Kinetic Assay Research Articles

Kinetic multiplex assay to assess biased signaling of clinical GPCR agonists.

Introduction: Creatinine is a key parameter for evaluating renal function. This study aims to assess the repeatability, reproducibility, measurement uncertainty, and method comparison for creatinine measurement by the Jaffé kinetic method on the Architect ci-2800, relying on three levels of controls (low, medium, high). Materials and Methods: Thirty fresh serum 7.17–74, 6 mg/L range measurements per level were carried out to determine repeatability, and a monitoring period of several days made it possible to evaluate reproducibility. Measurement uncertainty was estimated following the internal quality control + external evaluation of quality. The method comparison in order to estimate bias and the correlation coefficient was assessed by Bland–Altman analysis.. The results were interpreted according to limits set by the reference values (SFBC and RICOS), as well as the manufacturer. Results: Repeatability coefficient of variations (CVs) were 1.13 % (low), 1.05 % (medium), and 0.50 % (high), all below RICOS targets. Intermediate CVs over 30 days were 2.91 %, 2.02 % and 1.75 %. Expanded uncertainty (k = 2) ranged from 6.4 % to 7.2 % (RICOS ≤ 8.2 %). Regression gave y = 1.005 x – 0.365, r = 0.999, with a mean bias of 2.15 %. Conclusion: Creatinine measurement by the Jaffé kinetic method on the Architect ci-2800 shows excellent performance in terms of precision (repeatability, reproducibility, measurement uncertainty) and inter-instrument correlation. Quality criteria are generally satisfied, ensuring reliable use for routine clinical monitoring of renal function.

The current antibiotic arsenal for the treatment of Mycobacterium avium infections is limited and new treatment options are needed. This exploratory study assessed the in vitro activity of omadacycline against M. avium alone and combined with current guideline-recommended antibiotics and the ability of omadacycline to prevent the emergence of antibiotic resistance. The activity of omadacycline and comparator antibiotics against M. avium was studied by minimum inhibitory concentration (MIC) assays and time-kill kinetics assays to investigate time- and concentration-dependent activity patterns. Omadacycline's ability to prevent clarithromycin and amikacin resistance was investigated by phenotypic and molecular assays. The MIC of omadacycline was impacted by medium composition: a higher MIC was found in CAMHB (128 mg/L) versus Middlebrook broth (8 mg/L). MIC values were also impacted by omadacycline instability in solution over time and daily drug supplementation reduced the MIC to 1 mg/L. In time-kill assays, omadacycline demonstrated time- and concentration-dependent activity. Omadacycline-containing drug combinations resulted in varying degrees of enhanced activity, which was most apparent for omadacycline plus amikacin. Omadacycline prevented amikacin MICs from increasing and impeded the emergence of rrs mutations observed with amikacin alone. While enhanced activity of omadacycline plus clarithromycin was less pronounced, rrl mutations were not detected in the presence of omadacycline. Omadacycline demonstrated in vitro activity against M. avium, particularly in combination with amikacin. Emergence of clarithromycin and amikacin resistance seemed to be prevented by co-exposure to omadacycline. Further research is warranted to assess omadacycline's role in treating M. avium disease.

Enzymes are the molecular machines of life and play an indispensable role in numerous biotechnological and biomedical applications. Despite the availability of a few enzyme databases, their utility is often hindered by limitations such as infrequent updates, lack of standardization, and inadequate support for machine learning. In this study, we developed the Open Enzyme Database (OED), a community-wide repository and web-based infrastructure designed to facilitate the sharing and exploration of enzyme data. As a user-friendly platform, the first release of the OED provides extensively curated information on enzyme kinetic parameters, structural data, enzymatic reactions, assay conditions, and functional annotations. It also features cutting-edge artificial intelligence (AI) tools for enzyme property prediction, along with cheminformatics-powered algorithms for the identification of promising enzymes. This database should greatly accelerate research and applications in areas such as biocatalysis, systems biology, synthetic biology, and enzyme engineering, as well as AI. The resource is freely available at https://openenzymedb.platform.moleculemaker.org/.

ABSTRRACT Alpha-glucosidase inhibitors have been considered as the most effective agents in preventing hyperglycaemia and alternative targets for the treatment of Diabetes mellitus (DM). This study aimed to synthesise novel phenyl carbamoyl methoxy thiosemicarbazone derivatives and evaluate their potential as α-glucosidase inhibitors through biochemical assays, cytotoxicity screening, molecular docking, and molecular dynamics simulations. The tested derivatives exhibited a range of inhibitory potential, from moderate to strong as compared to acarbose. Derivative 7e revealed the least IC50 value among the tested compounds. 7e in the kinetic assay acted as a competitive inhibitor of the α-glucosidase. The cytotoxic effect 7e was assessed against the A549 and MDA-MB-453 cell lines. MD simulation revealed that 7e could affect the stability, flexibility, thermodynamics, and structure of α-glucosidase enzymes such as acarbose. Compound 7e demonstrates strong α-glucosidase inhibitory activity with low cytotoxicity in both cell lines, underscoring its potential as a lead candidate for antidiabetic drug development.

This study explored the potential of Raman spectroscopy to holistically monitor the glycolysis pathway kinetics as a function of time through the extracellular medium. Initially, the collinearity of individual metabolites of interest- glucose and lactic acid as a function of their concentration was tested followed by the sensitivity analysis of the approach by elucidating the limits of detection (0.85mM and 2.8mM) and quantification (2.5mM and 9.5mM) for glucose and lactic acid respectively in the biological range. In the process several datamining approaches were also explored. Finally, the A549 cell culture was used for kinetic spectral acquisition of the extracellular medium mimicking the kinetic glycolysis assay as a function of time under different pathway modulations. The spectra were resolved and fitted with a kinetically constrained-model (A→B→C) using the multivariate curve resolution- alternating least squares tool for all the modulated conditions to elucidate the pathway kinetics and the rate of change. The rate of change of the resolved components for the stimulated condition (k1: 0.005min-1, k2: 0.011min-1) was approximately twice as that of the control (k1: 0,045min-1; k2: 0.049min-1) while the inhibited condition (k1: 0.025min-1, k2: 0.017min-1) was substantially slower. The technique is superior to the targeted current gold standard kinetic assay approach, in that it is holistic in nature and has potential applications in drug discovery, bioprocessing, disease diagnostics, etc. Furthermore, this approach overcomes the limitations of the omics/multiomics approaches, limited to a snapshot of cellular metabolism. This study serves as a guideline for future, more complex subcellular kinetic spectroscopy experiments.

KDBI-RP: Kinetic Data of RNA-Protein Interactions Database.

Acetylcholinesterase immobilized on MIL-88B-NH2 MOF for rapid screening inhibitors from herbal medicines: Ligand fishing coupled with mass spectrometry.

β-Lactamases are the leading mechanism of resistance to β-lactam antibiotics in Enterobacterales. Here, we report the detection and characteristics of SED-2, a new member of the Citrobacter sedlakii class A chromosomal β-lactamase SED family. A C. sedlakii isolate recovered during routine screening was subjected to whole genome sequencing. A new variant of SED-1 was identified and named SED-2. blaSED-1 and blaSED-2 were cloned into E. coli DH10β. The donor C. sedlakii and the recombinant Escherichia coli-SED-1 and E. coli-SED-2 were further characterized. Antibiotic susceptibilities were determined using Sensititer. Kinetic assays were performed on purified SED-1 and SED-2. AlphaFold2 was used to predict the structure of SED-2 based on the primary amino acid sequence. blaSED-2 differed from blaSED-1 by six nucleotides, leading to one amino acid substitution (Q274R). Antibiotic susceptibility testing revealed that blaSED-1 and blaSED-2 conferred resistance to first-, second-, and third-generation cephalosporins but not to carbapenems. Kinetic studies of purified enzymes showed that SED-1 and SED-2 hydrolyzed ampicillin and cephalosporins but not meropenem. In comparison to SED-1, SED-2 had higher affinity and increased catalytic activity against the third-generation cephalosporins, especially ceftazidime. Moreover, compared with SED-1, SED-2 had a higher IC50 for clavulanic acid. 3D modeling of SED-2 predicted that the single amino acid substitution affected inhibitor-binding residues and led to a shift that destabilizes the enzyme active site, likely explaining the extended spectrum of activity. Our results highlight the diversity of β-lactamases naturally occurring in Citrobacter spp. We demonstrate here a rare example of a single amino acid substitution that simultaneously expanded the spectrum of β-lactamase activity by increasing affinity towards ceftazidime and reduced susceptibility to the β-lactamase inhibitor clavulanic acid.IMPORTANCEIn this work, we identified and characterized SED-2, a new member of the Citrobacter sedlakii class A chromosomal β-lactamase SED family. While only differing from the SED-1 by a single amino acid, the affinity of SED-2 towards the third-generation cephalosporin ceftazidime was dramatically increased. At the same time, its susceptibility to the β-lactamase inhibitor clavulanic acid was reduced. This is a rare example of a single amino acid substitution, occurring outside of the active site, simultaneously affecting two distinct enzyme functions.

ABSTRACTAimsAza-peptide aldehydes and ketones were developed as a new class of peptidyl analogues to inhibit the human constitutive (c)20S proteasome as alternative therapeutics to treat multiple myeloma (MM).Material and methodsEleven new aza-peptide aldehydes and ketones were designed based on their preference to bind at the ß5 catalytic subunit of c20S proteasome with benzyloxycarbonyl(Cbz)-Leu-Leu-Leu (MG132-like) and morpholinyl(Mp)-Homophenylalanyl(HPh)-Leu-Phe-Leu (Carfilzomib-like) sequences, synthesized, structurally characterized and evaluated for their inhibitory potency in competitive kinetic assays in vitro. Additionally, cell viability assays and molecular modeling experiments were designed and performed in support.ResultsAza-peptide aldehydes and ketones generated inhibitory activity with IC50 values in the µM range when tested at the ß5 catalytic subunit of the human c20S proteasome. Compound 1 was the most potent compound with an IC50 value of 2.3 ± 1.5 µM. When tested for concentration-dependent killing of three multiple myeloma, one leukemic and two normal natural killer (NK) cell lines, two compounds generated mid-µM EC50 values only for the cancer cells after 48 h.ConclusionsOverall, aza-peptide aldehydes and ketones are a new class of selective human c20S proteasome inhibitors with the potential for further development as alternative therapeutics for multiple myeloma.

Background/Objectives: Urinary angiotensin-converting enzyme (uACE) activity has long been regarded as a promising biomarker for kidney and cardiovascular diseases; however, its clinical applicability has been limited by the presence of endogenous urinary inhibitors and technically demanding assay protocols. We aimed to establish a fast and reproducible method for measuring uACE activity to identify the inhibitory compounds responsible for previous assay failures and to define practical preanalytical conditions suitable for routine laboratory implementation. Methods: A fluorescence-based kinetic assay was optimized for urine samples. Endogenous inhibitors were isolated by membrane filtration and chemically characterized, while the effect of sample dilution was evaluated as a simplified alternative for eliminating inhibitory interference. We assessed the stability of ACE activity under various storage conditions to support reliable measurement. Results: Urea (IC50 = 1.18 M), uric acid (IC50 = 3.61 × 10−3 M), and urobilinogen (IC50 = 2.98 × 10−4 M) were identified as the principal reversible inhibitors, jointly accounting for up to 90% suppression of uACE activity. Their inhibitory effect was effectively eliminated by a 128-fold dilution. ACE activity remained stable for 24 h at 25 °C but was completely lost after freezing. A strong positive correlation between uACE activity and creatinine concentration (r = 0.76, p < 0.0001) justified normalization. ACE activity-to-creatinine ratio turned out to be significantly lower in ACE inhibitor-treated patients than in untreated controls (6.49 vs. 36.69 U/mol, p < 0.0001). Conclusions: Our findings demonstrate that accurate measurement of uACE activity is feasible using a rapid dilution-based protocol. The normalized ACE activity can serve as a practical biomarker for detecting pharmacological ACE inhibition and monitoring therapy adherence in cardiovascular care and may also provide insight into renal pathophysiology such as tubular injury or local RAAS-related processes.

Physical stability of an active pharmaceutical ingredient (API) is a key consideration in the development of a pharmaceutical drug. Solution conditions such as pH, excipient concentrations, and storage temperatures can impact the physical stability of a therapeutic peptide in formulation. Optimizing these conditions is a critical activity in achieving a higher stability of a therapeutic peptide product. A Thioflavin T (ThioT) fluorescent reporter assay is widely used to measure the aggregation of peptide products. ThioT kinetic assays are used to predict the propensity of fibril formation by using ThioT curves for a peptide stored in a solution. However, there is no analytical relationship that can be used to relate the physical stability for different formulation conditions, resulting in execution of large-scale stability assays that require significant resources for pharmaceutical companies. Therefore, there is a need to develop new artificial intelligence (AI) methods to predict future ThioT curves in a fast and cost-effective manner. Here, we combined an experimental measure of time-varying conformational states from ThioT assays with AI models to predict peptide aggregation in different formulation conditions during drug development. We formulated the peptide aggregation problem as "language translation" in natural language processing, wherein the sequence of aggregation states at earlier time points was used to predict (or "translate") the aggregation states for future time points. We developed a new sequence-to-sequence long short-term memory (LSTM)-based recurrent neural network (RNN) model to predict entire ThioT curves at future time points (6 and 12 months) using data sets from initial and 1 month ThioT curves for different conditions. We achieved an excellent average mean absolute error (MAE) of 2.04 for the model, which was used to predict and experimentally validate ThioT curves for a 6 month time point. In contrast to the LSTM, the multilayer perceptron (MLP) baseline model showed a higher MAE of 5.17. However, at the 12 month time point, with limited training data, both models achieved comparable results with average MAEs of 4.25 and 4.45 for LSTM and MLP, respectively. Therefore, we conclude that LSTM models can be used to predict future ThioT curves only using the initial and 1 month ThioT curves as input. We believe that the use of recurrent neural network models will benefit the pharmaceutical industry to predict and explore the formulation landscape for future physical stability measurements of APIs based on short-term stability data.

To screen hereditary spherocytosis (HS), we first introduced the novel flow cytometric osmotic fragility test (FC-OFT) based on the two-point kinetic assay principle (FC-OFTKinetic). With the introduction of FC systems with automatic tube loaders, we updated the FC-OFT protocol to follow the endpoint assay principle (FC-OFTEndpoint). This study aims to evaluate the updated FC-OFT protocol (FC-OFTEndpoint) and compare its assay performance with that of FC-OFTKinetic. We investigated factors influencing the FC-OFTEndpoint assay, optimized its protocol, and defined the cutoff index using 152 negative or artificially positive control samples. We then compared the assay performance with that of FC-OFTKinetic in 25 patients with anemia, including 14 with spherocytosis-8, 4, 1, and 1 with HS, autoimmune hemolytic anemia, burn injury, and liver cirrhosis, respectively. To optimize FC-OFTEndpoint, we adopted phosphate-buffered saline as the red cell suspension medium, 50% deionized water for hypotonic osmotic pressure in adults, and a 3-min standby time. This FC-OFTEndpoint was more accurate than FC-OFTKinetic in identifying spherocytosis in the 25 patients with anemia (p = 0.0313). FC-OFTEndpoint is a viable alternative to conventional OFT or FC-OFTKinetic for HS screening in clinical laboratories, as automatic FC enhances assay performance. These findings warrant validation in future multicenter studies with larger sample sizes.

The discovery and synthesis of composite nanomaterials often rely on molecular self-assembly and crystallization, posing significant challenges due to the vast chemical space and the irreproducibility of experimental methods. We present a programmable robotic platform, controlled by the universal Chemical Description Language (χDL), that enables the solid-phase synthesis of composite nanomaterials. In addition to synthesis, the platform validates its catalytic performance through an automated workflow. This platform enables open-ended exploration of composition-morphology-activity relationships, with high accuracy and reproducibility, while also reducing synthesis time and cost. In this study, we are moving beyond the colloidal, plasmonic-focused systems previously explored in robotic platforms to the discovery, synthesis, and catalytic properties of CuO-based nanomaterials, such as CuO-Au and CuO-Ag2O NP heterostructures that show good reproducibility across repeated syntheses. Remarkably, even at very low metal loadings, as confirmed by ICP (Au wt % = 0.06%, Ag wt % = 0.03%), the heterostructures exhibited enhanced photodegradation efficiency of the dye Methyl Green (MG) compared with pristine CuO. The degradation yield increased from 45 ± 2% for pristine CuO to 57 ± 3% for CuO-Au and 65 ± 2% for CuO-Ag2O, as observed through real-time UV-vis spectroscopy. Additionally, a kinetic assay of the synthesis process provided insights into the self-assembly mechanism, highlighting the interactions between the core material (CuO NPs) and the surface coatings (Au or Ag2O). This work demonstrates a shift from traditional manual experimentation to programmable, data-driven workflows, highlighting both the progress and the remaining challenges in the automation of solid-phase nanomaterial synthesis in the field of materials science.

Amyloid-β (Aβ) fibrillation is a spontaneous, thermodynamic process governed by nucleation and elongation. While many studies have explored the ability of engineered nanomaterials (ENMs) to modulate Aβ fibrillation, such as inhibitors, promoters, and dual-modulators, the key physicochemical property of ENMs that determines this behavior remains unclear. In this study, we developed a comprehensive library of ENMs with well-controlled physicochemical properties, including surface charges, morphologies, and hydrophilicity, to systematically investigate their effects on Aβ40 fibrillation. We identified hydrophilicity as the primary determinant of ENM-mediated modulation, rather than surface charge or morphology. Thioflavin T (ThT) kinetics assays indicated that hydrophilic ENMs exhibited bidirectional modulation, both promoting and inhibiting fibrillation depending on concentration. This bidirectional effect results from a competition between accelerated nucleation and decelerated elongation. While hydrophobic ENMs exhibited only unidirectional inhibition from the initial nucleation phase, two-dimensional-NMR (2D-NMR) mechanism studies indicated that this difference resulted from specific interactions with Aβ40 residues. Hydrophilic ENMs targeted hydrophilic residues involved in elongation, including Arginine R5, Glycine G9, G25, G33, G37, and G38, Lysine K28, and Alanine A30, while hydrophobic ENMs bound to hydrophobic residues critical for nucleation, such as I31. These findings provide mechanistic insight into NP-peptide interactions and lay a foundation for the rational design of nanomaterials to modulate amyloid fibrillation.

Molecular characterisation of two novel KPC variants mediating ceftazidime-avibactam resistance in ST11 Klebsiella pneumoniae.

Islatravir (ISL, EFdA) is a nucleoside analog that inhibits HIV-1 reverse transcriptase (RT) translocation during viral replication. Its high potency stems from unique structural features: a 4'-ethynyl group that interacts with the hydrophobic pocket (containing A114, Y115, F160, M184, and D185) in HIV-1 RT, hindering translocation, and a 3'-hydroxyl group that mimics natural nucleosides for efficient incorporation. Recent phase 3 clinical trials, combining ISL with Doravirine (DOR), a non-nucleoside reverse transcriptase inhibitor, show that it is noninferior to existing treatments, offering a unique advantage due to their distinct resistance profiles. For instance, DOR-associated mutations, V106I/F227C, which confer >105-fold DOR resistance in clinics, unexpectedly boost ISL's potency by 2.3-fold in published cell-based resistance selection assays. In contrast, V106I alone does not affect Islatravir's potency, while F227C alone enhances it by 5.6-fold. To kinetically understand these findings, we used presteady-state kinetic assays to determine the kinetic parameters for EFdA 5'-triphosphate (EFdA-TP) and dATP incorporation. We found that the incorporation efficiency of EFdA-TP was 1.4-fold higher than that of dATP on an RNA template and 1.7-fold higher on a DNA template with the F227C mutant. However, this difference was only 1.1- to 1.3-fold higher with the F227C/V106I mutant. Our energy-minimized modeling revealed that these mutations remotely alter the hydrophobic 4'-ethynyl group-binding pocket's structure, surprisingly strengthening the pocket's binding interactions with EFdA-TP. Alongside this, the F227C mutation decreased dATP's binding affinity with both templates. Our data established a kinetic basis for the published cell-based resistance selection assay results, underscoring the significant potential of the ISL/DOR combination therapy in treating HIV-1 infected patients.

The development of synthetically-useful biocatalysts requires characterizing the behavior of an enzyme under conditions amenable to preparative-scale reactions. Whole cells harboring the catalyst of interest are often used in such reactions, as protein purification is laborious and expensive. However, monitoring reaction rates when using whole cells is challenging, as cellular debris precludes the use of a continuous assay. Herein, we describe an approach to continuous monitoring of whole cell reactions that is enabled by the use of an integrating cavity spectrophotometer. This approach was used to directly profile the kinetic behavior of a previously uncharacterized extradiol dioxygenase DfdB from Rhodococcus sp. YK2. Data obtained from these experiments were compared with traditional in vitro assays. In both whole cell and purified enzyme kinetic assays, similar enzymatic behaviors were observed, including substrate inhibition. Measured KM values and observed trends in catalytic efficiency were comparable for both types of enzyme preparation. Information provided by this analysis was leveraged to optimize preparative-scale whole cell reactions with DfdB, enabling the synthesis of two DfdB products in high yield. Importantly, this work showcases the potential for ICS-based methods to rapidly evaluate kinetic trends on analytical scale and thereby enables efficient reaction optimization for preparative-scale synthesis.

Abstract Eco‐friendly antimicrobial ZnO/NC@PVA films were fabricated by incorporating dual nanofillers, nanocrystalline cellulose (NC) and zinc oxide nanoparticles (ZnONPs), prepared from Codiaeum variegatum leaf extract, into a polyvinyl alcohol matrix (PVA) at varying ZnONPs concentrations. Advanced characterization techniques confirmed the successful integration of nanofillers into the PVA matrix. The inclusion of ZnONPs and NC significantly improved the thermal stability and mechanical strength of the films. Among the fabricated films, the ZnO/NC@PVA film containing 5 wt% ZnO displayed the highest light opacity (0.55 ± 0.79 mm−1) and lowest water vapor permeability (13.13 g/m2/day). Antibacterial activity of the developed films, evaluated by disk diffusion and kinetic assays, demonstrated effective inhibition of bacterial growth. Biocompatibility of films was confirmed using MCF‐10A epithelial cells. Furthermore, the performance of ZnO/NC@PVA films in preserving cherry tomatoes demonstrated their suitability for food packaging applications.

Clinical utility of the ATP hydrolysis assay for the diagnosis of complex V deficiency in cultured skin fibroblasts.