Affinity Probes Research Articles

Arv1; a "Mover and Shaker" of Subcellular Lipids.

The composition of eukaryotic membranes reflects a varied but precise amalgam of lipids. The genetic underpinning of how such diversity is achieved or maintained is surprisingly obscure, despite its clear metabolic and pathophysiological impact. The Arv1 protein is represented in all eukaryotes and was initially identified in the model eukaryote Sacccharomyces cerevisiae as a candidate transporter of lipids from the endoplasmic reticulum. Human Arv1 has been shown to directly bind cholesterol and fatty acid affinity probes. Murine in vivo studies point to a role for ARV1 in regulating obesity, glucose tolerance, insulin sensitivity and brain function. Multiple human ARV1 variants have been associated with epileptic encephalopathy, cerebellar ataxia, and severe intellectual deficits. We hypothesize that Arv1 acts as an energy independent, lipid scramblase at the endoplasmic reticulum thereby modulating membrane lipid asymmetry and thus the trafficking of sterols and the substituents of glycosyl-phosphatidylinositol and sphingolipid biosynthesis.

A label-free colorimetric aptasensor for rapid multiplex detection of leuco-malachite green and leuco-crystal violet.

Leuco-malachite green (LMG) and leuco-crystal violet (LCV) are widespread co-pollutants in aquatic products that pose a severe threat to human health. Therefore, it is urgent and challenging to develop rapid multiplex detection of LMG and LCV. Herein, the bispecific aptamer (A5b) for LMG and LCV was characterized. Its dissociation constant (Kd) was 2.03 ± 0.13 μM for LMG and 0.97 ± 0.06 μM for LCV. Then, we used A5b as a sensing probe to develop a label-free colorimetric aptasensor using gold nanoparticles (AuNPs) as an indicator and poly-diallyl dimethylammonium chloride (PDDA) as an aggregation inducer. In the absence of LMG and LCV, PDDA hybridized A5b via electrostatic interaction to form a "duplex" structure, which failed to induce the aggregation of AuNPs. However, in the presence of LMG or LCV, A5b specifically bound to LMG or LCV, releasing PDDA. The free PDDA then induced the aggregation of AuNPs. Consequently, a visible color change from red to blue was observed with increasing LMG/LCV concentration. This sensor exhibited excellent sensitivity and bispecificity, and a limit of detection (LOD) of 77.9 nM for LCV and 123.1 nM for LMG, with a linear concentration range of 1-6 μM for LCV and 1-8 μM for LMG. Additionally, the potential of this sensor for real-world applications was well demonstrated in crucian samples. Overall, this work provided a bispecific affinity probe for LMG and LCV for the first time and reported a label-free colorimetric aptasensor for multiplex detection of LMG and LCV in aquatic products.

Synthesis and Preliminary Evaluation of Tanshinone Mimic Conjugates for Mechanism of Action Studies.

Human antigen R (HuR) is an RNA binding protein (RBP) belonging to the ELAV (Embryonic Lethal Abnormal Vision) family, which stabilizes mRNAs and regulates the expression of multiple genes. Its altered expression or localization is related to pathological features such as cancer or inflammation. Dihydrotanshinone I (DHTS I) is a naturally occurring, tetracyclic ortho-quinone inhibitor of the HuR-mRNA interaction. Our earlier efforts led to the identification of a synthetic Tanshinone Mimic (TM) 2 with improved affinity for HuR. Here we report five new TM probes 3-5 bearing a detection-promoting moiety (either photo affinity probe - PAP or biotin) as a para-substituent on the phenyl-sulphonamide for mechanism of action (MoA) studies. Biological and biochemical assays were used to characterize the novel TM conjugates 3-5. They showed similar toxic activity in HuR-expressing triple-negative breast cancer MDA-MB-231 cells, with micromolar CC50s. REMSAs revealed that photoactivatable groups (4 a and 4 b), but not biotin (5 a and 5 b), prevented conjugates' ability to disrupt rHuR-RNA complexes. Further biochemical studies confirmed that biotinylated probes, in particular 5 a, can be used to isolate rM1 M2 from solutions, taking advantage of streptavidin-coated magnetic beads, thus being the most promising HuR inhibitor to be used for further MoA studies in cell lysates.

DNA Conformation-Regulated Hemin Switch for Lab-on-Chip Chemiluminescent Detection of an Antibody Secreted from Hybridoma Cells.

This work designed a DNA conformation-regulated hemin switch for rapid chemiluminescent (CL) detection of a monoclonal antibodies. This switch was performed with an affinity probe and an inhibition probe, which were conveniently prepared by hybridizing hemin-labeled DNA1 with KHL peptide-labeled DNA2 and binding biotin-labeled DNA3 to streptavidin, respectively. In the absence of the target antibody, streptavidin-DNA3 could hybridize with hemin-DNA1/KHL-DNA2 to release KHL-DNA2, which led to the loss of hemin activity due to the affinity hindrance of streptavidin-DNA3. After the KHL peptide was recognized by the target antibody, the strand replacement hybridization could be inhibited by the bound antibody, which retained the high catalytic activity of hemin overhung on the antibody-bound affinity probe for a CL reaction, leading to a "signal-on" process for CL antibody detection. Using a KHL-specific antibody, anti-proprotein convertase subtilisin/kexin type 9 antibody (PCSK9-Ab), as a target model and common L012-1,2,4-triazole-H2O2 CL system, the designed switch showed a detection range of 10 ng mL-1 to 1 μg mL-1 with a detection limit of 4.16 ng mL-1 (56.2 pM) and a short analytical time of 6.5 min. The proposed quick method could simply be used for lab-on-chip CL detection of PCSK9-Ab in situ-secreted from PCSK9-6E3 hybridoma cells, which showed an accuracy of 90.2% compared with the statistical results from general fluorescence imaging, providing a potential technique for screening specific hybridoma cells.

Chemical glycoproteomic profiling in rice seedlings reveals N-glycosylation in the ERAD-L machinery

As a ubiquitous and essential posttranslational modification occurring in both plants and animals, protein N-linked glycosylation regulates various important biological processes. Unlike the well-studied animal N-glycoproteomes, the landscape of rice N-glycoproteome remains largely unexplored. Here, by developing a chemical glycoproteomic strategy based on metabolic glycan labeling (MGL), we report a comprehensive profiling of the N-glycoproteome in rice seedlings. The rice seedlings are incubated with N-azidoacetylgalactosamine (GalNAz) - a monosaccharide analog containing a bioorthogonal functional group - to metabolically label N-glycans, followed by conjugation with an affinity probe via click chemistry for enrichment of the N-glycoproteins. Subsequent mass spectrometry analyses identify a total of 403 N-glycosylation sites and 673 N-glycosylated proteins, which are involved in various important biological processes. In particular, the core components of the endoplasmic reticulum (ER)-associated protein degradation (ERAD) machinery are N-glycosylated, and the N-glycosylation is important for the ERAD-L function. This work not only provides an invaluable resource for studying rice N-glycosylation, but also demonstrates the applicability of MGL in glycoproteomic profiling for crop species.

Detection Strategies for Sialic Acid and Sialoglycoconjugates.

Glycoconjugates are a vast class of biomolecules implicated in biological processes important for human health and disease. The structural complexity of glycoconjugates remains a challenge to deciphering their precise biological roles and for their development as biomarkers and therapeutics. Human glycoconjugates on the outside of the cell are modified with sialic (neuraminic) acid residues at their termini. The enzymes that install sialic acids are sialyltransferases (SiaTs), a family of 20 different isoenzymes. The removal and degradation of sialic acids is mediated by neuraminidase (NEU; sialidase) enzymes, of which there are four isoenzymes. In this review, we discuss chemical and biochemical approaches for the detection and analysis of sialoglycoconjugate (SGC) structures and their enzymatic products. The most common methods include affinity probes and synthetic substrates. Fluorogenic and radiolabelled substrates are also important tools for many applications, including screening for enzyme inhibitors. Strategies that give insight into the native substrate-specificity of enzymes that regulate SGCs (SiaT & NEU) are necessary to improve our understanding of the role of sialic acid metabolism in health and disease.

Integrated global proteomic and phosphoproteomic analysis of cisplatin-induced apoptosis in A549 cells

Protein phosphorylation, a widely occurring and significant post-translational modification, is integral to various biological processes. We previously utilized a protein affinity probe to identify genes damaged by cisplatin, revealing that it inflicts substantial damage on protein kinase and protein phosphatase genes. In this study, we investigated cisplatin-induced alterations in the global proteome and phosphoproteome of A549 cells. Employing Fe-IMAC beads and tyrosine phosphorylation enrichment antibodies, we identified 6944 protein groups and 18,274 phosphorylation sites on 4915 proteins across three biological replicates of both cisplatin-treated A549 cells and control cells. Among these, 730 tyrosine phosphorylation sites were identified—marking the most substantial discovery of such sites in A549 cells following cisplatin treatment. Bioinformatics analysis indicated that the proteins exhibiting significant phosphorylation level changes predominantly involved in RNA processing, modification, transcription, translation, and the spliceosome. This suggests that cisplatin-induced damage to protein kinases and phosphatases may disrupt the normal function of these proteins, consequently impairing DNA replication, RNA translation, and shearing, ultimately culminating in tumor cell death. Moreover, we cross-referenced our proteomic data with our previously obtained cisplatin-damaged genes, observing that the majority of down-regulated proteins derived from cisplatin-induced gene damage. The data are available on ProteomeXchange under the identifier PXD053902.

Novel Corrector for Variants of SLC6A8: A Therapeutic Opportunity for Creatine Transporter Deficiency.

Mutations in creatine transporter SLC6A8 cause creatine transporter deficiency (CTD), which is responsible for 2% of all cases of X-linked intellectual disability. CTD has no current treatments and has a high unmet medical need. Inspired by the transformational therapeutic impact of small molecule "correctors" for the treatment of cystic fibrosis, which bind to mutated versions of the CFTR ion channel to promote its trafficking to the cell surface, we sought to identify small molecules that could stabilize SLC6A8 as a potential treatment for CTD. We leveraged a novel chemoproteomic technology for ligand discovery, reactive affinity probe interaction discovery, to identify small-molecule fragments with photoaffinity handles that bind to SLC6A8 in a cellular environment. We synthesized a library of irreversible covalent analogs of these molecules to characterize in functional assays, which revealed molecules that could promote the trafficking of mutant SLC6A8 variants to the cell surface. Further medicinal chemistry was able to identify reversible drug-like small molecules that both promoted trafficking of the transporter and also rescued creatine uptake. When profiled across the 27 most prevalent SLC6A8 missense variants, we found that 10-20% of patient mutations were amenable to correction by our molecules. These results were verified in an endogenous setting using the CRISPR knock-in of selected missense alleles. We established in vivo proof-of-mechanism for correctors in a novel CTD mouse model with the P544L patient-defined variant knocked in to the SLC6A8 locus, where treatment with our orally bioavailable and brain penetrant tool corrector increased brain creatine levels in heterozygous female mice, validating correctors as a potential therapeutic approach for CTD.

ДОСЛІДЖЕННЯ ВЗАЄМОДІЇ ФЛУОРЕСЦЕНТНОГО ЗОНДУ 4’-ДОДЕЦИЛКАРБОКСИ-3-ГІДРОКСИФЛАВОНУ З ПОЛІВІНІЛПІРОЛІДОНОМ

The study examined the interaction of a new ESIPT fluorescent probe, which is based on 4`-dodecyl¬carboxy-substituted 3-hydroxyflavone, with polyvinylpyrrolidone (PVP) at varying polymer concentrations. Through fluorimetric titration, it was determined that the probe's affinity to the polymer relies on the concentration and morphology of PVP in the solution. At low polymer concentrations, a slight increase in fluorescence intensity and a slight hypsochromic shift were observed, indicating a weak probe-polymer interaction. A significant amplification of the probe signal was noticed in high concentration PVP solutions, suggesting the probe's penetration into the hydrophobic environment of the polymer matrix. It was observed that at high concentrations, PVP forms a dense spatial structure, increasing the probe's affinity to the polymer. Molecular docking calculations were utilized to investigate the molecular aspects of the probe-polymer interaction. The binding interactions of the probe ФЛ-С11СООН with PVP macromolecules of different sizes and spatial structures were studied. It was found that the spatial structure of the polymer plays a crucial role in the probe binding. The molecular docking calculations indicated that the probe exhibited weak binding (-4.5 kcal/mol) to an unfolded conformation of PVP100. Conversely, certain hydrophobic sites in a compact, globular matrix of PVP1440 were able to accommodate the probe ФЛ-С11СООН with a high binding affinity of -7.3 kcal/mol. The long-alkyl chain of the probe anchor within at PVP matrix. In summary, it was demonstrated that long-chain fatty acid-functionalized 3-hydroxyflavone derivatives have great potential as environment-sensitive fluorescent probes for studying the microscopic structure and morphology of synthetic polymers, macromolecules, and organized solutions.

CETZOLE Analogs as Potent Ferroptosis Inducers and Their Target Identification Using Covalent/Affinity Probes.

Ferroptosis is a recently discovered cell death mechanism triggered by iron-dependent elevation of reactive oxygen species leading to lipid membrane peroxidation. We previously reported the development of a new class of ferroptosis inducers referred to as CETZOLEs with CC50 values in the low micromolar range. Structure-activity relationship study of these compounds led to the development of more potent analogs with CC50 values in the nanomolar range. Cells exposed to these compounds displayed the hallmarks of ferroptosis including cell death through ROS accumulation. Cancer cells were found to be more sensitive to these compounds than normal cells. Proteomic studies using covalent and affinity probes led to the identification of cystathionine β-synthase, peroxiredoxins, ADP/ATP carriers, and glucose dehydrogenase as enriched proteins. The binding of CETZOLEs to these proteins as well as GPX4 was validated by Western blotting. This group of proteins is known to be associated with cellular antioxidant pathways.

Claudin18.2-Targeted SPECT/CT Imaging for Gastric Cancer: Preclinical Evaluation and Clinical Translation of the 99mTc-Labeled Nanobody (PHG102) Radiotracer.

Claudin18.2 (CLDN18.2) has emerged as a significant target in the treatment of advanced gastric cancer. The screening of patients positive for CLDN18.2 is crucial for the effective application of targeted therapies specific to CLND18.2. In this study, we developed a novel nanobody-based probe, [99mTc]Tc-PHG102, for use in nuclear medicine. We analyzed its radiochemical yield and stability to ensure accurate probe characterization. Additionally, we assessed the probe's affinity and specificity toward the CLDN18.2 target and evaluated its efficacy in the BGC82318.2 xenograft model for SPECT/CT imaging of gastric cancer. The binding of [99mTc]Tc-PHG102 to HEK-293T18.2 and BGC82318.2 cells was notably higher than its binding to HEK-293T18.1, HEK-293T, and BGC823 cells, with bound values of 12.87 ± 1.46%, 6.16 ± 0.34%, 1.25 ± 0.22%, 1.14 ± 0.26%, and 1.32 ± 0.07% AD, respectively. The binding ability of [99mTc]Tc-PHG102 was significantly different between CLDN18.2-positive and negative cells (P < 0.001). Imaging results demonstrated a time-dependent tumor accumulation of the radiotracer. Notably, at 0.5 h postinjection, rapid accumulation was observed with an average tumor uptake of 4.63 ± 0.81% ID/cc (n = 3), resulting in clear tumor visualization. By 1 h postinjection, as [99mTc]Tc-PHG102 was rapidly metabolized, a decrease in uptake by other organs was noted. Preliminary clinical imaging trials further confirmed the safety and effectiveness of the probe, indicating specificity for lesions expressing CLDN18.2 in gastric cancer and favorable in vivo metabolic properties. In conclusion, the nanobody-based probe [99mTc]Tc-PHG102 proves to be a safe and effective tool for detecting CLDN18.2 expression levels in gastric cancer tumors and for screening CLDN18.2-positive patients.

Versatility and stability of melamine foam-based biosensors (f-ELISA) using antibodies, nanobodies, and peptides as sensing probes

Macroporous three-dimensional (3D) framework structured melamine foam-based Enzyme-Linked Immunosorbent Assay (f-ELISA) biosensors were developed for rapid, reliable, sensitive, and on-site detection of trace amount of biomolecules and chemicals. Various ligands can be chemically immobilized onto the melamine foam, which brings in the possibility of working with antibodies, nanobodies, and peptides, respectively, as affinity probes for f-ELISA biosensors with improved stability. Different chemical reagents can be used to modify the foam materials, resulting in varied reactivities with antibodies, nanobodies, and peptides. As a result, the f-ELISA sensors produced from these modified foams exhibit varying levels of sensitivity and performance. This study demonstrated that the chemical reagents used for immobilizing antibodies, nanobodies, and peptides could affect the sensitivities of the f-ELISA sensors, and their storage stabilities under different temperatures varied depending on the sensing probes used, with f-ELISA sensors employing nanobodies as probes exhibiting the highest stability. This study not only showcases the versatility of the f-ELISA system but also opens new avenues for developing cost-effective, portable, and user-friendly diagnostic tools with optimized sensitivity and stability.

Dual-Probe Activity-Based Protein Profiling Reveals Site-Specific Differences in Protein Binding of EGFR-Directed Drugs.

Comparative, dose-dependent analysis of interactions between small molecule drugs and their targets, as well as off-target interactions, in complex proteomes is crucial for selecting optimal drug candidates. The affinity of small molecules for targeted proteins is largely dictated by interactions between amino acid side chains and these drugs. Thus, studying drug-protein interactions at an amino acid resolution provides a comprehensive understanding of the drug selectivity and efficacy. In this study, we further refined the site-specific activity-based protein profiling strategy (ABPP), PhosID-ABPP, on a timsTOF HT mass spectrometer. This refinement enables dual dose-dependent competition of inhibitors within a single cellular proteome. Here, a comparative analysis of two activity-based probes (ABPs), developed to selectively target the epidermal growth factor receptor (EGFR), namely, PF-06672131 (PF131) and PF-6422899 (PF899), facilitated the simultaneous identification of ABP-specific binding sites at a proteome-wide scale within a cellular proteome. Dose-dependent probe-binding preferences for proteinaceous cysteines, even at low nanomolar ABP concentrations, could be revealed. Notably, in addition to the intrinsic affinity of the electrophilic probes for specific sites in targeted proteins, the observed labeling intensity is influenced by several other factors. These include the efficiency of cellular uptake, the stability of the probes, and their intracellular distribution. While both ABPs showed comparable labeling efficiency for EGFR, PF131 had a broader off-target reactivity profile. In contrast, PF899 exhibited a higher labeling efficiency for the ERBB2 receptor and bound to catalytic cysteines in several other enzymes, which is likely to disrupt their catalytic activity. Notably, PF131 effectively labeled ADP/ATP translocase proteins at a concentration of just 1 nm, and we found this affected ATP transport. Analysis of the effect of PF131 and its parent inhibitor Afatinib on murine translocase SLC25A4 (ANT1)-mediated ATP transport strongly indicated that PF131 (10 μM) partially blocked ATP transport. Afatinib was less efficient at inhibiting ATP transport by SLC25A4 than PF131, and the reduction of ATP transport by Afatinib was not significant. Follow-up analysis is required to evaluate the affinity of these inhibitors for ADP/ATP translocase SLC25A4 in more detail. Additionally, the analysis of different binding sites within the EGF receptor and the voltage-dependent anion channel 2 revealed secondary binding sites of both probes and provided insights into the binding poses of inhibitors on these proteins. Insights from the PhosID-ABPP analysis of these two ABPs serve as a valuable resource for understanding drug on- and off-target engagement in a dose- and site-specific manner.

Addressing Heterogeneity in Direct Analysis of Extracellular Vesicles and Their Analogs by Membrane Sensing Peptides as Pan-Vesicular Affinity Probes.

Extracellular vesicles (EVs), crucial mediators of cell-to-cell communication, hold significant diagnostic potential due to their ability to concentrate protein biomarkers in bodily fluids. However, challenges in isolating EVs from biological specimens hinder their widespread use. The preferred strategy involves direct analysis, integrating isolation and analysis solutions, with immunoaffinity methods currently dominating. Yet, the heterogeneous nature of EVs poses challenges, as proposed markers may not be as universally present as thought, raising concerns about biomarker screening reliability. This issue extends to EV-mimics, where conventional methods may lack applicability. Addressing these challenges, the study reports on Membrane Sensing Peptides (MSP) as pan-vesicular affinity ligands for both EVs and their non-canonical analogs, streamlining capture and phenotyping through Single Molecule Array (SiMoA). MSP ligands enable direct analysis of circulating EVs, eliminating the need for prior isolation. Demonstrating clinical translation, MSP technology detects an EV-associated epitope signature in serum and plasma, distinguishing myocardial infarction from stable angina. Additionally, MSP allow analysis of tetraspanin-lacking Red Blood Cell-derived EVs, overcoming limitations associated with antibody-based methods. Overall, the work underlines the value of MSP as complementary tools to antibodies, advancing EV analysis for clinical diagnostics and beyond, and marking the first-ever peptide-based application in SiMoA technology.

Synthesis and characterization of 64Cu-labeled Geldanamycin derivative for imaging HSP90 expression in breast cancer

Heat shock protein 90 (HSP90) plays a crucial role in cancer cell growth and metastasis by stabilizing overexpressed signaling proteins. Inhibiting HSP90 has emerged as a promising anti-cancer strategy. In this study, we aimed to develop and characterize a HSP90-targeted molecular imaging probe, [64Cu]Cu-DOTA-BDA-GM, based on a specific HSP90 inhibitor, geldanamycin (GM), for PET imaging of cancers. GM is modified at the C-17 position with 1,4-butane-diamine (BDA) and linked to 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) for 64Cu radiolabeling. We evaluated the probe's specific binding to HSP90-expressing cells using Chinese hamster ovary (CHO) cells and breast cancer cells including MDA-MB-231, MDA-MB-435S, MCF7, and KR-BR-3 cell lines. A competition study with non-radioactive GM-BDA yielded an IC50 value of 1.35 ± 0.14 nM, underscoring the probe's affinity for HSP90. In xenograft models of MDA-MB-231 breast cancer, [64Cu]Cu-DOTA-BDA-GM showcased targeted tumor localization, with significant radioactivity observed up to 18 h post-injection. Blocking studies using unlabeled GM-BDA and treatment with the anticancer drug Vorinostat (SAHA), which can affect the expression and activity of numerous proteins, such as HSPs, confirmed the specificity and sensitivity of the probe in cancer targeting. Additionally, PET/CT imaging in a lung metastasis mouse model revealed increased lung uptake of [64Cu]Cu-DOTA-BDA-GM in metastatic sites, significantly higher than in non-metastatic lungs, illustrating the probe's ability to detect metastatic breast cancer. In conclusion, [64Cu]Cu-DOTA-BDA-GM represents a sensitive and specific approach for identifying HSP90 expression in breast cancer and metastases, offering promising implications for clinical diagnosis and monitoring.

Developing new Hydrazine Carbothioamide Molecules for Selective Colorimetric/Fluorometric Detection of Environmentally Essential Hg2+ and Ag+ Metal Ions in Mixed Aqueous Media.

A novel colorimetric and fluorogenic probe L based on hydrazine carbothioamide and 1,8-naphthalimide moieties has been designed and synthesized for the hypersensitive detection of Hg2+ or Ag+ ions. The observed probe L showed colorimetric and fluorometric responses for these studies when Hg2+ or Ag+ was added to the DMSO - HEPES buffer solution (pH = 7). An interference test with other metal ions was determined, and the high selectivity of Hg2+ and Ag+ did not interfere with other metal ions in colorimetric and fluorogenic methods. The possible mechanism of binding of these metal ions and the probe L 1:1 complex was determined by H1 NMR. Additionally, the reversibility of the affinity of probe L with mercury (Hg2+) and silver (Ag+) ions was investigated by adding Na2EDTA. The naked eye detected the "Off-On" type fluorescence sensor in the presence of Hg2+ and EDTA. The tested test strip kits provided a strong probability of probe L with high response and rapid, sensitive detection with Hg2+ ion, which may be suitable for practical use.

Distinguishing methicillin-resistant Staphylococcus aureus from methicillin-sensitive strains by combining Fe3O4 magnetic nanoparticle-based affinity mass spectrometry with a machine learning strategy

Pathogenic bacteria, including drug-resistant variants such as methicillin-resistant Staphylococcus aureus (MRSA), can cause severe infections in the human body. Early detection of MRSA is essential for clinical diagnosis and proper treatment, considering the distinct therapeutic strategies for methicillin-sensitive S. aureus (MSSA) and MRSA infections. However, the similarities between MRSA and MSSA properties present a challenge in promptly and accurately distinguishing between them. This work introduces an approach to differentiate MRSA from MSSA utilizing matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) in conjunction with a neural network-based classification model. Four distinct strains of S. aureus were utilized, comprising three MSSA strains and one MRSA strain. The classification accuracy of our model ranges from ~ 92 to ~ 97% for each strain. We used deep SHapley Additive exPlanations to reveal the unique feature peaks for each bacterial strain. Furthermore, Fe3O4 MNPs were used as affinity probes for sample enrichment to eliminate the overnight culture and reduce the time in sample preparation. The limit of detection of the MNP-based affinity approach toward S. aureus combined with our machine learning strategy was as low as ~ 8 × 103 CFU mL−1. The feasibility of using the current approach for the identification of S. aureus in juice samples was also demonstrated.Graphical

Colabind: A Cloud-Based Approach for Prediction of Binding Sites Using Coarse-Grained Simulations with Molecular Probes.

Binding site prediction is a crucial step in understanding protein-ligand and protein-protein interactions (PPIs) with broad implications in drug discovery and bioinformatics. This study introduces Colabind, a robust, versatile, and user-friendly cloud-based approach that employs coarse-grained molecular dynamics simulations in the presence of molecular probes, mimicking fragments of drug-like compounds. Our method has demonstrated high effectiveness when validated across a diverse range of biological targets spanning various protein classes, successfully identifying orthosteric binding sites, as well as known druggable allosteric or PPI sites, in both experimentally determined and AI-predicted protein structures, consistently placing them among the top-ranked sites. Furthermore, we suggest that careful inspection of the identified regions with a high affinity for specific probes can provide valuable insights for the development of pharmacophore hypotheses. The approach is available at https://github.com/porekhov/CG_probeMD.

Chiral Discrimination of Acyclic Secondary Amines by 19F NMR.

Chiral aliphatic amine compounds exhibit a range of physiological activities, making them highly sought-after in the pharmaceutical industry and biological research. One notable obstacle in studying these compounds stems from the pronounced steric hindrance surrounding the nitrogen atom. This characteristic often leads to a weak affinity of acyclic secondary amines for molecular probes, making their chiral discrimination intricate. In response to this challenge, our research has unveiled a novel 19F-labeled probe adept at recognizing and distinguishing between enantiomers of these acyclic secondary amines. By strategically incorporating a single fluorine atom as the 19F label, we have managed to diminish the steric hindrance at the binding site. This alteration bolsters the probe's affinity toward bulkier analytes. As a testament to its effectiveness, we have successfully employed our probe in the chiral analysis of relevant pharmaceuticals, accurately determining their enantiocomposition.

A fluorescent "Turn-ON" probe with rapid and differential response to HSA and BSA: quantitative detection of HSA in urine.

The present study provides insight into the differential response of a benzimidazole-malononitrile fluorescent "Turn-ON" probe on interaction with two structurally similar proteins, BSA and HSA. Compound 6 shows more sensitivity towards the two SAs, which is completely lost in the case of compound 7, synthesized by substitution on 6. The aggregates of compound 6 show absorption maxima at 385 nm and weak emission maxima at 565 nm. Compound 6 forms a new emission band at 475 nm on gradual addition of BSA (200 μM) along with a slight increase in the emission band at 565 nm. However, on addition of HSA (50 μM), a new band at 475 nm is formed. In contrast to BSA, in the case of HSA, 50% quenching is observed in the emission band of compound 6 at 565 nm. The new band formed on the interaction of 6 with BSA shows four-fold more enhancement compared to HSA. Furthermore, the mechanism of interaction of 6 with serum albumin has been investigated through lifetime-fluorescence analysis, site-selective drug experiments, dynamic light scattering, FE-SEM, FT-IR, etc. Molecular docking studies and site marker drug displacement experiments reveal differential interactions of 6 towards the two structurally similar proteins. Aggregates of 6 with an average hydrodynamic size of 100-190 nm are disassembled on adding BSA and HSA, and the size of the serum albumin and 6 complex decreases to 10-20 nm, revealing the ligand's encapsulation in the serum albumin cavity. Practical applicability for the quantitative detection of HSA in human urine samples is also demonstrated. The high binding affinity, sensitivity, selectivity and differential response of probe 6 towards two serum albumins (HSA and BSA) and significant quantification of HSA in urine samples shows the potential ability of this probe in medical applications.