1B1, in which Q541 and L545 play the most important roles in DCF transport. Replacement of TM10 in OATP1B1 with its OATP1B3 counterpart led to OATP1B1's complete intracellular retention. Q541 and L545 may interact with DCF directly via hydrogen bonding and hydrophobic interactions. The decrease of DCF uptake by Q541A and L545S was due to their reduced binding affinity for DCF as compared with OATP1B1. In addition, Q541 and L545 are also crucial for the transport of estradiol-17β-glucuronide (E17βG) but not for the transport of estrone-3-sulfate (E3S), indicating different interaction modes between DCF/E17βG and E3S in OATP1B1. Taken together, Q541 and L545 in TM10 are critical for OATP1B1-mediated DCF uptake, but their effect is substrate-dependent. SIGNIFICANCE STATEMENT: The key TMs and amino acid residues for the selective transport of DCF by OATP1B1 were identified. TM10 is crucial for the surface expression and function of OATP1B1. Within TM10, Q541 and L545 played the most significant roles and affected the function of OATP1B1 in a substrate-dependent manner. This information is crucial for a better understanding of the mechanism of the multispecificity of OATP1B1 and as a consequence the mechanism of OATP1B1-mediated drug-drug interactions.

Resistance to Acetyl Coenzyme A Carboxylase (ACCase) Inhibitor in Lolium multiflorum: Effect of Multiple Target-Site Mutations

Italian ryegrass (Lolium multiflorum Lam.) is a persistent weed species that poses significant management challenges in key agricultural crops such as wheat, corn, cotton, and soybean. This study investigated the prevalence of resistance to ACCase inhibitor herbicides, specifically diclofop and pinoxaden, among field-collected Italian ryegrass populations. The survey revealed widespread resistance to diclofop and emerging cross-resistance to pinoxaden. To elucidate the physiological mechanism of ACCase herbicide resistance, we investigated mutations in the carboxyl-transferase (CT) domain of the ACCase enzyme, a critical region for herbicide sensitivity. Using dCAPS assays and CT domain sequencing, several known resistance-conferring mutations were detected in diclofop survivors, including I1781L, W2027C, I2041N, D2078G, and C2088R. Additionally, other mutations such as L1701M, E1874A, N1878H, G1946E/Q, V1992D, and E2039D were identified. To understand the functional role of these mutations in herbicide resistance, homology modeling was performed using AutoDock Vina for selected mutation combinations. The computational analysis revealed that all mutations and their combinations resulted in reduced binding affinity with diclofop and pinoxaden compared to the wild-type ACCase CT domain. Computational binding energy predictions indicated that the G1946E mutation and the L1701M + I1781L + E1874A + N1878H combination exhibited the lowest affinities for diclofop and pinoxaden, respectively. This study provides valuable insights into the molecular basis of ACCase inhibitor resistance in Italian ryegrass. However, further research is needed to validate the functional significance of each new substitution and its combinations in conferring herbicide resistance.

Effects of SARS-CoV-2 Main Protease Mutations at Positions L50, E166, and L167 Rendering Resistance to Covalent and Noncovalent Inhibitors.

SARS-CoV-2 propagation under nirmatrelvir and ensitrelvir pressure selects for main protease (MPro) drug-resistant mutations E166V (DRM2), L50F/E166V (DRM3), E166A/L167F (DRM4), and L50F/E166A/L167F (DRM5). DRM2-DRM5 undergoes N-terminal autoprocessing to produce mature MPro with dimer dissociation constants (Kdimer) 2-3 times larger than that of the wildtype. Co-selection of L50F restores catalytic activity of DRM2 and DRM4 from ∼10 to 30%, relative to that of the wild-type enzyme, without altering Kdimer. Binding affinities and thermodynamic profiles that parallel the drug selection pressure, exhibiting significant decreases in affinity through entropy/enthalpy compensation, were compared with GC373. Reorganization of the active sites due to mutations observed in the inhibitor-free DRM3 and DRM4 structures as compared to MProWT may account for the reduced binding affinities, although DRM2 and DRM3 complexes with ensitrelvir are almost identical to MProWT-ensitrelvir. Chemical reactivity changes of the mutant active sites due to differences in electrostatic and protein dynamics effects likely contribute to losses in binding affinities.

7447 Rare KSR2 Variants: The Effect of Glycine and N-acetylcysteine (GlyNAC) Supplementation

Abstract Disclosure: J. Youn: None. Y. Carcamo-Bahena: None. E. Lartigue: None. S. Sisley: None. Kinase suppressor of ras 2 (KSR2) is an intracellular scaffolding protein involved in multiple signaling pathways. Of note, disruption of the kinase domain of KSR2 in humans and mice causes obesity by reducing metabolic rate, suggesting its important role in energy homeostasis. In the present study, we identified two variants (P189L, T290A) with mutations located in the proximal CA2 region from children with severe, early-onset obesity. Since variants in this region have not been confirmed to cause obesity, we generated plasmid constructs containing these KSR2 mutations by site directed mutagenesis. The HEK293 cells transfected with KSR2 plasmid construct containing P189L or T290A had reduced binding affinity with AMPK and phosphorylation of AMPK without affecting KSR2 protein integrity. These variants also significantly reduced cellular metabolic rates measured by Seahorse analysis in C2C12 cells, suggesting that KSR2 variants in the proximal CA2 region can result in impaired KSR2 function, subsequently leading to a decrease in cellular metabolic rate. In addition, these KSR2 variants decreased protein levels of NRF-2, a transcription factor of cytoprotective genes regulating the antioxidant glutathione (GSH) pathway compared to wild type KSR2 which augmented NRF2 levels. We also observed lower levels of total GSH and GSH/Glutathione disulfide (GSSG) ratio and higher levels of thiobarbituric acid reactive substances (TBARS), an indicator of oxidative stress, indicating an imbalance of antioxidant and oxidant with proximal KSR2 variants. However, treatment of cells with Glycine and N-acetylcysteine (GlyNAC) reversed the detrimental effects of KSR2 variants by normalizing protein expression levels of NRF-2, GSH/GSSG ratios and TBARS levels. Taken together, we show strong evidence that two new KSR2 variants, P189L and T290A, cause disruption of KSR2 function. Additionally, we also report a novel role of KSR2 in maintaining redox homeostasis suggesting that increased oxidative stress following GSH deficiency may play an important role in the development of obesity from KSR2 variants. Presentation: 6/1/2024

Cryo-EM investigation of ryanodine receptor type 3.

Ryanodine Receptor isoform 3 (RyR3) is a large ion channel found in the endoplasmic reticulum membrane of many different cell types. Within the hippocampal region of the brain, it is found in dendritic spines and regulates synaptic plasticity. It controls myogenic tone in arteries and is upregulated in skeletal muscle in early development. RyR3 has a unique functional profile with a very high sensitivity to activating ligands, enabling high gain in Ca2+-induced Ca2+ release. Here we solve high-resolution cryo-EM structures of RyR3 in non-activating and activating conditions, revealing structural transitions that occur during channel opening. Addition of activating ligands yields only open channels, indicating an intrinsically high open probability under these conditions. RyR3 has reduced binding affinity to the auxiliary protein FKBP12.6 due to several sequence variations in the binding interface. We map disease-associated sequence variants and binding sites for known pharmacological agents. The N-terminal region contains ligand binding sites for a putative chloride anion and ATP, both of which are targeted by sequence variants linked to epileptic encephalopathy.

Insights into lipid-modified recognition of Apolipoprotein E3 to extra-cellular domain of TREM2 associated with Alzheimer’s disease

E3 genotype of apolipoprotein E (ApoE) and triggering receptor expressed on myeloid cells 2 (TREM2) are critical genetic risk factors for late-onset Alzheimer’s diseases (AD) that influences the formation of plaques and neurofibrillary tangles associated with AD pathogenesis. In addition, variation in genetic association of ApoE and TREM2 in presence of lipids have been advocated to potentially regulate different aspects of AD pathology by facilitating the clearance of amyloid beta (Aβ) from brain. However, the proteinopathy and functional mechanism underlying their molecular interaction in presence of lipid remains poorly understood. Herein, to shed inherent insights into the mode of ApoE3-TREM2 interaction in both lipid and aqueous medium, we have used a combination of different state-of-the-art computational tools including knowledge-based protein–protein docking and molecular dynamics (MD) simulations. Remarkably, we find the direct involvement of regulatory complementarity-determining region-2 (CDR2) loop of TREM2 in interaction with the major hinge region and helix H4 of ApoE3. We further anticipate that ApoE3 attains distinct conformational states and forms an open and extended cavity-like structure in ApoE3-TREM2 complex in presence of lipids. Our data also exhibits reduced hydrophobicity in the ApoE3 binding interface of TREM2, which sets a baseline for reduced binding affinity towards ApoE3. Therefore, we hypothesize that this decline in hydrophobic index might have resulted due to structural variations in the H4 helix and major hinge region leading to altered TREM2 conformation in presence of lipids. Altogether, our findings demonstrate the effect of phospholipids on the structural dynamics and conformation of ApoE3, which contributes to our understanding of how APOE and TREM2 influences the development of AD. However, further experimental studies are necessary to validate and explore our findings which will open up new avenues towards development of novel therapeutic strategies for AD.

Genetic and in-silico approaches for investigating the mechanisms of ciprofloxacin resistance in Salmonella typhi: Mutations, extrusion, and antimicrobial resistance

Salmonella enterica serovar Typhi spreads typhoid infection in humans through the consumption of contaminated food and water. Poor sanitation plays a pivotal role in its dissemination. Over time, the bacterium has acquired resistance to many promising antibiotics, posing a growing global health concern and hindering the achievement of sustainable development goals. This study aims to elucidate the molecular complexity of fluoroquinolone resistance, a first-line treatment for typhoid infection. To achieve this aim, 80 clinical isolates were collected from various diagnostic laboratories. These isolates were confirmed based on morphological characteristics and biochemical tests. Multidrug-resistant (MDR) and extensively drug-resistant (XDR) isolates were identified using the Kirby-Bauer disc diffusion method. The mechanism of ciprofloxacin resistance was investigated by sequencing the quinolone resistance-determining region (QRDR) genes and identifying the presence of the qnrS1 gene. As a result of this study, 60 % of isolates showed resistance to ciprofloxacin. At the same time, the qnrS1 gene was present in all the selected strains while mutation analysis identified significant mutation in QRDR of DNA gyrase subunit A (gyrA) and Topoisomerase IV (parC) gene. The combinatorial effect was further investigated by downloading 286 draft genomes. The Mutation analysis reveals significant mutations at gyrA S83F, gyrA D87N, gyrA S83Y, gyrB S464F, parC S80I, and parE L416F. Additionally, docking analysis indicates reduced binding affinity and altered solvent accessibility, which show the structural changes at mutation sites. This study provides crucial insights that mutation reduces the binding affinity while qnrS1 acts as a transport channel to extrude the ciprofloxacin. In the future, further validation through experimental mutagenesis is recommended, for targeted therapeutic interventions against the mounting threat of antibiotic-resistant S. Typhi.

Downregulation of APN1 and ABCC2 mutation in Bt Cry1Ac-resistant Trichoplusia ni are genetically independent.

The resistance to the insecticidal protein Cry1Ac from the bacterium Bacillus thuringiensis (Bt) in the cabbage looper, Trichoplusia ni, has previously been identified to be associated with a frameshift mutation in the ABC transporter ABCC2 gene and with altered expression of the aminopeptidase N (APN) genes APN1 and APN6, shown as missing of the 110-kDa APN1 (phenotype APN1¯) in larval midgut brush border membrane vesicles (BBMV). In this study, genetic linkage analysis identified that the APN1¯ phenotype and the ABCC2 mutation in Cry1Ac-resistant T. ni segregated independently, although they were always associated under Cry1Ac selection. The ABCC2 mutation and APN1¯ phenotype were separated into two T. ni strains respectively. Bioassays of the T. ni strains with Cry1Ac determined that the T. ni with the APN1¯ phenotype showed a low level resistance to Cry1Ac (3.5-fold), and the associated resistance is incompletely dominant in the background of the ABCC2 mutation. Whereas the ABCC2 mutation-associated resistance to Cry1Ac is at a moderate level, and the resistance is incompletely recessive in the genetic background of downregulated APN1. Analysis of Cry1Ac binding to larval midgut BBMV indicated that the midgut in larvae with the APN1¯ phenotype had reduced binding affinity for Cry1Ac, but the number of binding sites remained unchanged, and the midgut in larvae with the ABCC2 mutation had both reduced binding affinity and reduced number of binding sites for Cry1Ac. The reduced Cry1Ac binding to BBMV from larvae with the ABCC2 mutation or APN1¯ phenotype correlated with the lower levels of resistance.IMPORTANCEThe soil bacterium Bacillus thuringiensis (Bt) is an important insect pathogen used as a bioinsecticide for pest control. Bt genes coding for insecticidal proteins are the primary transgenes engineered into transgenic crops (Bt crops) to confer insect resistance. However, the evolution of resistance to Bt proteins in insect populations in response to exposure to Bt threatens the sustainable application of Bt biotechnology. Cry1Ac is a major insecticidal toxin utilized for insect control. Genetic mechanisms of insect resistance to Cry1Ac are complex and require to be better understood. The resistance to Cry1Ac in Trichoplusia ni is associated with a mutation in the ABCC2 gene and also associated with the APN expression phenotype APN1¯. This study identified the genetic independence of the APN1¯ phenotype from the ABCC2 mutation and isolated and analyzed the ABCC2 mutation-associated and APN1¯ phenotype-associated resistance traits in T. ni to provide new insights into the genetic mechanisms of Cry1Ac resistance in insects.

In Silico Screening, Molecular Dynamics Simulation and Binding Free Energy Identify Single-Point Mutations That Destabilize p53 and Reduce Binding to DNA.

Considering p53's pivotal role as a tumor suppressor protein, proactive identification and characterization of potentially harmful p53 mutations are crucial before they appear in the population. To address this, four computational prediction tools-SIFT, Polyphen-2, PhD-SNP, and MutPred2-utilizing sequence-based and machine-learning algorithms, were employed to identify potentially deleterious p53 nsSNPs (nonsynonymous single nucleotide polymorphisms) that may impact p53 structure, dynamics, and binding with DNA. These computational methods identified three variants, namely, C141Y, C238S, and L265P, as detrimental to p53 stability. Furthermore, molecular dynamics (MD) simulations revealed that all three variants exhibited heightened structural flexibility compared to the native protein, especially the C141Y and L265P mutations. Consequently, due to the altered structure of mutant p53, the DNA-binding affinity of all three variants decreased by approximately 1.8 to 9.7 times compared to wild-type p53 binding with DNA (14 μM). Notably, the L265P mutation exhibited an approximately ten-fold greater reduction in binding affinity. Consequently, the presence of the L265P mutation in p53 could pose a substantial risk to humans. Given that p53 regulates abnormal tumor growth, this research carries significant implications for surveillance efforts and the development of anticancer therapies.

A Rare Mutation in MESP1 Increases the Risk of Congenital Heart Disease

Objective: In this study, we aim to screen MESP1 variants in congenital heart disease (CHD), and explore the biological functions of these variants. Methods: Targeted sequencing was used to screen MESP1 variants in a cohort of patients with CHD and healthy controls from Shandong Province, China, and confirm the variants with Sanger sequencing. Dual-luciferase reporter assay was used to assess the effects of MESP1 variants on the expression of downstream target genes in HEK293T and H9C2 cells. Binding affinity with NKX2-5 between variants and wild-type MESP1 was detected in HEK293T cells using an electrophoretic gel mobility shift assay (EMSA). The teratogenic effects of the MESP1 variants compared to the wild-type were explored with zebrafish embryo microinjection. Results: We identified two rare mutations, namely c.359T>C (p.L120P) and c.526A>T (p.T176S), in MESP1. The impact of these mutations was investigated using dual-luciferase reporter assay. Our findings demonstrate that compared to the wild-type, the mutation c.359T>C (p.L120P) significantly inhibits the expression of downstream target genes, whereas the mutation c.526A>T (p.T176S) does not exhibit altered activity. Further insights from EMSA revealed that the mutation c.359T>C (p.L120P) displays a reduced binding affinity with NKX2-5. Notably, our investigation involving zebrafish embryo microinjection unveiled a significant increase in the rate of malformation associated with the mutation c.359T>C (p.L120P). Conclusions: Two rare mutations in MESP1 were identified in the CHD cohort. Our in vitro and in vivo studies showed that MESP1 c.359T>C (p.L120P) is a loss-of-function mutation that may increase the risk of CHD.

Protein-Templated Click Ligation Reaction Triggered by Protein-Split Aptamer Interactions.

DNA-templated reactions have found wide applications in sensing and drug discovery. However, this strategy has been limited to the use of nucleic acids as templating elements to direct the proximity effect. Herein, we describe a versatile protein-templated split aptamer click ligation reaction (PT-SpA-CLR) in which the protein template-induced covalent proximity ligation of split aptamer elements enables translating protein/aptamer binding events into the output of ligated DNA products. A ligation yield of >80% is observed for three model protein templates, including VEGF165, PDGF-BB, and SARS-CoV-2 S1. The ligation reaction compensates for the weakness of reduced binding affinity resulting from splitting the aptamer, as evidenced by an approximately 2-fold lower dissociation constant than the non-ligated system. This newly developed PT-SpA-CLR strategy is further integrated with colorimetric or fluorescent reporting mechanisms to achieve easy-to-use and low-cost biosensors utilizing ligation to produce a fully active G-quadruplex or an RNA-cleaving DNAzyme to report protein binding. Both assays can achieve specific detection of an intended protein target with a limit of detection at the picomolar level even when challenged in biological samples. The combined PT-SpA-CLR and versatile sensing strategies offer attractive universal platforms for efficient detection of protein biomarkers.

Engineering SuFEx-derived macrocyclic polysulfates for superior dynamic iodine capture at high temperatures

Efficient dynamic capture of radioiodine vapor from reprocessing of spent nuclear fuel under industrial conditions (∼423.15 K) is critically required due to its detrimental environmental and health impacts. However, conventional adsorbents often suffer from reduced binding affinity and structural degradation at elevated temperatures. To address these challenges, we have engineered a series of advanced polysulfate networks using sulfur (VI) fluoride exchange (SuFEx) click chemistry. These innovative networks integrate supramolecular macrocycle calix[4]pyrrole (C[4]P) and nitrogen-rich co-monomers as building blocks, and utilize polar sulfate bonds as important linkers, which provide availability of adsorptive sites and enhance the structural stability, thereby affording unexpected iodine capture performance at high temperatures. Among these SuFEx-derived materials, C[4]P-Azo exhibits exceptional iodine affinity through cooperative interactions involving multiple binding motifs, achieving an impressive iodine uptake capacity of 0.45 g g−1 at 423.15 K under dynamic adsorption conditions, setting a new benchmark in this field. Comprehensive characterizations and DFT calculations elucidate that advanced linker sulfate bonds enabling the marriage of C[4]P and N-rich monomers, play pivotal roles in facilitating iodine adsorption and maintaining structural integrity. These findings may pave the way for designing robust macrocyclic polysulfate-based adsorbents for dynamic radioiodine capture under industrial conditions.

Structural basis for the evolution and antibody evasion of SARS-CoV-2 BA.2.86 and JN.1 subvariants

The Omicron subvariants of SARS-CoV-2, especially for BA.2.86 and JN.1, have rapidly spread across multiple countries, posing a significant threat in the ongoing COVID-19 pandemic. Distinguished by 34 additional mutations on the Spike (S) protein compared to its BA.2 predecessor, the implications of BA.2.86 and its evolved descendant, JN.1 with additional L455S mutation in receptor-binding domains (RBDs), are of paramount concern. In this work, we systematically examine the neutralization susceptibilities of SARS-CoV-2 Omicron subvariants and reveal the enhanced antibody evasion of BA.2.86 and JN.1. We also determine the cryo-EM structures of the trimeric S proteins from BA.2.86 and JN.1 in complex with the host receptor ACE2, respectively. The mutations within the RBDs of BA.2.86 and JN.1 induce a remodeling of the interaction network between the RBD and ACE2. The L455S mutation of JN.1 further induces a notable shift of the RBD–ACE2 interface, suggesting the notably reduced binding affinity of JN.1 than BA.2.86. An analysis of the broadly neutralizing antibodies possessing core neutralizing epitopes reveals the antibody evasion mechanism underlying the evolution of Omicron BA.2.86 subvariant. In general, we construct a landscape of evolution in virus-receptor of the circulating Omicron subvariants.

Exploring pathogenic SNPs and estrogen receptor alpha interactions in breast cancer: An in silico approach

The estrogen receptor 1 gene (ESR1) plays a crucial role in breast and mammary development in humans. Alterations such as gene amplification, genomic rearrangements, and missense mutations in the ESR1 gene are reported to increase the risk of breast cancer in humans. The purpose of this study is to analyze the missense mutations and molecular modeling of ESR1, focusing on the pathogenic SNP H516N, for a better understanding of disease risk and future benefits for therapeutic benefits. This SNP was selected based on its location in the binding pocket of ESR1 and its predicted impact on drug binding. The in silico analysis was performed by applying various computational approaches to identify highly pathogenic SNPs in the binding pocket of ESR1. The effect of the SNP was explored through docking and intra-molecular interaction studies. All SNPs in ESR1 were identified followed by the identification of the highly pathogenic variant located in the binding pocket of ESR1. The mutant model of the pathogenic SNP H516N was generated, and hydroxytamoxifen was docked with the wild-type and the mutant model. The mutant model lost the formation of stable hydrogen bonds with the active site residues and hydroxytamoxifen, which may result in reduced binding affinity and therefore, will predict the patient's response to estrogenic inhibitors.

Potentilla tormentilla Extract Loaded Gel: Formulation, In Vivo and In Silico Evaluation of Anti-Inflammatory Properties.

The objective of the study was to develop a novel topical gel by mixing Potentilla tormentilla ethanolic extract, thermosensitive poloxamer 407, and carbomer 940 and evaluating its stability and rheological behavior. The irritation potential of the gel was evaluated in accordance with the Organization for Economic Cooperation and Development Guidelines 404. The potential anti-inflammatory effects of the developed gel were evaluated in vivo in rats using the carrageenan-induced paw edema test. Moreover, the in silico binding affinity for chlorogenic and ellagic acid, as dominant components in the extract, against cyclooxygenase (COX) 1 and 2 was also determined. Our findings suggest that the gel containing Potentilla tormentilla extract remained stable throughout the observation period, exhibited pseudoplastic behavior, and caused no irritation in rats, thus being considered safe for topical treatment. Additionally, the developed gel showed the capability to reduce rat paw edema, which highlights significant anti-inflammatory potential. In silico analysis revealed that chlorogenic and ellagic acid exhibited a reduced binding affinity against COX-1 but had a similar inhibitory effect on COX-2 as flurbiprofen, which was confirmed by molecular dynamics results. The study proposes the possible application of Potentilla tormentilla ethanolic extract gel for the alleviation of localized inflammatory diseases; however, future clinical evaluation is required.

Deciphering the impact of F23L mutation on the aggregation propensity of human islet amyloid polypeptide using molecular simulations

The self-aggregation of 37-residue human islet amyloid polypeptide (hIAPP) into β-sheet rich oligomers and amyloid fibrils contributes to the loss of pancreatic β-cell mass in type 2 diabetes (T2D). A previous study highlighted that the F23L mutation in the amyloidogenic core (Ser20–Ser29) of hIAPP slows down its self-aggregation by a factor of 2.6. However, the underlying molecular mechanism behind slower aggregation and reduced cytotoxicity of hIAPP on F23L mutation remains obscure. Thus, the structural and conformational changes in F23L–hIAPP that lead to slower aggregation and lower cytotoxicity were examined by molecular dynamics (MD) simulations. The MD simulations depicted that F23L mutation marginally increases the helical content from 10.20 ± 1.94 to 11.50 ± 2.18 % accompanied by a noteworthy decrease in aggregation-prone β-sheet from 14.00 ± 1.58 to 0.80 ± 0.37 %. Notably, FEL conformations depicted a complete disappearance of β-sheet content in F23L–hIAPP as compared to 9–20 % in wt–hIAPP in agreement with the notably reduced toxicity of F23L–hIAPP aggregates against cultured INS-1 832/13 cells. Interestingly, dihedral principal component analysis (dPCA) depicted noteworthy compression in the conformational sub-space and a lower trace value of hIAPP on F23L mutation, which indicates lower conformational fluctuations in F23L–hIAPP. F23L mutation alters the conformational landscape of hIAPP dimer by lowering interchain contacts, and hydrogen bonds as well as reducing binding affinity between chains A-B of hIAPP dimer. Importantly, MD simulations illuminated the key role of Phe23 in mediating intramolecular interactions leading to aggregation-prone structures that promote hIAPP aggregation. The key insights regarding structural transitions and conformational preferences of hIAPP on F23L mutation illuminated from this work will benefit the design and optimization of potential inhibitors targeting Phe23-mediated interactions to block hIAPP aggregation in T2D.

Characterization of a Novel Monoclonal Antibody with High Affinity and Specificity against Aflatoxins: A Discovery from Rosetta Antibody-Ligand Computational Simulation.

Aflatoxin B1 (AFB1) accumulates in crops, where it poses a threat to human health. To detect AFB1, anti-AFB1 monoclonal antibodies have been developed and are widely used. While the sensitivity and specificity of these antibodies have been extensively studied, information regarding the atomic-level docking of AFB1 (and its derivatives) with these antibodies is limited. Such information is crucial for understanding the key interactions that are required for high affinity and specificity in aflatoxin binding. First, a 3D comparative model of anti-AFB1 antibody (Ab-4B5G6) was predicted from the sequence using RosettaAntibody. We then utilized RosettaLigand to dock AFB1 onto ten homology models, producing a total of 10,000 binding modes. Interestingly, the best-scoring mode predicted strong interactions involving four sites within the heavy chain: ALA33, ASN52, HIS95, and TRP99. Importantly, these strong binding interactions exclusively involve the variable domain of the heavy chain. The best-scoring mode with AFB1 was also obtained through AF multimer combined with RosettaLigand, and two interactions at TRP and HIS were consistent with those found by Rosetta antibody-ligand computational simulation. The role of tryptophan in π interactions in antibodies was confirmed through mutation experiments, and the resulting mutant (W99A) exhibited a >1000-fold reduction in binding affinity for AFB1 and analogs, indicating the effect of tryptophan on the stability of CDR-H3 region. Additionally, we evaluated the binding of two glycolic acid-derived molecular derivatives (with impaired hydrogen bonding potential), and these derivatives (AFB2-GA and AFG2-GA) demonstrated a very weak binding affinity for Ab-4B5G6. The heavy chain was successfully isolated, and its sensitivity and specificity were consistent with those of the intact antibody. The homology models of variable heavy (VH) single-domain antibodies were established by RosettaAntibody, and the docking analysis revealed the same residues, including Ala, His, and Trp. Compared to the potential binding mode of fragment variable (FV) region, the results from a model of VH indicated that there are seven models involved in hydrophobic interaction with TYR32, which is usually referred to as polar amino acid and has both hydrophobic and hydrophilic features depending on the circumstances. Our work encompasses the entire process of Rosetta antibody-ligand computational simulation, highlighting the significance of variable heavy domain structural design in enhancing molecular interactions.