Identification of novel allosteric sites of SARS-CoV-2 papain-like protease (PLpro) for the development of COVID-19 antivirals.

Coronaviruses such as SARS-CoV-2 encode a conserved papain-like protease (PLpro) that is crucial for viral replication and immune evasion, making it a prime target for antiviral drug development. In this study, three surface pockets on SARS-CoV-2 PLpro that may function as sites for allosteric inhibition were computationally identified. To evaluate the effects of these pockets on proteolytic activity, 52 residues were separately mutated to alanine. In Pocket 1, located between the Ubl and thumb domains, the introduction of alanine at T10, D12, T54, Y72, or Y83 reduced PLpro activity to <12% of that of WT. In Pocket 2, situated at the interface of the thumb, fingers, and palm domains, Q237A, S239A, H275A, and S278A inactivated PLpro. Finally, introducing alanine at five residues in Pocket 3, between the fingers and palm domains, inactivated PLpro: S212, Y213, Y251, K254, and Y305. Pocket 1 has a higher druggability score than Pockets 2 and 3. MD simulations showed that interactions within and between domains play critical roles in PLpro activity and thermal stability. The essential residues in Pockets 1 and 2 participate in a combination of intra- and inter-domain interactions. By contrast, the essential residues in Pocket three predominantly participate in inter-domain interactions. The most promising targets for therapeutic development are Pockets one and 3, which have the highest druggability score and the largest number of essential residues, respectively. Non-competitive inhibitors targeting these pockets may be antiviral agents against COVID-19 and related coronaviruses.

pH profiles of 3-chymotrypsin-like protease (3CLpro) from SARS-CoV-2 elucidate its catalytic mechanism and a histidine residue critical for activity.

3-Chymotrypsin-like protease (3CLpro) is a promising drug target for coronavirus disease 2019 and related coronavirus diseases because of the essential role of this protease in processing viral polyproteins after infection. Understanding the detailed catalytic mechanism of 3CLpro is essential for designing effective inhibitors of infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Molecular dynamics studies have suggested pH-dependent conformational changes of 3CLpro, but experimental pH profiles of SARS-CoV-2 3CLpro and analyses of the conserved active-site histidine residues have not been reported. In this work, pH-dependence studies of the kinetic parameters of SARS-CoV-2 3CLpro revealed a bell-shaped pH profile with 2 pKa values (6.9 ± 0.1 and 9.4 ± 0.1) attributable to ionization of the catalytic dyad His41 and Cys145, respectively. Our investigation of the roles of conserved active-site histidines showed that different amino acid substitutions of His163 produced inactive enzymes, indicating a key role of His163 in maintaining catalytically active SARS-CoV-2 3CLpro. By contrast, the H164A and H172A mutants retained 75% and 26% of the activity of WT, respectively. The alternative amino acid substitutions H172K and H172R did not recover the enzymatic activity, whereas H172Y restored activity to a level similar to that of the WT enzyme. The pH profiles of H164A, H172A, and H172Y were similar to those of the WT enzyme, with comparable pKa values for the catalytic dyad. Taken together, the experimental data support a general base mechanism of SARS-CoV-2 3CLpro and indicate that the neutral states of the catalytic dyad and active-site histidine residues are required for maximum enzyme activity.

Key Allosteric and Active Site Residues of SARS-CoV-2 3CLpro Are Promising Drug Targets.

The main protease of SARS-CoV-2, 3-chymotrypsin-like protease (3CLpro), is a prominent target for antiviral development due to its essential role in the viral life cycle. Research has largely focused on competitive inhibitors of 3CLpro that target the active site. However, allosteric sites distal to the peptide substrate-binding region are also potential targets for the design of reversible noncompetitive inhibitors. Computational analyses have examined the importance of key contacts at allosteric sites of 3CLpro, but these contacts have not been validated experimentally. In this work, four druggable pockets spanning the surface of SARS-CoV-2 3CLpro were predicted: pocket 1 is the active site, whereas pockets 2, 3, and 4 are located away from the active site at the interface of domains II and III. Site-directed alanine mutagenesis of selected residues with important structural interactions revealed that 7 of 13 active site residues (N28, R40, Y54, S147, Y161, D187 and Q192) and 7 of 12 allosteric site residues (T111, R131, N133, D197, N203, D289 and D295) are essential for maintaining catalytically active and thermodynamically stable 3CLpro. Alanine substitution at these key amino acid residues inactivated or reduced the activity of 3CLpro. In addition, the thermodynamic stability of 3CLpro decreased in the presence of some of these mutations. This work provides experimental validation of essential contacts in the active and allosteric sites of 3CLpro that could be targeted with competitive and noncompetitive inhibitors as new therapeutics against COVID-19.

An Improved In-Motion Coarse Alignment Method for SINS/GPS Integration with Initial Velocity Error Suppression.

The integrated system with the strapdown inertial navigation system (SINS) and the global positioning system (GPS) is the most popular navigation mode. It has been used in many navigation fields. Before the integrated system works properly, it must determine the initial attitude for SINS. In SINS/GPS-integrated systems, the navigational velocity can be used to carry out the initial alignment when the system is installed in the in-motion vehicle. However, the initial velocity errors are not considered in the current popular in-motion alignment methods for SINS/GPS integration. It is well-known that the initial velocity errors must exist when the initial velocity is obtained from the GPS outputs. In this paper, an improved method was proposed to solve this problem. By analyzing the original observation vectors in the in-motion coarse alignment method, an average operation was used to construct the intermediate vectors, and the new observation vector can be calculated by subtracting the intermediate vector from the original observation vector. Then, the initial velocity errors can be eliminated from the newly constructed observation vector. Thus, the interferences of the initial velocity errors for the initial alignment process can be suppressed. The simulation and field tests are designed to verify the performance of the proposed method. The tests results showed that the proposed method can obtain the higher accurate results than the current methods when the initial velocity is considered. Additionally, the results of the proposed method were similar to the current methods when the initial velocity errors were not considered. This shows that the initial velocity errors were eliminated effectively by the proposed method, and the alignment accuracy were not decreased.

Dimethyl sulfoxide reduces the stability but enhances catalytic activity of the main SARS-CoV-2 protease 3CLpro.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for coronavirus disease 2019 (COVID-19), one of the most challenging global pandemics of the modern era. Potential treatment strategies against COVID-19 are yet to be devised. It is crucial that antivirals that interfere with the SARS-CoV-2 life cycle be identified and developed. 3-Chymotrypsin-like protease (3CLpro) is an attractive antiviral drug target against SARS-CoV-2, and coronaviruses in general, because of its role in the processing of viral polyproteins. Inhibitors of 3CLpro activity are screened in enzyme assays before further development of the most promising leads. Dimethyl sulfoxide (DMSO) is a common additive used in such assays and enhances the solubility of assay components. However, it may also potentially affect the stability and efficiency of 3CLpro but, to date, this effect had not been analyzed in detail. Here, we investigated the effect of DMSO on 3CLpro-catalyzed reaction. While DMSO (5%-20%) decreased the optimum temperature of catalysis and thermodynamic stability of 3CLpro, it only marginally affected the kinetic stability of the enzyme. Increasing the DMSO concentration up to 20% improved the catalytic efficiency and peptide-binding affinity of 3CLpro. At such high DMSO concentration, the solubility and stability of peptide substrate were improved because of reduced aggregation. In conclusion, we recommend 20% DMSO as the minimum concentration to be used in screens of 3CLpro inhibitors as lead compounds for the development of antiviral drugs against COVID-19.

Good-Practice Non-Radioactive Assays of Inorganic Pyrophosphatase Activities.

Inorganic pyrophosphatase (PPase) is a ubiquitous enzyme that converts pyrophosphate (PPi) to phosphate and, in this way, controls numerous biosynthetic reactions that produce PPi as a byproduct. PPase activity is generally assayed by measuring the product of the hydrolysis reaction, phosphate. This reaction is reversible, allowing PPi synthesis measurements and making PPase an excellent model enzyme for the study of phosphoanhydride bond formation. Here we summarize our long-time experience in measuring PPase activity and overview three types of the assay that are found most useful for (a) low-substrate continuous monitoring of PPi hydrolysis, (b) continuous and fixed-time measurements of PPi synthesis, and (c) high-throughput procedure for screening purposes. The assays are based on the color reactions between phosphomolybdic acid and triphenylmethane dyes or use a coupled ATP sulfurylase/luciferase enzyme assay. We also provide procedures to estimate initial velocity from the product formation curve and calculate the assay medium's composition, whose components are involved in multiple equilibria.

Resurrecting the phoenix: When an assay fails.

Understanding protein-small-molecule interactions is a critical component of rational drug-design. Structure-activity relationship (SAR)-guided medicinal chemistry is informed by the biological outcome, as assessed by biochemical activity or cellular effect, of chemical modifications on small molecules. The effectiveness of SAR is reliant on the sturdiness and durability of assay design and the quality of information garnered from assays. Lack of quality data at this step can lead to obstruction of the drug discovery pipeline with profound implications for the timelines of introducing a drug into the market. Hence, it would not be an overstatement to consider biochemical/biological assays as the backbone of drug-discovery. Enzyme assays can fail for many different reasons, with the enzyme and the substrate being the principal players. Lack of clarity can hamper progress and can lead to mounting costs and potentially losing competitive advantage. Although each assay is unique and requires a specific approach to troubleshoot the problem at hand, there are general guidelines that can be followed to maximize the chances of success. This review is a step-by-step attempt at reintroducing fundamental biochemical concepts within the context of an enzyme assay, delineating probable causes for failure and potential approaches to get an assay back up and running.

Model for assessment of the velocity and force at the start of sprint race.

A mathematical model was developed for the assessment of the starting velocity and initial velocity and force of a 100-m sprint, based on a non-homogeneous differential equation with the air resistance proportional to the velocity, and the initial conditions for [Formula: see text], [Formula: see text]The use of this model requires the measurement of reaction time and segmental velocities over the course of the race. The model was validated by comparison with the data obtained from 100-m sprints of men: Carl Lewis (1988), Maurice Green (2001) and Usain Bolt (2009), and women: Florence Griffith-Joyner, Evelyn Ashford and Drechsler Heike (1988) showing a high level of agreement. Combined with the previous work of the authors, the present model allows for the assessment of important physical abilities, such as the exertion of a high starting force, development of high starting velocity and, later on, maximisation of the peak running velocity. These data could be of importance for practitioners to identify possible weaknesses and refine training methods for sprinters and other athletes whose performance depend on rapid movement initiations.

Analysis of phosphofructokinase-1 activity as affected by pH and ATP concentration.

A key player in energy metabolism is phosphofructokinase-1 (PFK1) whose activity and behavior strongly influence glycolysis and thus have implications in many areas. In this research, PFK1 assays were performed to convert F6P and ATP into F-1,6-P and ADP for varied pH and ATP concentrations. PFK1 activity was assessed by evaluating F-1,6-P generation velocity in two ways: (1) directly calculating the time slope from the first two or more datapoints of measured product concentration (the initial-velocity method), and (2) by fitting all the datapoints with a differential equation explicitly representing the effects of ATP and pH (the modeling method). Similar general trends of inhibition were shown by both methods, but the former gives only a qualitative picture while the modeling method yields the degree of inhibition because the model can separate the two simultaneous roles of ATP as both a substrate of reaction and an inhibitor of PFK1. Analysis based on the model suggests that the ATP affinity is much greater to the PFK1 catalytic site than to the inhibitory site, but the inhibited ATP-PFK1-ATP complex is much slower than the uninhibited PFK1-ATP complex in product generation, leading to reduced overall reaction velocity when ATP concentration increases. The initial-velocity method is simple and useful for general observation of enzyme activity while the modeling method has advantages in quantifying the inhibition effects and providing insights into the process.

Advanced hybrid attention-based deep learning network with heuristic algorithm for adaptive CT and PET image fusion in lung cancer detection.

Lung cancer is one of the most deadly diseases in the world. Lung cancer detection can save the patient's life. Despite being the best imaging tool in the medical sector, clinicians find it challenging to interpret and detect cancer from Computed Tomography (CT) scan data. One of the most effective ways for the diagnosis of certain malignancies like lung tumours is Positron Emission Tomography (PET) imaging. So many diagnosis models have been implemented nowadays to diagnose various diseases. Early lung cancer identification is very important for predicting the severity level of lung cancer in cancer patients. To explore the effective model, an image fusion-based detection model is proposed for lung cancer detection using an improved heuristic algorithm of the deep learning model. Firstly, the PET and CT images are gathered from the internet. Further, these two collected images are fused for further process by using the Adaptive Dilated Convolution Neural Network (AD-CNN), in which the hyperparameters are tuned by the Modified Initial Velocity-based Capuchin Search Algorithm (MIV-CapSA). Subsequently, the abnormal regions are segmented by influencing the TransUnet3+. Finally, the segmented images are fed into the Hybrid Attention-based Deep Networks (HADN) model, encompassed with Mobilenet and Shufflenet. Therefore, the effectiveness of the novel detection model is analyzed using various metrics compared with traditional approaches. At last, the outcome evinces that it aids in early basic detection to treat the patients effectively.

Inhibitors of the aminoglycoside 6'-N-acetyltransferase type Ib [AAC(6')-Ib] identified by in silico molecular docking.

AAC(6')-Ib is an important aminoglycoside resistance enzyme to target with enzymatic inhibitors. An in silico screening approach was used to identify potential inhibitors from the ChemBridge library. Several compounds were identified, of which two of them, 4-[(2-{[1-(3-methylphenyl)-4,6-dioxo-2-thioxotetrahydro-5(2H)-pyrimidinylidene]methyl}phenoxy)methyl]benzoic acid and 2-{5-[(4,6-dioxo-1,3-diphenyl-2-thioxotetrahydro-5(2H)-pyrimidinylidene)methyl]-2-furyl}benzoic acid, showed micromolar activity in inhibiting acetylation of kanamycin A. These compounds are predicted to bind the aminoglycoside binding site of AAC(6')-Ib and exhibited competitive inhibition against kanamycin A.

A Simulation and an Experimental Study of Space Harpoon Low-Velocity Impact, Anchored Debris.

The space harpoon is a rigid-flexible, coupled debris capture method with a simple, reliable structure and a high adaptability to the target. For the process of impacting and embedding the harpoon into the target plate, the effect of friction at a low-velocity impact is studied, and the criteria for effective embedding of the harpoon and the corresponding launch velocity are determined. A simulation model of the dynamics of the harpoon and the target plate considering tangential friction is established, and the reliability of the numerical simulation model is verified by comparing the impact test, focusing on the kinetic energy change and embedding length during the impact of the harpoon. The results show that the frictional effect in the low-velocity impact is more obvious for the kinetic energy consumption of the harpoon itself, and the effective embedding of the harpoon into the anchored target ranges from 50~90 mm, corresponding to a theoretical launch initial velocity between 88.4~92.5 m/s.

Monoclonal antibody on-rate constant determined from time-course data of ligand binding by capture ELISA: Evaluation of eight data analysis methods.

We describe an ELISA method with which to determine monoclonal antibody (mAb) on-rate constants (k+1) based on time-course data of ligand (L) binding to plate-bound mAb. The assay was performed in pseudo-first order kinetic conditions ([L] > > [mAb]) and at various starting ligand concentrations. Time-course initial velocity was analyzed by several methods to derive the pseudo-first order (kobs) and second order (k+1) association rate constants of the antibody; the methods included i) an exponential first order rate equation, ii) reaction half-time from the Michaelis-Menten relationship, iii) the Vmax/Km tangent of the time-course curve, iv) Boeker's extrapolated-vo method, v-vi) modified Hanes-Woolf and Lineweaver-Burk linear plots, vii) a LOS plot, and viii) initial velocity gradient. Due to k+1 value dispersion associated with the methods of analysis, the on-rate constant of mAb SIM 253-19 anti-cholera toxin was estimated as an average value of 1.79 ± 0.11 × 106 M-1 s-1, 95% CL (1.68-1.89) and 5.8 (%CV [coefficient of variation]), which is similar to the k+1 obtained by surface plasmon resonance, 1.60 ± 0.17 × 106 M-1 s -1 (mean ± half range). This kinetic ELISA is a sensitive, quantitative method by which to determine antibody association rate constants.

A guide to the Michaelis-Menten equation: steady state and beyond.

The modern definition of enzymology is synonymous with the Michaelis-Menten equation instituted by Leonor Michaelis and Maud Menten. Most textbooks, or chapters within, discussing enzymology start with the derivation of the equation under the assumption of rapid equilibrium (as done by Michaelis-Menten) or steady state (as modified by Briggs and Haldane) conditions to highlight the importance of this equation as the bedrock on which interpretation of enzyme kinetic results is dependent. However, few textbooks or monographs take the effort of placing the equation within its right historical context and discuss the assumptions that have gone into its institution. This guide will dwell on these in substantial detail. Further, this guide will attempt to instil a sense of appreciation for the mathematical curve rectangular hyperbola, its unique attributes and how ubiquitous the curve is in biological systems. To conclude, this guide will discuss the limitations of the equation, and the method it embodies, and trace the journey of how investigators are attempting to move beyond the steady-state approach and the Michaelis-Menten equation into full progress curve, pre-steady state and single-turnover kinetic analysis to obtain greater insights into enzyme kinetics and catalysis.

Development of a competitive inhibition kinetic ELISA to determine the inhibition constant (Ki) of monoclonal antibodies.

Antibody-antigen interactions are mediated by the same molecular recognition mechanisms as those of an enzyme and its substrate. On this basis, we developed a competitive inhibition kinetic ELISA to measure monoclonal antibody (mAb) inhibition constants. Serially diluted samples of ligand (mAb) and inhibitor (soluble antigen) were incubated to equilibrium in ELISA plates coated with a fixed concentration of antigen (receptor). Plates were washed, and bound mAb measured with antiglobulin-peroxidase. Initial velocity data of receptor-bound mAb at various ligand and inhibitor concentrations were analyzed with enzyme linear competitive inhibition methods by non-linear regression (NLR), linear transformations (Cornish-Bowden, Lineweaver-Burk, Hanes-Woolf, Dixon, Cortés [1/i0.5 vs. Vi/Vmax], Ascenzi [Ks/Vmax/Ks,0/Vmax vs. [I]]) and NLR IC50 plots, to derive mAb inhibition constants (Ki). We obtained similar mAb Ki and Kd values by ELISA and surface plasmon resonance, which confirmed the accuracy of the ELISA method. This competitive inhibition ELISA is a simple (it requires no labeling or prior knowledge of antibody concentration), sensitive (it detects Ki values in the low nanomolar range by conventional colorimetry), and reproducible method with which to calculate mAb inhibition constants.

External Versus Two Different Internal Foci of Attention in Long-Distance Throwing.

The present study examined the influence of attentional focus on performance during a long-distance throwing task. Twelve participants executed three maximum-effort, long-distance baseball throwing attempts in three focus conditions: internal focus on wrist flexion (wrist internal focus), internal focus on the separation between pelvis and upper torso orientations (torso internal focus), and external focus on the ball path (external focus). Compared with the external focus and torso internal focus conditions, performance was poorer in the wrist internal focus condition. Performances were not different in the torso internal and external focus conditions. In addition, attentional focus affected the release angle of the ball but not its initial velocity. Our results reveal that the body part targeted for internal focus of attention and the forcefulness of the motor activity can be as important to motor performance as whether the attention is internal or external.

Nearly 50 years in the making: defining the catalytic mechanism of the multifunctional enzyme, pyruvate carboxylase.

Numerous steady-state kinetic studies have examined the complex catalytic reaction mechanism of the multifunctional enzyme, pyruvate carboxylase (PC). Through initial velocity, product inhibition, isotopic exchange and alternate substrate experiments, early investigators established that PC catalyzes the MgATP-dependent carboxylation of pyruvate by HCO3 (-) through a nonclassical sequential Bi Bi Uni Uni reaction mechanism. This review surveys previous steady-state kinetic investigations of PC and evaluates the proposed hypotheses concerning the overall catalytic mechanism, nonlinear kinetics and active site coupling in the context of recent structural and mutagenic analyses of this multifunctional enzyme. The determination several PC holoenzyme structures have aided in corroborating the proposed molecular mechanisms by which catalysis occurs and established the inextricable link between the dynamic protein motions and complex kinetic mechanisms associated with PC activity. Unexpectedly, the conclusions drawn from these early steady-state kinetic investigations have consistently proven to be in fundamental agreement with our current understanding of PC catalysis, which is a testament to the overarching sophistication of the methods pioneered by Michaelis and Menten and further developed by Northrop, Cleland and others.

Detection ofα1 antitrypsin activity by chromogenic substrate assay with initial veloci-ty of enzymatic reaction / 军事医学

Objective To detect the activity of α1 antitrypsin(AAT) with initial velocity of enzymatic reaction in order to detect the activity of samples in the process of separating and purifying plasma protein ,chromogenic substrate assay was optimized.Methods The effect of trypsin concentration and reaction time on enzymatic reaction was acquired by the kinetic monitoring mode of the microplate reader .Initial velocity was calculated to confirm the largest concentration of trypsin which was saturated by substrate .AAT was incubated with trypsin and absorbance produced by enzymatic reaction of remaining trypsin and substrate could reflect the activity of AAT .A standard curve was established with △D fitting with the activity of AAT standard.The activity of related samples was detected and the precision and accuracy of the method were evaluated . Results Trypsin concentration was 0.0625 mg/ml.Within 20 minutes, enzymatic reaction was with initial velocity .The range of the standard curve was 200-1200 IU/ml.Correlation coefficient was more than 0.99.The activity of Cohn Ⅳ, samples of pre-processing and elution were (720.59 ±18.63), (601.84 ±19.18),and (568.09 ±24.83)IU/ml, respec-tively.The relative standard deviation was less than 10%. Sample recovery rate was 90%-110%.Conclusion The optimized chromogenic substrate assay greatly improves accuracy and precision .The method can be used for the detec-tion of AAT activity of samples in laboratories and workshops .