Simulated complexes formed from a set of postsynaptic proteins suggest a localised effect of a hypomorphic Shank mutation.

BACKGROUND: The postsynaptic density is an elaborate protein network beneath the postsynaptic membrane involved in the molecular processes underlying learning and memory. The postsynaptic density is built up from the same major proteins but its exact composition and organization differs between synapses. Mutations perturbing protein: protein interactions generally occurring in this network might lead to effects specific for cell types or processes, the understanding of which can be especially challenging. RESULTS: In this work we use systems biology-based modeling of protein complex distributions in a simplified set of major postsynaptic proteins to investigate the effect of a hypomorphic Shank mutation perturbing a single well-defined interaction. We use data sets with widely variable abundances of the constituent proteins. Our results suggest that the effect of the mutation is heavily dependent on the overall availability of all the protein components of the whole network and no trivial correspondence between the expression level of the directly affected proteins and overall complex distribution can be observed. CONCLUSIONS: Our results stress the importance of context-dependent interpretation of mutations. Even the weakening of a generally occurring protein: protein interaction might have well-defined effects, and these can not easily be predicted based only on the abundance of the proteins directly affected. Our results provide insight on how cell-specific effects can be exerted by a mutation perturbing a generally occurring interaction even when the wider interaction network is largely similar.

Nitrilotriacetic Acid Functionalized Microgels for Efficient Immobilization of Hyaluronan Synthase.

Enzymes play a vital role in synthesizing complex biological molecules like hyaluronic acid (HA). Immobilizing enzymes on support materials is essential for their efficient use and reuse in multiple cycles. Microgels, composed of cross-linked, highly swollen polymer networks, are ideal for enzyme uptake owing to their high porosity. This study demonstrates the immobilization of His6-tagged hyaluronan synthase from Pasteurella multocida (PmHAS) onto nitrilotriacetic acid functionalized microgels using different bivalent ions (Ni2+, Co2+, Mn2+, Mg2+, and Fe2+) via metal affinity binding. The results indicate that using Ni2+ yields the microgels with the highest enzyme uptake and HA formation. The immobilized PmHAS enables repetitive enzymatic production, producing high molecular weight HAs with decreasing dispersities in each step. Furthermore, the highest reported yield of HA with high molecular weight for immobilized PmHAS is achieved. This system establishes a foundation for continuous HA formation, with future works potentially enhancing PmHAS stability through protein engineering.

Berberine analog of chloramphenicol exhibits a distinct mode of action and unveils ribosome plasticity.

Chloramphenicol (CHL) is an antibiotic targeting the peptidyl transferase center in bacterial ribosomes. We synthesized a new analog, CAM-BER, by substituting the dichloroacetyl moiety of CHL with a positively charged aromatic berberine group. CAM-BER suppresses bacterial cell growth, inhibits protein synthesis in vitro, and binds tightly to the 70S ribosome. Crystal structure analysis reveals that the bulky berberine group folds into the P site of the peptidyl transferase center (PTC), where it competes with the formyl-methionine residue of the initiator tRNA. Our toe-printing data confirm that CAM-BER acts as a translation initiation inhibitor in stark contrast to CHL, a translation elongation inhibitor. Moreover, CAM-BER induces a distinct rearrangement of conformationally restrained nucleotide A2059, suggesting that the 23S rRNA plasticity is significantly higher than previously thought. CAM-BER shows potential in avoiding CHL resistance and presents opportunities for developing novel berberine derivatives of CHL through medicinal chemistry exploration.

Single-Step Purification of Catalase Enzyme From Human Blood Erythrocytes Using Affinity Chromatography Technique.

In this study, we aimed to isolate and purify catalase from human blood erythrocytes by using a newly synthesized affinity gel. The synthesized ω-amino hexyl agarose-1,2,3-triazole-5-carboxylic acid affinity gel was analyzed by FT-IR. Then, different buffer, pH, and ionic strength parameters were optimized to determine the equilibration, washing, and elution buffer conditions. The catalase was purified from human blood erythrocytes with a specific activity of 45.58 EU/mg, purification fold of 529.50, and a yield of 0.416% using the synthesized new affinity gel. The purity and molecular weight of the enzyme were analyzed by SDS-PAGE, and a single band at 60 kDa was observed for catalase. The optimum reaction temperature of the catalase was found to be 30°C, while the thermal stability temperature was 60°C. The Km and Vmax of the enzyme for hydrogen peroxide were calculated at 0.125 mM and 2500 U mL-1, respectively.

Improving assay feasibility and biocompatibility of 3D cyclic olefin copolymer microwells by superhydrophilic modification via ultrasonic spray deposition of polyvinyl alcohol.

Sample partitioning is a crucial step towards digitization of biological assays on polymer microfluidic platforms. However, effective liquid filling into microwells and long-term hydrophilicity remain a challenge in polymeric microfluidic devices, impeding the applicability in diagnostic and cell culture studies. To overcome this, a method to produce permanent superhydrophilic 3-dimensional microwells using cyclic olefin copolymer (COC) microfluidic chips is presented. The COC substrate is oxidized using UV treatment followed by ultrasonic spray coating of polyvinyl alcohol solution, offering uniform and long-term coating of high-aspect ratio microfeatures. The coated COC surfaces are UV-cured before bonding with a hydrophobic pressure-sensitive adhesive to drive selective filling into the wells. The surface hydrophilicity achieved using this method remains unchanged (water contact angle of 9°) for up to 6 months and the modified surface is characterized for physical (contact angle & surface energy, morphology, integrity of microfeatures and roughness), chemical composition (FTIR, Raman spectroscopy) and coating stability (pH, temperature, time). To establish the feasibility of the modified surface in biological applications, PVA-coated COC microfluidic chips are tested for DNA sensing (digital LAMP detection of CMV), and biocompatibility through protein adsorption and cell culture studies (cell adhesion, viability, and metabolic activity). Kidney and breast cells remained viable for the duration of testing (7 days) on this modified surface, and the coating did not affect the protein content, morphology or quality of the cultured cells. The ultrasonic spray coated system, coating with 0.25 % PVA for 15 cycles with 0.12 A current after UV oxidation, increased the surface energy of the COC (naturally hydrophobic) from 22.04 to 112.89 mJ/m2 and improved the filling efficiency from 40 % (native untreated COC) to 94 % in the microwells without interfering with the biocompatibility of the surface, proving to be an efficient, high-throughput and scalable method of microfluidic surface treatment for diagnostic and cell growth applications.

A Single-step Generation of AlissAID-based Conditional Knockdown Strains Using Nanobody that Targets GFP or mCherry in Budding Yeast.

The Auxin-inducible degron (AID) system is a genetic tool that induces rapid target protein depletion in an auxin-dependent manner. Recently, two advanced AID systems-the super-sensitive AID and AID 2-were developed using an improved pair of synthetic auxins and mutated TIR1 proteins. In these AID systems, a nanomolar concentration of synthetic auxins is sufficient as a degradation inducer for target proteins. However, despite these advancements, AID systems still require the fusion of an AID tag to the target protein for degradation, potentially affecting its function and stability. To address this limitation, we developed an affinity linker-based super-sensitive AID (AlissAID) system using a single peptide antibody known as a nanobody. In this system, the degradation of GFP- or mCherry-tagged target proteins is induced in a synthetic auxin (5-Ad-IAA)-dependent manner. Here, we introduce a simple method for generating AlissAID strains targeting GFP or mCherry fusion proteins in budding yeasts. Key features • AlissAID system enables efficient degradation of the GFP or mCherry fusion proteins in a 5-Ad-IAA-depending manner. • Transforming the pAlissAID plasmids into strains with GFP- or mCherry- tagged proteins.

Recombinant AAV Purification.

Purification of rAAV is a crucial unit operation of the AAV production process. It enables the capture of AAV and removal of contaminants such as host cell proteins, host cell DNA, and other cell culture-related impurities. Here we describe the purification of rAAV produced in insect cells Sf9/rBEV by immuno-affinity capture chromatography. The method is fully scale-amenable unlike other traditional purification methods based on ultracentrifugation. The method reported herein has two main steps: (1) the clarification of cell lysate by depth filtration and (2) the selective capture and single-step purification of AAV via immune-affinity chromatography. This purification method has been successfully implemented to purify the majority of wild-type AAV serotypes.

Design and characterization of neutral linker-based bis-intercalator via computer simulations: Balancing DNA binding and cellular uptake.

Bis-intercalators play a significant role in altering the DNA structure, affecting its stability, and potentially influencing various cellular processes. These compounds have gained considerable attention in medicinal chemistry and biochemistry due to their potential applications in cancer therapy, where they may interfere with DNA replication and transcription, leading to anticancer effects. Traditionally, these molecules often possess a high positive charge to enhance their affinity for the negatively charged DNA. However, due to a high positive charge, their cellular uptake is compromised, along with their enhanced potential off-target effects. In this study, we utilized bis-intercalator TOTO and replaced the charged linker segment (propane-1,3-diammonium) with a neutral peroxodisulphuric acid linker. Using molecular modeling and computer simulations (500 ns, 3 replicas), we investigated the potential of the designed molecule as a bis-intercalator and compared the properties with the control bis-intercalator bound to DNA. We observed that the designed bis-intercalator exhibited improved DNA binding (as assessed through MM-PBSA and Delphi methods) and membrane translocation permeability. With an overall reduced charge, significantly less off-target binding of the designed molecule is also anticipated. Consequently, bis-intercalators based on peroxodisulphuric linkers can potentially target DNA effectively, and their role in the future design of bis-intercalators is foreseen.

Design and synthesis of novel site-specific antibody-drug conjugates that target TROP2.

The approval of Trodelvy® validates TROP2 as a druggable but challenging target for antibody-drug conjugates (ADCs) to treat metastatic triple-negative breast cancer (mTNBC). Here, based on the TROP2-targeted antibody sacituzumab, we designed and developed several site-specific ADC candidates, which employ MMAE (monomethyl auristatin E) as the toxin, via IgG glycoengineering or affinity-directed traceless conjugation. Systematic evaluation of these site-specific ADCs in homogeneity, hydrophilicity, stability, and antitumor efficiency was conducted. The results indicate that the site-specific ADCs gsADC 3b made from one-step glycoengineering exhibit good aggregation stability and in vivo efficacy, providing a new format of ADCs that target TROP2.

Bivalent affinity binding-inspired PPARγ immobilization with selective conformation and improved ligand-binding activity.

Functional protein immobilization forms the basis for bio-detections. A series of one-point, site-specific immobilization methods have been developed, however, it still remains as a challenge how to avoid the proteins to move in all directions as well as conveniently regenerate the bio-devices. Herein, we have developed a bivalent affinity binding-inspired method for PPARγ immobilization using DNA aptamer and nickel-nitrilotriacetic acid (Ni2+-NTA) chelation. The specific DNA aptamer (Apt 2) was selected by an on-column systematic evolution of ligands by exponential enrichment (SELEX) method with affinity of (1.57 ± 0.15) × 105 M-1, determined by isothermal titration calorimetry (ITC). Apt 2 and nickel-nitrilotriacetic acid (Ni2+-NTA) were modified on macroporous silica gels via L-α-allylglycine as a linker. They respectively interacted with PPARγ and 6×His tag via bivalent affinity binding for the receptor immobilization. After comprehensive surface characterization, PPARγ was proved to be successful immobilized. Chromatographic studies revealed that the immobilized PPARγ has conformation selectivity, which discriminated agonist and antagonist of the receptor. Ligand-binding parameters (affinity and rate constant) of four agonists (rosiglitazone, pioglitazone, troglitazone, and magnolol) with PPARγ were determined. Troglitazone showed the lowest dissociation rate constant. The binding affinities (3.28 × 107, 1.91 × 106, 2.25 × 107, and 2.43 × 107 M-1) were highly consistent with the data obtained using purified receptor in solution (2.16 × 107, 4.52 × 106, 1.20 × 107, and 1.56 × 107 M-1), offering reliable bio-detection method for PPARγ and its ligands. Due to the biocompatibility of nuclear receptor with DNA, it is conceivable that the bivalent affinity-based method will be a general method for the immobilization of other nuclear receptors, which may provide selective conformation and improved ligand-binding activity for the receptors.

Software for Predicting Binding Free Energy of Protein-Protein Complexes and Their Mutants.

Protein-protein binding affinity prediction is important for understanding complex biochemical pathways and to uncover protein interaction networks. Quantitative estimation of the binding affinity changes caused by mutations can provide critical information for protein function annotation and genetic disease diagnoses. The binding free energies of protein-protein complexes can be predicted using several computational tools. This chapter is a summary of software developed for the prediction of binding free energies for protein-protein complexes and their mutants.

Development and validation of extraction and clean-up procedures for UPLC-MS/MS analysis of aflatoxins in spices.

UPLC-MS/MS analytical conditions for the analysis of aflatoxins in spices were optimized and validated in this study. Liquid-liquid partition-based protocols for the cleaning up of extracts using common organic solvents such as acetonitrile, hexane, and ethyl acetate were developed and validated. The developed liquid-liquid partition methods were compared with immuno-affinity column and QuEChERS clean-up methods for the UPLC-MS/MS analysis of aflatoxins in 8 spices. The reduction of lipophilic components using the partition with hexane is particularly useful in spices like red pepper that have higher levels of fatty acids, carotenoids, sterols, triterpenoids, etc. The subsequent partitioning with ethyl acetate considerably reduced the matrix interference from the polar components and increased the sensitivity. The cleaning up of spice extracts using liquid-liquid partition techniques resulted in limits of quantification (LOQ) of 2-5 µgL-1 in UPLC-MS/MS analysis. Trueness, repeatability, and reproducibility of the methods were in acceptable ranges. The accuracy of the developed methods was further verified by analyzing aflatoxins in naturally incurred samples of spices and comparing the results with those obtained from the immuno-affinity column cleanup-HPLC-FD method.

A deep attention model for wide-genome protein-peptide binding affinity prediction at a sequence level.

Peptides are pivotal in numerous biological activities by engaging in up to 40 % of protein-protein interactions in many cellular processes. Due to their exceptional specificity and effectiveness, peptides have emerged as promising candidates for drug design. However, accurately predicting protein-peptide binding affinity remains a challenging. Aiming at the problem, we develop a prediction model PepPAP based on convolutional neural network and multi-head attention, which relies solely on sequence features. These features include physicochemical properties, intrinsic disorder, sequence encoding, and especially interface propensity which is extracted from 16,689 non-redundant protein-peptide complexes. Notably, the adopted regression stratification cross-validation scheme proposed in our previous work is beneficial to improve the prediction for the cases with extreme binding affinity values. On three benchmark test datasets: T100, a series of peptides targeting to PDZ domain and CXCR4, PepPAP shows excellent performance, outperforming the existing methods and demonstrating its good generalization ability. Furthermore, PepPAP has good results in binary interaction prediction, and the analysis of the feature space distribution visualization highlights PepPAP's effectiveness. To the best of our knowledge, PepPAP is the first sequence-based deep attention model for wide-genome protein-peptide binding affinity prediction, and holds the potential to offer valuable insights for the peptide-based drug design.

Affinity Chromatography for Anti-Glucosylated Adhesin Antibody Purification: Depletion of Nonspecific Anti-Protein Antibodies and Antibody Recovery with Unconventional Elution Solutions.

Antibodies from sera of a multiple sclerosis (MS) patient subpopulation preferentially recognize the hyperglucosylated adhesin protein HMW1ct(Glc) of the pathogen Haemophilus influenzae. This protein is the first example of an N-glucosylated native antigen candidate, potentially triggering pathogenic antibodies in MS. Specific antibodies in patients' sera can be isolated exploiting their biospecific interaction with antigens by affinity chromatography. Herein, the proteins HMW1ct and HMW1ct(Glc) were first immobilized on appropriately functionalized supports and further used to purify antibodies directly from MS patients sera. We describe a protocol to obtain an antibody fraction specifically recognizing the glusosylated residues on the HMW1ct(Glc) adhesin protein depleting antibodies to the unglucosylated HMW1ct sequence. Different elution solutions have been tested to recover the purified antibody fraction, strongly bound to the immobilized HMW1ct(Glc) adhesin protein.

Assessment of the Antigen-Binding Capacity and Separation of Extracellular Vesicles Coated with Antigen-Specific Antibody Light Chains.

Many researchers are interested in the possibility of manipulating the targeting specificity of extracellular vesicles (EVs) for their use as physiological delivery vehicles for drugs and bioactive molecules. Our studies demonstrated the possibility of directing EVs toward the desired acceptor cell by coating them with antigen-specific antibody light chains. Here, we describe the methods for detection of the presence of antibody light chains on the EV surface, proving their ability to specifically bind the antigen and for separating the antigen-binding EV subpopulation.

Detection of the GH analog somatrogon in sports drug testing: Immunological approaches and LC-HRMS/MS.

Due to the presumed lipolytic and anabolic properties, the misuse of human growth hormone (hGH) and its synthetic analogs in sports is prohibited both in- and out-of-competition. Within this research project, the detectability of somatrogon, a recombinant fusion glycoprotein of 22 kDa hGH and the C-terminal peptide (CTP) of the human chorionic gonadotropin (hCG) ß-subunit, with current WADA-approved doping control assays for hGH and hCG was investigated. For that purpose, cross-reactivity tests and a somatrogon administration study were conducted, and only "Kit 2" of the GH isoform differential immunoassays proved applicable to the detection of somatrogon administration in serum. In urine, the immunoassay specific for total hCG yielded presumptively positive findings for several post-administration samples, which can probably be attributed to the presence of an immunoreactive fragment of the hCG ß-subunit. As the detectability of somatrogon with these approaches was found to be limited, a highly specific detection assay (LOD: 10 ng/mL) for the drug in serum samples was developed by using affinity purification with GH receptor (GHR)-conjugated magnetic beads, proteolytic digestion, and liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS). Following optimization, the approach was comprehensively characterized, and authentic post-administration serum samples were successfully analyzed as proof-of-concept, indicating a detection window of at least 96 h. Consequently, the presented method can be employed to confirm the presence of somatrogon in serum samples, where only "Kit 2" of the currently used immunoassay kits yielded an abnormally high Rec/Pit ratio.

MuToN Quantifies Binding Affinity Changes upon Protein Mutations by Geometric Deep Learning.

Assessing changes in protein-protein binding affinity due to mutations helps understanding a wide range of crucial biological processes within cells. Despite significant efforts to create accurate computational models, predicting how mutations affect affinity remains challenging due to the complexity of the biological mechanisms involved. In the present work, a geometric deep learning framework called MuToN is introduced for quantifying protein binding affinity change upon residue mutations. The method, designed with geometric attention networks, is mechanism-aware. It captures changes in the protein binding interfaces of mutated complexes and assesses the allosteric effects of amino acids. Experimental results highlight MuToN's superiority compared to existing methods. Additionally, MuToN's flexibility and effectiveness are illustrated by its precise predictions of binding affinity changes between SARS-CoV-2 variants and the ACE2 complex.

Drug repurposing for obsessive-compulsive disorder using deep learning-based binding affinity prediction models.

Obsessive-compulsive disorder (OCD) is a chronic psychiatric disease in which patients suffer from obsessions compelling them to engage in specific rituals as a temporary measure to alleviate stress. In this study, deep learning-based methods were used to build three models which predict the likelihood of a molecule interacting with three biological targets relevant to OCD, SERT, D2, and NMDA. Then, an ensemble model based on those models was created which underwent external validation on a large drug database using random sampling. Finally, case studies of molecules exhibiting high scores underwent bibliographic validation showcasing that good performance in the ensemble model can indicate connection with OCD pathophysiology, suggesting that it can be used to screen molecule databases for drug-repurposing purposes.

Binding Affinity Determination in Drug Design: Insights from Lock and Key, Induced Fit, Conformational Selection, and Inhibitor Trapping Models.

Binding affinity is a fundamental parameter in drug design, describing the strength of the interaction between a molecule and its target protein. Accurately predicting binding affinity is crucial for the rapid development of novel therapeutics, the prioritization of promising candidates, and the optimization of their properties through rational design strategies. Binding affinity is determined by the mechanism of recognition between proteins and ligands. Various models, including the lock and key, induced fit, and conformational selection, have been proposed to explain this recognition process. However, current computational strategies to predict binding affinity, which are based on these models, have yet to produce satisfactory results. This article explores the connection between binding affinity and these protein-ligand interaction models, highlighting that they offer an incomplete picture of the mechanism governing binding affinity. Specifically, current models primarily center on the binding of the ligand and do not address its dissociation. In this context, the concept of ligand trapping is introduced, which models the mechanisms of dissociation. When combined with the current models, this concept can provide a unified theoretical framework that may allow for the accurate determination of the ligands' binding affinity.

The Immunotherapy of Acute Myeloid Leukemia: A Clinical Point of View.

The potential of the immune system to eradicate leukemic cells has been consistently demonstrated by the Graft vs. Leukemia effect occurring after allo-HSCT and in the context of donor leukocyte infusions. Various immunotherapeutic approaches, ranging from the use of antibodies, antibody-drug conjugates, bispecific T-cell engagers, chimeric antigen receptor (CAR) T-cells, and therapeutic infusions of NK cells, are thus currently being tested with promising, yet conflicting, results. This review will concentrate on various types of immunotherapies in preclinical and clinical development, from the point of view of a clinical hematologist. The most promising therapies for clinical translation are the use of bispecific T-cell engagers and CAR-T cells aimed at lineage-restricted antigens, where overall responses (ORR) ranging from 20 to 40% can be achieved in a small series of heavily pretreated patients affected by refractory or relapsing leukemia. Toxicity consists mainly in the occurrence of cytokine-release syndrome, which is mostly manageable with step-up dosing, the early use of cytokine-blocking agents and corticosteroids, and myelosuppression. Various cytokine-enhanced natural killer products are also being tested, mainly as allogeneic off-the-shelf therapies, with a good tolerability profile and promising results (ORR: 20-37.5% in small trials). The in vivo activation of T lymphocytes and NK cells via the inhibition of their immune checkpoints also yielded interesting, yet limited, results (ORR: 33-59%) but with an increased risk of severe Graft vs. Host disease in transplanted patients. Therefore, there are still several hurdles to overcome before the widespread clinical use of these novel compounds.