scholarly journals A two-point IC50 method for evaluating the biochemical potency of irreversible enzyme inhibitors

2020 ◽  
Author(s):  
Petr Kuzmič
Keyword(s):  
Binding Affinity ◽  
Enzyme Inhibitors ◽  
Chemical Reactivity ◽  
Single Point ◽  
Reaction Times ◽  
Kinetic Mechanism ◽  
Enzyme Concentration ◽  
Inactivation Rate Constant ◽  
Covalent Inhibitors ◽  
Simplifying Assumptions

AbstractIrreversible (covalent) enzyme inhibitors cannot be unambiguously ranked for biochemical potency by using IC50 values determined at a single point in time, because the same IC50 value could originate either from relatively low initial binding affinity accompanied by high chemical reactivity, or the other way around. To disambiguate the potency ranking of covalent inhibitors, here we describe a data-analytic procedure relying on two separate IC50 values, determined at two different reaction times. In the case of covalent inhibitors following the two-step kinetic mechanism E + I ⇌ E I → EI, the two IC50 values alone can be used to estimate both the inhibition constant (Ki) as a measure of binding affinity and the inactivation rate constant (kinact) as a measure of chemical reactivity. In the case of covalent inhibitors following the one-step kinetic mechanism E + I → EI, a simple algebraic formula can be used to estimate the covalent efficiency constant (kinact/Ki) from a single experimental value of IC50. The two simplifying assumptions underlying the method are (1) zero inhibitor depletion, which implies that the inhibitor concentrations are always significantly higher than the enzyme concentration; and (2) constant reaction rate in the uninhibited control assay. The newly proposed method is validated by using a simulation study involving 64 irreversible inhibitors with covalent efficiency constants spanning seven orders of magnitude.

scholarly journals Deciding Between One-Step and Two-Step Irreversible Inhibition Mechanisms on the Basis of “Kobs” Data: A Statistical Approach

2020 ◽  
Author(s):  
Petr Kuzmic
Keyword(s):  
Drug Discovery ◽  
Statistical Approach ◽  
Enzyme Inhibitors ◽  
Chemical Reactivity ◽  
Kinetic Mechanism ◽  
Irreversible Inhibitor ◽  
Experimental Drugs ◽  
Covalent Inhibitors ◽  
Kinetic Mechanisms ◽  
One Step

Covalent (irreversible) enzyme inhibitors are an important group of actual or potential therapeutics. For example, Aspirin is an irreversible inhibitor of the cyclooxygenase enzyme. Evaluating covalent inhibitors in the drug discovery is exceptionally challenging, because their overall inhibitory potency consists of two separate but intertwined contributions: (1) initial binding affinity and (2) chemical reactivity. It is especially difficult to reliably asses the kinetic mechanism of inhibition. This paper describes an objective statistical approach that can be used to decide between two alternate kinetic mechanisms of covalent enzyme inhibition, from kinetic experiments based on the standard "kobs" method [Copeland (2013) "Evaluation of Enzyme Inhibitors in Drug Discovery", section 9.1]. The two alternatives are either a two-step kinetic mechanism, which involves a reversibly formed noncovalent intermediate, or a one-step kinetic mechanism, proceeding in a single bimolecular step. The proposed statistical toolkit uses four independent methods to arrive at a reliable mechanistic conclusion. The results are illustrated by using recently published experimental data on the inhibition of two different protein kinases by the experimental drugs ibrutinib (PCI-32765) and acalabrutinib [Hopper <i>et al.</i> (2020) <i>J. Pharm. Exp. Therap.</i> <b>372</b>, 331–338].

scholarly journals Deciding Between One-Step and Two-Step Irreversible Inhibition Mechanisms on the Basis of “Kobs” Data: A Statistical Approach

2020 ◽  
Author(s):  
Petr Kuzmic
Keyword(s):  
Drug Discovery ◽  
Statistical Approach ◽  
Enzyme Inhibitors ◽  
Chemical Reactivity ◽  
Kinetic Mechanism ◽  
Irreversible Inhibitor ◽  
Experimental Drugs ◽  
Covalent Inhibitors ◽  
Kinetic Mechanisms ◽  
One Step

Covalent (irreversible) enzyme inhibitors are an important group of actual or potential therapeutics. For example, Aspirin is an irreversible inhibitor of the cyclooxygenase enzyme. Evaluating covalent inhibitors in the drug discovery is exceptionally challenging, because their overall inhibitory potency consists of two separate but intertwined contributions: (1) initial binding affinity and (2) chemical reactivity. It is especially difficult to reliably asses the kinetic mechanism of inhibition. This paper describes an objective statistical approach that can be used to decide between two alternate kinetic mechanisms of covalent enzyme inhibition, from kinetic experiments based on the standard "kobs" method [Copeland (2013) "Evaluation of Enzyme Inhibitors in Drug Discovery", section 9.1]. The two alternatives are either a two-step kinetic mechanism, which involves a reversibly formed noncovalent intermediate, or a one-step kinetic mechanism, proceeding in a single bimolecular step. The proposed statistical toolkit uses four independent methods to arrive at a reliable mechanistic conclusion. The results are illustrated by using recently published experimental data on the inhibition of two different protein kinases by the experimental drugs ibrutinib (PCI-32765) and acalabrutinib [Hopper <i>et al.</i> (2020) <i>J. Pharm. Exp. Therap.</i> <b>372</b>, 331–338].

scholarly journals Deciding Between One-Step and Two-Step Irreversible Inhibition Mechanisms on the Basis of “Kobs” Data: A Statistical Approach

2020 ◽  
Author(s):  
Petr Kuzmic
Keyword(s):  
Drug Discovery ◽  
Statistical Approach ◽  
Enzyme Inhibitors ◽  
Chemical Reactivity ◽  
Kinetic Mechanism ◽  
Irreversible Inhibitor ◽  
Experimental Drugs ◽  
Covalent Inhibitors ◽  
Kinetic Mechanisms ◽  
One Step

Covalent (irreversible) enzyme inhibitors are an important group of actual or potential therapeutics. For example, Aspirin is an irreversible inhibitor of the cyclooxygenase enzyme. Evaluating covalent inhibitors in the drug discovery is exceptionally challenging, because their overall inhibitory potency consists of two separate but intertwined contributions: (1) initial binding affinity and (2) chemical reactivity. It is especially difficult to reliably asses the kinetic mechanism of inhibition. This paper describes an objective statistical approach that can be used to decide between two alternate kinetic mechanisms of covalent enzyme inhibition, from kinetic experiments based on the standard "kobs" method [Copeland (2013) "Evaluation of Enzyme Inhibitors in Drug Discovery", section 9.1]. The two alternatives are either a two-step kinetic mechanism, which involves a reversibly formed noncovalent intermediate, or a one-step kinetic mechanism, proceeding in a single bimolecular step. The proposed statistical toolkit uses four independent methods to arrive at a reliable mechanistic conclusion. The results are illustrated by using recently published experimental data on the inhibition of two different protein kinases by the experimental drugs ibrutinib (PCI-32765) and acalabrutinib [Hopper <i>et al.</i> (2020) <i>J. Pharm. Exp. Therap.</i> <b>372</b>, 331–338].

Standardized reaction times used to describe the mechanism of enzyme-induced gelation in whey protein systems

2000 ◽  
Vol 67 (3) ◽  
pp. 403-413 ◽  
Author(s):  
RICHARD IPSEN ◽  
JEANETTE OTTE ◽  
STIG B. LOMHOLT ◽  
KARSTEN B. QVIST
Keyword(s):  
Whey Protein ◽  
Reaction Times ◽  
Enzyme Concentration ◽  
Protein Isolate ◽  
Effect Of Temperature ◽  
Degree Of Hydrolysis ◽  
Thermally Induced ◽  
Constant Degree ◽  
Increasing Temperature ◽  
Rate Limiting

Whey protein isolate (WPI), either untreated or pretreated at 80 °C for 30 min, was incubated with a proteinase from Bacillus licheniformis until a gel was formed. Standardized reaction times, directly linked to the degree of hydrolysis, were obtained from plots of the relative amount of peptides released v. reaction time obtained under different conditions (enzyme concentration, temperature, pH, NaCl addition). This provided a connection between the gelation profile and the degree of hydrolysis. In the case of untreated WPI, gelation occurred at lower degrees of proteolysis when the enzyme concentration was decreased, demonstrating that a rate-limiting aggregation process occurred at the same time as the proteolysis in a manner similar to the renneting of milk. This was not the case for preheated WPI, when gelation was found to take place at a constant degree of proteolysis, independent of the enzyme concentration. In this case, the mechanism could be described by assuming the thermally induced aggregates present in this substrate had progressively more stabilizing peptide segments shaved off, resulting in increased attraction between individual aggregates that ultimately led to gelation. Results obtained at 40–60 °C supported this, as we found no effect of temperature on the degree of proteolysis at gelation for the untreated WPI, whereas the degree of proteolysis decreased with increasing temperature when heated WPI was hydrolysed. The effect of pH and NaCl addition on the process was to reduce repulsion between the aggregating species so that gelation was induced at a decreased degree of proteolysis.

Targeting histidine for developing a new generation of covalent enzyme inhibitors

2021 ◽  
Vol 17 ◽  
Author(s):  
Donald Poirier
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Drug Development ◽  
Chemical Bond ◽  
Amino Acid Residue ◽  
Enzyme Inhibitors ◽  
Irreversible Inhibitors ◽  
Covalent Inhibitors ◽  
Targeted Inhibitors ◽  
New Generation

: Despite the significant number of irreversible inhibitors developed over the years, strong prejudices remain for this type of therapeutic molecule, particularly in the area of drug development. New generations of covalent targeted inhibitors are, however, in development, and interest is increasingly growing. In fact, the new generation of covalent inhibitors has a weakly reactive species (warhead) that is able, in a particular context, to selectively form a chemical bond with a given amino acid residue, which can be irreversible or reversible. In addition to new selective warheads, new amino acids are also targeted. In the following text, we will focus on covalent targeted inhibitors that selectively alkylate histidine.

scholarly journals Investigation of Some Antiviral N-Heterocycles as COVID 19 Drug: Molecular Docking and DFT Calculations

2020 ◽  
Vol 21 (11) ◽  
pp. 3922 ◽  
Author(s):  
Mohamed Hagar ◽  
Hoda A. Ahmed ◽  
Ghadah Aljohani ◽  
Omaima A. Alhaddad
Keyword(s):  
Dipole Moment ◽  
Molecular Docking ◽  
Dft Calculations ◽  
Binding Affinity ◽  
Electrostatic Potential ◽  
Density Functional ◽  
Molecular Electrostatic Potential ◽  
Hydrophobic Interactions ◽  
Chemical Reactivity ◽  
Main Protease

The novel coronavirus, COVID-19, caused by SARS-CoV-2, is a global health pandemic that started in December 2019. The effective drug target among coronaviruses is the main protease Mpro, because of its essential role in processing the polyproteins that are translated from the viral RNA. In this study, the bioactivity of some selected heterocyclic drugs named Favipiravir (1), Amodiaquine (2), 2′-Fluoro-2′-deoxycytidine (3), and Ribavirin (4) was evaluated as inhibitors and nucleotide analogues for COVID-19 using computational modeling strategies. The density functional theory (DFT) calculations were performed to estimate the thermal parameters, dipole moment, polarizability, and molecular electrostatic potential of the present drugs; additionally, Mulliken atomic charges of the drugs as well as the chemical reactivity descriptors were investigated. The nominated drugs were docked on SARS-CoV-2 main protease (PDB: 6LU7) to evaluate the binding affinity of these drugs. Besides, the computations data of DFT the docking simulation studies was predicted that the Amodiaquine (2) has the least binding energy (−7.77 Kcal/mol) and might serve as a good inhibitor to SARS-CoV-2 comparable with the approved medicines, hydroxychloroquine, and remdesivir which have binding affinity −6.06 and −4.96 Kcal/mol, respectively. The high binding affinity of 2 was attributed to the presence of three hydrogen bonds along with different hydrophobic interactions between the drug and the critical amino acids residues of the receptor. Finally, the estimated molecular electrostatic potential results by DFT were used to illustrate the molecular docking findings. The DFT calculations showed that drug 2 has the highest of lying HOMO, electrophilicity index, basicity, and dipole moment. All these parameters could share with different extent to significantly affect the binding affinity of these drugs with the active protein sites.

scholarly journals Comparison of Mind Control Techniques: An Assessment of Reaction Times

2016 ◽  
Vol 15 (4) ◽  
pp. 596-600
Author(s):  
Varun Malhotra ◽  
Neera Goel ◽  
Usha Dhar ◽  
Rinku Garg ◽  
Yogish Tripathi
Keyword(s):  
Reaction Time ◽  
Single Point ◽  
Medical Science ◽  
Reaction Times ◽  
Devotional Music ◽  
Mind Control ◽  
Visual Reaction ◽  
Time Test ◽  
The Mind ◽  
Reaction Time Test

Background: Every activity requires a certain amount of concentration and no effective action may be performed without deep concentration. Businessman or artists or students in school must know the art of focusing all powers of attention on a single point in order to succeed in their respective vocation.Methods: We wanted to find the best technique to increase the concentration scientifically. We thus, endeavored to study and compare the reaction times in maneuvers of anuloma viloma pranayama, kapalbhatti pranayama, gayatri chanting and exercise. Reaction time test was taken online before anuloma viloma pranayama, kapalbhatti pranayama, gayatri chanting and exercise and compared after.Results: Reaction times decreased significantly and was least during gayatri mantra. Concentration as seen by a decrease in visual reaction time denotes first a withdrawal of attention from objects of distraction and then focusing all attention upon one thing at a time. Just 30 minutes of physical activity each day offers substantial benefits to your health.Conclusions: Pranayama or devotional music chanting also decreases fatigue keeps the mind alert, and active.Bangladesh Journal of Medical Science Vol.15(4) 2016 p.596-600

scholarly journals In Silico Insights into HIV-1 Vpu-Tetherin Interactions and Its Mutational Counterparts

2019 ◽  
Vol 7 (6) ◽  
pp. 74
Author(s):  
Patil Sneha ◽  
Urmi Shah ◽  
Seetharaman Balaji
Keyword(s):  
Cell Surface ◽  
Binding Affinity ◽  
Hydrophobic Interactions ◽  
Single Point ◽  
Resistance Mechanism ◽  
Point Mutations ◽  
Protein Docking ◽  
Host Protein ◽  
Single Point Mutations ◽  
Hiv 1

Tetherin, an interferon-induced host protein encoded by the bone marrow stromal antigen 2 (BST2/CD317/HM1.24) gene, is involved in obstructing the release of many retroviruses and other enveloped viruses by cross-linking the budding virus particles to the cell surface. This activity is antagonized in the case of human immunodeficiency virus (HIV)-1 wherein its accessory protein Viral Protein U (Vpu) interacts with tetherin, causing its downregulation from the cell surface. Vpu and tetherin connect through their transmembrane (TM) domains, culminating into events leading to tetherin degradation by recruitment of β-TrCP2. However, mutations in the TM domains of both proteins are reported to act as a resistance mechanism to Vpu countermeasure impacting tetherin’s sensitivity towards Vpu but retaining its antiviral activity. Our study illustrates the binding aspects of blood-derived, brain-derived, and consensus HIV-1 Vpu with tetherin through protein–protein docking. The analysis of the bound complexes confirms the blood-derived Vpu–tetherin complex to have the best binding affinity as compared to other two. The mutations in tetherin and Vpu are devised computationally and are subjected to protein–protein interactions. The complexes are tested for their binding affinities, residue connections, hydrophobic forces, and, finally, the effect of mutation on their interactions. The single point mutations in tetherin at positions L23Y, L24T, and P40T, and triple mutations at {L22S, F44Y, L37I} and {L23T, L37T, T45I}, while single point mutations in Vpu at positions A19H and W23Y and triplet of mutations at {V10K, A11L, A19T}, {V14T, I18T, I26S}, and {A11T, V14L, A15T} have revealed no polar contacts with minimal hydrophobic interactions between Vpu and tetherin, resulting in reduced binding affinity. Additionally, we have explored the aggregation potential of tetherin and its association with the brain-derived Vpu protein. This work is a possible step toward an understanding of Vpu–tetherin interactions.

Origins of the Activity of PAL and LAP Enzyme Inhibitors:  Toward Ab Initio Binding Affinity Prediction

2005 ◽  
Vol 127 (6) ◽  
pp. 1658-1659 ◽  
Author(s):  
Edyta Dyguda ◽  
Jolanta Grembecka ◽  
W. Andrzej Sokalski ◽  
Jerzy Leszczyński
Keyword(s):  
Ab Initio ◽  
Binding Affinity ◽  
Enzyme Inhibitors ◽  
Binding Affinity Prediction ◽  
Affinity Prediction

scholarly journals mmCSM-NA: accurately predicting effects of single and multiple mutations on protein–nucleic acid binding affinity

2021 ◽  
Vol 3 (4) ◽  
Author(s):  
Thanh Binh Nguyen ◽  
Yoochan Myung ◽  
Alex G C de Sá ◽  
Douglas E V Pires ◽  
David B Ascher
Keyword(s):  
Nucleic Acid ◽  
Binding Affinity ◽  
Single Point ◽  
Point Mutations ◽  
Multiple Point ◽  
Missense Mutations ◽  
Nucleic Acid Binding ◽  
Single Point Mutations ◽  
Protein Nucleic Acid ◽  
Nucleic Acid Interactions

Abstract While protein–nucleic acid interactions are pivotal for many crucial biological processes, limited experimental data has made the development of computational approaches to characterise these interactions a challenge. Consequently, most approaches to understand the effects of missense mutations on protein-nucleic acid affinity have focused on single-point mutations and have presented a limited performance on independent data sets. To overcome this, we have curated the largest dataset of experimentally measured effects of mutations on nucleic acid binding affinity to date, encompassing 856 single-point mutations and 141 multiple-point mutations across 155 experimentally solved complexes. This was used in combination with an optimized version of our graph-based signatures to develop mmCSM-NA (http://biosig.unimelb.edu.au/mmcsm_na), the first scalable method capable of quantitatively and accurately predicting the effects of multiple-point mutations on nucleic acid binding affinities. mmCSM-NA obtained a Pearson's correlation of up to 0.67 (RMSE of 1.06 Kcal/mol) on single-point mutations under cross-validation, and up to 0.65 on independent non-redundant datasets of multiple-point mutations (RMSE of 1.12 kcal/mol), outperforming similar tools. mmCSM-NA is freely available as an easy-to-use web-server and API. We believe it will be an invaluable tool to shed light on the role of mutations affecting protein–nucleic acid interactions in diseases.

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