Sulfate adenylyl transferase kinetics and mechanisms of metabolic inhibitors of microbial sulfate respiration

Sulfate analog oxyanions that function as selective metabolic inhibitors of dissimilatory sulfate reducing microorganisms (SRM) are widely used in ecological studies and industrial applications. As such, it is important to understand the mode of action and mechanisms of tolerance or adaptation to these compounds. Different oxyanions vary widely in their inhibitory potency and mechanism of inhibition, but current evidence suggests that the sulfate adenylyl transferase/ATP sulfurylase (Sat) enzyme is an important target. We heterologously expressed and purified the Sat from the model SRM, Desulfovibrio alaskensis G20. With this enzyme we determined the turnover kinetics (kcat, KM) for alternative substrates (molybdate, selenate, arsenate, monofluorophosphate, and chromate) and inhibition constants (KI) for competitive inhibitors (perchlorate, chlorate, and nitrate). These measurements enable the first quantitative comparisons of these compounds as substrates or inhibitors of a purified Sat from a respiratory sulfate reducer. We compare predicted half-maximal inhibitory concentrations (IC50) based on Sat kinetics with measured IC50 values against D. alaskensis G20 growth and discuss our results in light of known mechanisms of sensitivity or resistance to oxyanions. This analysis helps with the interpretation of recent adaptive laboratory evolution studies and illustrates the value of interpreting gene-microbe-environment interactions through the lens of enzyme kinetics.

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Sulfate adenylyl transferase kinetics and mechanisms of metabolic inhibitors of microbial sulfate respiration

ISME Communications ◽

10.1038/s43705-021-00069-1 ◽

2021 ◽

Vol 1 (1) ◽

Author(s):

Hans K. Carlson ◽

Matthew D. Youngblut ◽

Steven A. Redford ◽

Adam J. Williamson ◽

John D. Coates

Keyword(s):

Industrial Applications ◽

Metabolic Inhibitors ◽

Current Evidence ◽

Ecological Studies ◽

Adaptive Laboratory Evolution ◽

Inhibitory Potency ◽

Sulfate Reducing ◽

Important Target ◽

Mechanism Of Inhibition ◽

Competitive Inhibitors

AbstractSulfate analog oxyanions that function as selective metabolic inhibitors of dissimilatory sulfate reducing microorganisms (SRM) are widely used in ecological studies and industrial applications. As such, it is important to understand the mode of action and mechanisms of tolerance or adaptation to these compounds. Different oxyanions vary widely in their inhibitory potency and mechanism of inhibition, but current evidence suggests that the sulfate adenylyl transferase/ATP sulfurylase (Sat) enzyme is an important target. We heterologously expressed and purified the Sat from the model SRM, Desulfovibrio alaskensis G20. With this enzyme we determined the turnover kinetics (kcat, KM) for alternative substrates (molybdate, selenate, arsenate, monofluorophosphate, and chromate) and inhibition constants (KI) for competitive inhibitors (perchlorate, chlorate, and nitrate). These measurements enable the first quantitative comparisons of these compounds as substrates or inhibitors of a purified Sat from a respiratory sulfate reducer. We compare predicted half-maximal inhibitory concentrations (IC50) based on Sat kinetics with measured IC50 values against D. alaskensis G20 growth and discuss our results in light of known mechanisms of sensitivity or resistance to oxyanions. This analysis helps with the interpretation of recent adaptive laboratory evolution studies and illustrates the value of interpreting gene–microbe–environment interactions through the lens of enzyme kinetics.

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Design, Synthesis, and Biological Evaluation of Pyridazinones Containing the (2-Fluorophenyl) Piperazine Moiety as Selective MAO-B Inhibitors

Molecules ◽

10.3390/molecules25225371 ◽

2020 ◽

Vol 25 (22) ◽

pp. 5371

Author(s):

Muhammed Çeçen ◽

Jong Min Oh ◽

Zeynep Özdemir ◽

Saliha Ebru Büyüktuncel ◽

Mehtap Uysal ◽

...

Keyword(s):

Biological Evaluation ◽

Inhibitory Potency ◽

Design Synthesis ◽

Docking Simulations ◽

Mao B ◽

Ic50 Value ◽

Competitive Inhibitors ◽

Mao A ◽

Ic50 Values ◽

Synthesis And Biological Evaluation

Twelve pyridazinones (T1–T12) containing the (2-fluorophenyl) piperazine moiety were designed, synthesized, and evaluated for monoamine oxidase (MAO) -A and -B inhibitory activities. T6 was found to be the most potent MAO-B inhibitor with an IC50 value of 0.013 µM, followed by T3 (IC50 = 0.039 µM). Inhibitory potency for MAO-B was more enhanced by meta bromo substitution (T6) than by para bromo substitution (T7). For para substitution, inhibitory potencies for MAO-B were as follows: -Cl (T3) > -N(CH3)2 (T12) > -OCH3 (T9) > Br (T7) > F (T5) > -CH3 (T11) > -H (T1). T6 and T3 efficiently inhibited MAO-A with IC50 values of 1.57 and 4.19 µM and had the highest selectivity indices (SIs) for MAO-B (120.8 and 107.4, respectively). T3 and T6 were found to be reversible and competitive inhibitors of MAO-B with Ki values of 0.014 and 0.0071, respectively. Moreover, T6 was less toxic to healthy fibroblast cells (L929) than T3. Molecular docking simulations with MAO binding sites returned higher docking scores for T6 and T3 with MAO-B than with MAO-A. These results suggest that T3 and T6 are selective, reversible, and competitive inhibitors of MAO-B and should be considered lead candidates for the treatment of neurodegenerative disorders like Alzheimer’s disease.

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In Vitro and In Silico Kinetic Studies of Patented 1,7-diEthyl and 1,7-diMethyl Aminoalkanol Derivatives as New Inhibitors of Acetylcholinesterase

International Journal of Molecular Sciences ◽

10.3390/ijms23010270 ◽

2021 ◽

Vol 23 (1) ◽

pp. 270

Author(s):

Błażej Grodner ◽

Mariola Napiórkowska ◽

Dariusz Pisklak

Keyword(s):

Acetylcholinesterase Inhibitors ◽

Kinetic Studies ◽

Docking Studies ◽

Inhibitory Potency ◽

Main Compound ◽

Competitive Inhibitors ◽

Ic50 Values ◽

Visible Effect ◽

Derivatives Of

Two aminoalkanol derivatives of 1,7-diEthyl-8,9-diphenyl-4azatricyclo (5.2.1.02,6) dec-8-ene-3,5,10-trione and two derivatives of 1,7-diMethyl-8,9-diphenyl-4-azatricyclo (5.2.1.02.6) dec-8-ene-3,5,10-trione were evaluated in vitro for their inhibition efficacy of acetylcholinesterase. The Km, Vmax, slope angles of Lineweaver–Burk plots, Ki and IC50 values showed that all four aminoalkanol derivatives are competitive inhibitors of acetylcholinesterase whose inhibitory potency depends, to a varying extent, on the nature of the four different substituents present in the main compound structure. Studies have shown that the most potent acetylcholinesterase inhibitors are derivatives containing isopropylamine and/or methyl substituents in their structure. In contrast, dimethylamine and/or ethyl substituents seem to have a weaker, albeit visible, effect on the inhibitory potency of acetylcholinesterase. Additionally, docking studies suggest that studied compounds binds with the peripheral anionic site and not enter into the catalytic pocket due to the presence of the sterically extended substituent.

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Effect of Copper on the Mitochondrial Carnitine/Acylcarnitine Carrier Via Interaction with Cys136 and Cys155. Possible Implications in Pathophysiology

Molecules ◽

10.3390/molecules25040820 ◽

2020 ◽

Vol 25 (4) ◽

pp. 820

Author(s):

Nicola Giangregorio ◽

Annamaria Tonazzi ◽

Lara Console ◽

Mario Prejanò ◽

Tiziana Marino ◽

...

Keyword(s):

Conformational Changes ◽

Dose Response ◽

Liver Mitochondria ◽

Rat Liver Mitochondria ◽

Response Analysis ◽

Native Protein ◽

Mechanism Of Inhibition ◽

Ic50 Values ◽

Effect Of Copper ◽

Substrate Protection

The effect of copper on the mitochondrial carnitine/acylcarnitine carrier (CAC) was studied. Transport function was assayed as [3H]carnitine/carnitine antiport in proteoliposomes reconstituted with the native protein extracted from rat liver mitochondria or with the recombinant CAC over-expressed in E. coli. Cu2+ (as well as Cu+) strongly inhibited the native transporter. The inhibition was reversed by GSH (reduced glutathione) or by DTE (dithioerythritol). Dose-response analysis of the inhibition of the native protein was performed from which an IC50 of 1.6 µM for Cu2+ was derived. The mechanism of inhibition was studied by using the recombinant WT or Cys site-directed mutants of CAC. From the dose-response curve of the effect of Cu2+ on the recombinant protein, an IC50 of 0.28 µM was derived. Inhibition kinetics revealed a non-competitive type of inhibition by Cu2+. However, a substrate protection experiment indicated that the interaction of Cu2+ with the protein occurred in the vicinity of the substrate-binding site. Dose-response analysis on Cys mutants led to much higher IC50 values for the mutants C136S or C155S. The highest value was obtained for the C136/155S double mutant, indicating the involvement of both Cys residues in the interaction with Cu2+. Computational analysis performed on the WT CAC and on Cys mutants showed a pattern of the binding energy mostly overlapping the binding affinity derived from the dose-response analysis. All the data concur with bridging of Cu2+ with the two Cys residues, which blocks the conformational changes required for transport cycle.

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Discovery and Design of Novel Small Molecule GSK-3 Inhibitors Targeting the Substrate Binding Site

International Journal of Molecular Sciences ◽

10.3390/ijms21228709 ◽

2020 ◽

Vol 21 (22) ◽

pp. 8709

Author(s):

Ido Rippin ◽

Netaly Khazanov ◽

Shirley Ben Joseph ◽

Tania Kudinov ◽

Eva Berent ◽

...

Keyword(s):

Binding Site ◽

Small Molecule ◽

Substrate Binding ◽

Pharmacophore Model ◽

Drug Induced ◽

Substrate Binding Site ◽

Zinc Database ◽

Competitive Inhibitors ◽

Ic50 Values ◽

Critical Binding

The serine/threonine kinase, GSK-3, is a promising drug discovery target for treating multiple pathological disorders. Most GSK-3 inhibitors that were developed function as ATP competitive inhibitors, with typical limitations in specificity, safety and drug-induced resistance. In contrast, substrate competitive inhibitors (SCIs), are considered highly selective, and more suitable for clinical practice. The development of SCIs has been largely neglected in the past because the ambiguous, undefined nature of the substrate-binding site makes them difficult to design. In this study, we used our previously described structural models of GSK-3 bound to SCI peptides, to design a pharmacophore model and to virtually screen the “drug-like” Zinc database (~6.3 million compounds). We identified leading hits that interact with critical binding elements in the GSK-3 substrate binding site and are chemically distinct from known GSK-3 inhibitors. Accordingly, novel GSK-3 SCI compounds were designed and synthesized with IC50 values of~1–4 μM. Biological activity of the SCI compound was confirmed in cells and in primary neurons that showed increased β-catenin levels and reduced tau phosphorylation in response to compound treatment. We have generated a new type of small molecule GSK-3 inhibitors and propose to use this strategy to further develop SCIs for other protein kinases.

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In silico-guided engineering of Pseudomonas putida towards growth under micro-oxic conditions

Microbial Cell Factories ◽

10.1186/s12934-019-1227-5 ◽

2019 ◽

Vol 18 (1) ◽

Cited By ~ 7

Author(s):

Linde F. C. Kampers ◽

Ruben G. A. van Heck ◽

Stefano Donati ◽

Edoardo Saccenti ◽

Rita J. M. Volkers ◽

...

Keyword(s):

Pseudomonas Putida ◽

In Silico ◽

Redox Balance ◽

Industrial Applications ◽

Strictly Aerobic ◽

Class Iii ◽

Adaptive Laboratory Evolution ◽

Laboratory Evolution ◽

Atp Production ◽

Lactobacillus Lactis

Abstract Background Pseudomonas putida is a metabolically versatile, genetically accessible, and stress-robust species with outstanding potential to be used as a workhorse for industrial applications. While industry recognises the importance of robustness under micro-oxic conditions for a stable production process, the obligate aerobic nature of P. putida, attributed to its inability to produce sufficient ATP and maintain its redox balance without molecular oxygen, severely limits its use for biotechnology applications. Results Here, a combination of genome-scale metabolic modelling and comparative genomics is used to pinpoint essential $$\text {O}_{2}$$ O 2 -dependent processes. These explain the inability of the strain to grow under anoxic conditions: a deficient ATP generation and an inability to synthesize essential metabolites. Based on this, several P. putida recombinant strains were constructed harbouring acetate kinase from Escherichia coli for ATP production, and a class I dihydroorotate dehydrogenase and a class III anaerobic ribonucleotide triphosphate reductase from Lactobacillus lactis for the synthesis of essential metabolites. Initial computational designs were fine-tuned by means of adaptive laboratory evolution. Conclusions We demonstrated the value of combining in silico approaches, experimental validation and adaptive laboratory evolution for microbial design by making the strictly aerobic Pseudomonas putida able to grow under micro-oxic conditions.

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Targeting cancer stem cells from a metabolic perspective

Experimental Biology and Medicine ◽

10.1177/1535370220909309 ◽

2020 ◽

Vol 245 (5) ◽

pp. 465-476 ◽

Cited By ~ 1

Author(s):

Yao-An Shen ◽

Siao-Cian Pan ◽

I Chu ◽

Ruo-Yun Lai ◽

Yau-Huei Wei

Keyword(s):

Stem Cells ◽

Cancer Stem Cells ◽

Defense Mechanism ◽

Therapeutic Targets ◽

Metabolic Inhibitors ◽

Current Evidence ◽

Special Focus ◽

Self Renewal ◽

Atp Production ◽

Anticancer Treatment

The process of cancer development and progression is driven by distinct subsets of cancer stem cells (CSCs) that contribute the self-renewal capacity as the major impetus to the metastatic dissemination and main impediments in cancer treatment. Given that CSCs are so scarce in the tumor mass, there are debatable points on the metabolic signatures of CSCs. As opposed to differentiated tumor progenies, CSCs display exquisite patterns of metabolism that, depending on the type of cancer, predominately rely on glycolysis, oxidative metabolism of glutamine, fatty acids, or amino acids for ATP production. Metabolic heterogeneity of CSCs, which attributes to differences in type and microenvironment of tumors, confers CSCs to have the plasticity to cope with the endogenous mitochondrial stress and exogenous microenvironment. In essence, CSCs and normal stem cells are like mirror images of each other in terms of metabolism. To achieve reprogramming, CSCs not only need to upregulate their metabolic engine for self-renewal and defense mechanism, but also expedite the antioxidant defense to sustain the redox homeostasis. In the context of these pathways, this review portrays the connection between the metabolic features of CSCs and cancer stemness. Identification of the metabolic features in conferring resistance to anticancer treatment dictated by CSCs can enhance the opportunity to open up a new therapeutic dimension, which might not only improve the effectiveness of cancer therapies but also annihilate the whole tumor without recurrence. Henceforth, we highlight current findings of potential therapeutic targets for the design of alternative strategies to compromise the growth, drug resistance, and metastasis of CSCs by altering their metabolic phenotypes. Perturbing the versatile skills of CSCs by barricading metabolic signaling might bring about plentiful approaches to discover novel therapeutic targets for clinical application in cancer treatments. Impact statement This minireview highlights the current evidence on the mechanisms of pivotal metabolic pathways that attribute to cancer stem cells (CSCs) with a special focus on developing metabolic strategies of anticancer treatment that can be exploited in preclinical and clinical settings. Specific metabolic inhibitors that can overwhelm the properties of CSCs may impede tumor recurrence and metastasis, and potentially achieve a permanent cure of cancer patients.

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Synthesis and Evaluation of Novel 2H-Benzo[e]-[1,2,4]thiadiazine 1,1-Dioxide Derivatives as PI3Kδ Inhibitors

Molecules ◽

10.3390/molecules24234299 ◽

2019 ◽

Vol 24 (23) ◽

pp. 4299

Author(s):

Gong ◽

Tang ◽

Liu ◽

Liu

Keyword(s):

Fluorine Atom ◽

Indole Derivative ◽

High Selectivity ◽

Hydrophobic Pocket ◽

Inhibitory Potency ◽

Methyl Group ◽

Arylboronic Acids ◽

Lead Compounds ◽

Ic50 Values ◽

Quinazolinone Derivatives

In previous work, we applied the rotation-limiting strategy and introduced a substituent at the 3-position of the pyrazolo [3,4-d]pyrimidin-4-amine as the affinity element to interact with the deeper hydrophobic pocket, discovered a series of novel quinazolinones as potent PI3Kδ inhibitors. Among them, the indole derivative 3 is one of the most selective PI3Kδ inhibitors and the 3,4-dimethoxyphenyl derivative 4 is a potent and selective dual PI3Kδ/γ inhibitor. In this study, we replaced the carbonyl group in the quinazolinone core with a sulfonyl group, designed a series of novel 2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide derivatives as PI3Kδ inhibitors. After the reduction of nitro group in N-(2,6-dimethylphenyl)-2-nitrobenzenesulfonamide 5 and N-(2,6-dimethylphenyl)-2-nitro-5-fluorobenzenesulfonamide 6, the resulting 2-aminobenzenesulfonamides were reacted with trimethyl orthoacetate to give the 3-methyl-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide derivatives. After bromination of the 3-methyl group, the nucleophilic substitution with the 3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine provided the respective iodide derivatives, which were further reacted with a series of arylboronic acids via Suzuki coupling to furnish the 2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide derivatives 15a–J and 16a–d. In agreement with the quinazolinone derivatives, the introduction of a 5-indolyl or 3,4-dimethoxyphenyl at the affinity pocket generated the most potent analogues 15a and 15b with the IC50 values of 217 to 266 nM, respectively. In comparison with the quinazolinone lead compounds 3 and 4, these 2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide derivatives exhibited much decreased PI3Kδ inhibitory potency, but maintained the high selectivity over other PI3K isoforms. Unlike the quinazolinone lead compound 4 that was a dual PI3Kδ/γ inhibitor, the benzthiadiazine 1,1-dioxide 15b with the same 3,4-dimethoxyphenyl moiety was more than 21-fold selective over PI3Kγ. Moreover, the introducing of a fluorine atom at the 7-position of the 2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide core, in general, was not favored for the PI3Kδ inhibitory activity. In agreement with their high PI3Kδ selectivity, 15a and 15b significantly inhibited the SU-DHL-6 cell proliferation.

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Inhibition of neuronal nitric oxide synthase by 6-nitrocatecholamines, putative reaction products of nitric oxide with catecholamines under oxidative stress conditions

Biochemical Journal ◽

10.1042/bj3560105 ◽

2001 ◽

Vol 356 (1) ◽

pp. 105-110 ◽

Cited By ~ 4

Author(s):

Anna PALUMBO ◽

Giuseppe ASTARITA ◽

Marco d'ISCHIA

Keyword(s):

Nitric Oxide ◽

Nitric Oxide Synthase ◽

Neuronal Nitric Oxide Synthase ◽

Enzyme Activation ◽

H2o2 Production ◽

Reaction Products ◽

Competitive Inhibitors ◽

Cytochrome C Reduction ◽

Ic50 Values ◽

Oxide Synthase

6-Nitrodopamine and 6-nitronoradrenaline (6-nitronorepinephrine), putative products of the nitric oxide (NO)-dependent nitration of dopamine and noradrenaline, are reported to be reversible, competitive inhibitors of neuronal nitric oxide synthase (nNOS) with Ki values of 45 and 52μM respectively. The nitrocatecholamines inhibited H2O2 production in the absence of l-arginine and tetrahydrobiopterin (BH4) (the IC50 values for 6-nitrodopamine and 6-nitronoradrenaline were 85 and 55μM respectively) but without affecting cytochrome c reduction. The apparent Ki values for nitrocatecholamine inhibition of enzyme activation by BH4 were 18μM for 6-nitrodopamine and 40μM for 6-nitronoradrenaline. Both nitrocatecholamines antagonized the dimerization of nNOS induced by BH4 and by l-arginine, the effect being reversed by BH4 (more than 10μM) and l-arginine (e.g. 100μM). Overall, these results suggest that nitrocatecholamines interfere with nNOS activity by binding to the enzyme in the proximity of the substrate and BH4-binding sites near the haem group.

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In SilicoDesign and Synthesis of Targeted Curcumin Derivatives as Xanthine Oxidase Inhibitors

Current Drug Targets ◽

10.2174/1389450120666181122100511 ◽

2019 ◽

Vol 20 (5) ◽

pp. 593-603

Author(s):

Neelam Malik ◽

Priyanka Dhiman ◽

Anurag Khatkar

Keyword(s):

Xanthine Oxidase ◽

Inhibitory Activity ◽

Pyrimidine Ring ◽

Inhibitory Potency ◽

Curcumin Derivatives ◽

Xanthine Oxidase Inhibitors ◽

Excess Production ◽

Ic50 Values ◽

Inhibitory Potential ◽

Potent Inhibitory Activity

Background: Curcumin is a well-known pharmacophore and some of its derivatives are shown to target xanthine oxidase (XO) to alleviate disorders caused by the excess production of uric acid. Objective: Curcumin based derivatives were designed, synthesized and evaluated for their antioxidant and xanthine oxidase inhibitory potential. Method: In this report, we designed and synthesized two series of curcumin derivatives modified by inserting pyrazole and pyrimidine ring to central keto group. The synthesized compounds were evaluated for their antioxidant and xanthine oxidase inhibitory potential. Results: Results showed that pyrazole analogues of curcumin produced excellent XO inhibitory potency with the IC50 values varying from 06.255 µM to 10.503 µM. Among pyrimidine derivatives compound CU3a1 having ortho nitro substitution exhibited more potent xanthine oxidase inhibitory activity than any other curcumin derivative of this series. Conclusion: Curcumin derivatives CU5b1, CU5b2, CU5b3, and CU3a1 showed a potent inhibitory activity against xanthine oxidase along with good antioxidant potential.

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