Uncompetitive Inhibition Kinetics Research Articles

Alert to US physicians: DHEA, widely used as an OTC androgen supplement, may exacerbate COVID-19.

Androgens play a fundamental role in the morbidity and mortality of COVID-19, inducing both the ACE-2 receptor to which SARS-CoV-2 binds to gain entry into the cell, and TMPRS22, the transmembrane protease that primes the viral spike protein for efficient infection. The United States stands alone among developed nations in permitting one androgen, oral DHEA, to be freely available OTC and online as a 'dietary supplement'. DHEA is widely used by males in the US to offset the age-related decline in circulating androgens. This fact may contribute to the disparate statistics of COVID-19 morbidity and mortality in this country. In regulatory antithesis, every other developed nation regulates DHEA as a controlled substance. DHEA is an extremely potent inhibitor of glucose-6-phosphate dehydrogenase (G6PD), with uniquely unstable uncompetitive inhibition kinetics. This has particular relevance to COVID-19 because G6PD-deficient human cells have been demonstrated to be exceptionally sensitive to infection by human coronavirus. Because DHEA is lipophilic and freely passes into cells, oral DHEA bypasses the normal controls regulating androgen biology and uncompetitive G6PD inhibition. DHEA's status as a 'dietary supplement' means that no clinical trials demonstrating safety have been performed, and, in the absence of physician supervision, no data on adverse events have been collected. During the current pandemic, the unrestricted availability of oral DHEA as a 'dietary supplement' cannot be considered safe without proof from placebo-controlled clinical trials that it is not contributing to the severity of COVID-19. US physicians may therefore wish to query their patients' use of DHEA.

Studies on filarial GST as a target for antifilarial drug development—in silico and in vitro inhibition of filarial GST by substituted 1,4-naphthoquinones

Eleven 1,4-naphthoquinone analogues with different amino substitutions at position 3 of the quinone ring earlier reported for macrofilaricidal activity were selected and screened against purified cytosolic GST isolated from the bovine filarial worm Setaria digitata and IC(50) values were determined. Of the 11 compounds tested, 8 showed good inhibition against S. digitata GST. The IC(50) values of the most effective macrofilaricidal compounds-11 [2-(4-methylpiperazin-1-yl)naphthalene-1,4-dione] and 9 {2-[(1,3-dimethylbutyl)amino]naphthalene -1,4-dione}-were 0.872 and 0.994 mM, respectively. Compounds 9 and 11 were further studied for type of enzyme inhibition and found to exhibit competitive and uncompetitive inhibition kinetics, respectively, with respect to substrate GSH. All 11 compounds were in agreement with Lipinski's rule of five and passed through the FAFDrugs ADME/tox filter. Molecular docking was carried out using the modeled 3D structure of wbGST PDB ID:1SFM as receptor and substituted naphthoquinones as ligands using AutoDock 4.0. The binding energy of nine compounds varied from -9.15 to -6.58 Kcal mol(-1), whereas compounds 8 and 10 did not show any binding to the receptor. Among the compounds studied, compound 7 {2-[3-(diethylamino) propyl]aminonaphthalene-1,4-dione} showed maximum affinity towards wbGST as it exhibited the lowest binding energy, followed by compounds 11 and 9. However compound 7 was not macrofilaricidal while 11 and 9 exhibited macrofilaricidal activity. The results of in silico and in vitro studies with the synthesized 1,4 -naphthoquinone analogues on filarial GST and in vitro macrofilaricidal activity against adult bovine filarial worm S. digitata open up a promising biochemical target for antifilarial drug development.

Design, development, synthesis, and docking analysis of 2′-substituted triclosan analogs as inhibitors forPlasmodium falciparumEnoyl-ACP reductase

A structure-based approach has been adopted to develop 2'-substituted analogs of triclosan. The Cl at position 2' in ring B of triclosan was chemically substituted with other functional groups like NH(2), NO(2) and their inhibitory potencies against PfENR were determined. The binding energies of the 2' substituted analogs of triclosan for enoyl-acyl carrier protein reductase (ENR) of Plasmodium falciparum were determined using Autodock. Based on the autodock results, we synthesized the potential compounds. The IC(50) and inhibition constant (K(i)) of 2' substituted analogs of triclosan were determined against purified PfENR. Among them, two compounds, 2-(2'-Amino-4'-chloro-phenoxy)-5-chloro-phenol (compound 4) and 5-chloro-2-(4'-chloro-2'-nitro-phenoxy)-phenol) (compound 5) exhibited good potencies. Compound 4 followed uncompetitive inhibition kinetics with crotonoyl CoA and competitive with NADH. It was shown to have an IC(50) of 110 nM; inhibition constant was 104 nM with the substrate and 61 nM with the cofactor. IC(50) of compound 5 was determined to be 229 nM. Compounds 4 and 5 showed significant inhibition of the parasite growth in P. falciparum culture.

Modeling and optimization IV: Investigation of reaction kinetics and kinetic constants using a program in which artificial neural network (ANN) was integrated

Abstract This work describes an application of artificial neural networks (ANNs) to determine kinetics of enzymatic reactions and to estimate kinetic constants. A model enzymatic reaction, the hydrolysis of maltose catalyzed by amyloglucosidase, was performed in a batch reactor and time courses were obtained. The artificial neural network was trained with the data of seven time courses and the other eight time courses were used for testing the network. The trained network was integrated in a script coded in MATLAB®, which is used for the selection of the proper kinetic model and its constants. The kinetics of the reaction was also investigated using the conventional method and the results were compared. The results of both methods imply that the uncompetitive inhibition kinetics was valid for the amyloglucosidase reaction and the kinetic constants (Vmax, Km and Ki) were 1.48 μmol maltose/min/mg enzyme, 1.91 mM, and 71.42 mM for the model equation from developed program and 1.38 μmol maltose/min/mg enzyme, 1.96 mM, and 94.34 mM for the model equation obtained from the conventional method. The usability of the model equation in a real engineering problem was also tested by a numerical solution of a differential equation obtained from the batch reactor. The time courses obtained from the developed program and conventional method were compared with the experimentally obtained time courses. The results indicate that the time courses obtained from the developed program fit more properly to the experimental data than that from conventional method.

Nitrification modelling in biofilms under inhibitory conditions

A biofilm model has been developed for simulating nitrification in biofilms under inhibitory conditions. Nitrification inhibition has been modelled using uncompetitive inhibition kinetics. Inhibition kinetic experiments were performed by varying the bulk concentrations of inhibitory compound, aniline. Two sets of results were obtained with a nitrifying biofilm that was unacclimated to aniline and another which was acclimated to aniline. Fitting of the nitrification inhibition biofilm model to the experimental results yielded the nitrification inhibition constant, K i, for aniline. Both the experiments yielded a value of about 3 mg/L for K i, which was similar to that obtained during nitrification inhibition experiments with suspended growth process carried out in an earlier study. The nitrification inhibition biofilm model is general and can be applied to nitrification inhibition with other toxic compounds.

Modeling of nitrification inhibition with aniline in suspended-growth processes.

Nitrification inhibition due to aniline was investigated in completely mixed suspended-growth, batch, and continuous processes. Synthetic wastewater was used with aniline as the carbon source. The experiments were conducted at aniline concentrations inhibitory to nitrifier organisms. In the batch tests, degradation took place rapidly (within 4 to 6 hours) for initial aniline concentrations below 100 mg/L, with nitrification picking up as soon as the aniline concentration decreased to less than 3 to 4 mg/L. For initial aniline concentrations of 250 mg/L and higher, complete nitrification did not take place even though the aniline concentration decreased to less than 3 to 4 mg/L. This observation indicated nitrifier inhibition due to aniline. In the continuous experiments, a hydraulic residence time of 8 to 24 hours and a solids retention time of 8 to 24 days were maintained. Complete nitrification took place at bulk aniline concentrations less than 0.5 mg/L, while at higher aniline concentrations, nitrification inhibition took place. The inhibitory effect of aniline on the nitrification process was modeled using uncompetitive inhibition kinetics. Modeling of batch processes yielded the value for the inhibition constant for aniline, Ki, as 3.3 mg/L. Modeling of continuous processes yielded criteria for stable process operation for nitrification under inhibitory conditions, which were also confirmed through experimental results.

Biochemical Characterization of Human GMP Synthetase

GMP synthetase (EC 6.3.5.2) plays a key role in the de novo synthesis of guanine nucleotides. It is a potential target for immunosuppressive therapy. Recently, the human enzyme was purified to homogeneity (Hirst, M., Haliday, E., Nakamura, J., and Lou, L. (1994) J. Biol. Chem. 269, 23830-23837). We now report the characterization of this enzyme in terms of its biochemical and kinetic properties. We found that there are distinct features of the human enzyme that has not been reported for GMP synthetase from other sources. There are two variant forms of human GMP synthetase. Their catalytic properties are very similar, although their isoelectric points are different. They most likely represent post-translational modification variants. Magnesium ion is required for enzyme activity, and the requirement is beyond levels needed for ATP chelation. Magnesium appears to be an essential activator and there may be more than one binding site. Interaction of GMP synthetase with xanthosine 5'-monophosphate (XMP), a substrate, exhibits sigmoidal kinetics with a Hill coefficient of 1.48 +/- 0.07. This positive cooperativity is not due to ligand-induced oligomerization, since GMP synthetase remains a monomer in the presence of XMP and other substrates. Decoyinine, a selective inhibitor of GMP synthetase, inhibits the human enzyme reversibly with uncompetitive inhibition kinetics toward glutamine and XMP and non-competitive kinetics toward ATP.

Effect of etomoxiryl-CoA on different carnitine acyltransferases

The effects of etomoxiryl-CoA on purified carnitine acyltransferases and on carnitine acyltransferases of rat heart mitochondria and rat liver microsomes were determined. At nanomolar concentrations, the data agreed with that of other investigators who have shown that etomoxiryl-CoA must be binding to a high affinity site with specific inhibition of mitochondrial carnitine palmitoyltransferase (CPT o). Micromolar amounts of etomoxiryl-CoA inhibited both short- and long-chain carnitine acyltransferases. The concentrations of etomoxiryl-CoA required for 50% inhibition of the different carnitine acetyltransferases and microsomal and peroxisomal carnitine octanoyltransferase were in the low micromolar range. Mixed-type and uncompetitive inhibition kinetics were obtained, depending on the source of purified enzyme. When purified rat heart CPT was incubated with etomoxiryl-CoA, it increased the K 0.5 and decreased the Hill coefficient for acyl-CoA. Both proteins and phospholipids of mitochondria and microsomes formed covalent adducts of [ 3H]etomoxir, with the predominant labeling in phospholipids. None of the purified enzymes formed covalent adducts when incubated with [ 3H]etomoxiryl-CoA, in contrast to intact mitochondria or microsomes. The major 3H-labeled protein for rat heart mitochondria had a molecular weight of 81,000 ± 4000, and the major proteins from microsomes had a molecular weight of 51,000–57,000. Malonyl-CoA prevented most of the tritium incorporation into the 81,000 Da protein of mitochondria, but it had little effect on incorporation of tritiated etomoxir into the 51,000–57,000 Da proteins of microsomes. When 50 μM etomoxiryl-CoA was added to microsomes and to mitochondria that had been incubated with radioactive etomoxiryl-CoA, much of the radioactive etomoxir disappeared from the major microsomal proteins, but virtually none was displaced from the mitochondrial protein. Thus, at least two different types of covalent etomoxir complexes were formed. This pulse-chase experiment showed that the mitochondrial protein-etomoxir complex was not turned over, consistent with other data showing that etomoxir inhibited carnitine palmitoyltransferase. In contrast, the major protein-etomoxir complex in microsomes was turned over during the pulse-chase experiment.

Relationship between the inhibition constant ( KI) and the concentration of inhibitor which causes 50 per cent inhibition ( I50) of an enzymatic reaction

A theoretical analysis has been made of the relationship between the inhibition constant ( K I ) of a substance and the ( I 50) value which expresses the concentration of inhibitor required to produce 50 per cent inhibition of an enzymic reaction at a specific substrate concentration. A comparison has been made of the relationships between K I and I 50 for monosubstrate reactions when noncompetitive or uncompetitive inhibition kinetics apply, as well as for bisubstrate reactions under conditions of competitive, noncompetitive and uncompetitive inhibition kinetics. Precautions have been indicated against the indiscriminate use of I 50 values in agreement with the admonitions previously described in the literature. The analysis described shows K I does not equal I 50 when competitive inhibition kinetics apply; however, K I is equal to I 50 under conditions of either noncompetitive or uncompetitive kinetics.