DOCSLIB.ORG

Pdf (734.95 K)

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Pdf (734.95 K) Biochemistry Letters, 11(20) 2016, pages 259-274 Scientific Research & Studies Center-Faculty of Science- Zagazig University- Egypt Biochemistry Letters Journal home page: Interactions of Monoamine Oxidase from Human And Rat Liver Mitochondria with (S)-2-[4-(3-Flouro-Benzyloxy)-Benzylamino]-Propionamide Zakia Ben Ramadan*1 , Keith Tipton2 , Safaa khater3 1-Department of Biochemistry, Faculty of Medicine University of Tripoli-Libya. 2-Department of Biochemistry, Trinity College, Dublin 2, Ireland . 3- Department of Biochemistry, Zagazig University –Egypt. ARTICLE INFO ABSTRACT Article history: Background:The interactions of the anticonvulsant (S)-2-[4- Received : (3-Flouro-Benzyloxy) Benzylamino]- Propionamide (FCE Accepted : 26743) with MAO-A and MAO -B from human and rat liver Available online : mitochondria has been studied. Objective: This compound is an alanine derivative that differs from the parent anticonvulsant Keywords: Monoamine compound, 2-n-pentylamino acetamide in that alaninamide oxidase-A and-B (MAO-A& residue replaces the glycinamide moiety, and in the presence of MAO -B), kinetics of inhibition, a phenyl ring substituted in the para-position by a 3-flouro- (S)-2-[4-(3-flourobenzyloxy)- benzyloxy group. Results: The results indicated that, The Ki benzylamino] propionamide, values for rat and human MAO-A were > 1000 times higher (FCE 26743), Inhibition than their corresponding values for rat and human MAO- Studies. Consistent with the IC50values that showed this compound to be > 1000 times more potent as an inhibitor of MAO-B than of MAO-A of the same species. There was an increase in the strength of inhibition when MAO-B was incubated with this compound. This was confirmed by the extended time-courses studies which showed a rather rapid increase in the degree of inhibition of MAO-B, despite incubation being continued for a total of 4h. This is consistent with FCE 26743 being a reversible inhibitor for MAO-A and a slow- binding reversible inhibitor of MAO-B.FCE 26743 was found to be a time- independent weak inhibitor of MAO-A from both rat and human preparations. However, there was a significant difference in the inhibitory potency of FCE 26743 with MAO- B from the two species, the Ki value for rat MAO-B was about two times higher than that for human MAOB. Conclusion: The FCE 26743 was not metabolized (in vitro) by either human or rat liver mitochondrial MAO-A or -B. Without enzyme- inhibitor preincubation FCE 26743 was found to be a competitive inhibitor of both MAO-A and MAO-B from rat and human liver mitochondria. © 2016 Publisher All rights reserved. INTRODUCTION where the active inhibitory species is formed through the action of MAO itself, Monoamine Monoamine oxidase inhibitors, hydrazines oxidase (MAO) inhibitors, although not used that used clinically include phenelzine, as first line antidepressants, due to risk of the phenylhydrazine, nialamide and isocarboxazid, drug’s interaction with diet or other drugs, act as enzyme activated irreversible inhibitors continue to have a niche in the treatment of Corresponding author: psychiatric and neurological disorders. Interest mean ± SD, after 2min of preincubation] and in these drugs has also increased markedly in to weakly inhibit MAO-A [IC50 = 0.37 ± recent years following numerous reports of 0.035 mM]. These workers also showed that their neuroprotective actions in vivo and in the results of in vitro preincubation studies and vitro (3).The potent anticonvulsant compound of experiments involving the in vitro and "ex FCE 26743, The alanine derivative (S)-2-[4- vivo" dilution of brain homogenates (flourobenzyloxy)-benzylamino] experiments were consistent with FCE 26743 propionamide belongs to a series of analogues acting as an irreversible MAO-B inhibitor. In of milacemide (2-(n-pentylamino) acetamide) contrast, the time-course of "ex vivo" (4). a glycine derivative with atypicalanti- inhibition of rat brain MAO-B with this epileptic and potential psychotropic properties compound was a typical of short acting and differs from milacemide in that inhibitor. Additional experiments are needed alaninamide residue replaces the glycinamide to establish whether FCE 26743 acts as a moiety, and in the presence of a phenyl ring slow-binding inhibitor, or is metabolized to substituted in the para-position by a 3-flouro- some degree by MAO-B to form an adduct benzyloxy group. It has been suggested in the with the enzyme. literature that the anticonvulsant compound It has been suggested that molecules that, milacemide (2-n-pentylaminoacetamide) acts in addition to their anticonvulsant properties, as a pro-drug delivering glycine into the possess potent MAO-inhibitory activities central nervous system (5,6) and this is a might decrease the oxidative stress associated prerequisite for milacemide's anticonvulsant with epilepsy as a consequence of hydrogen activity. It has been shown that milacemide peroxide formation impairment (8,14). In this functions as a suicide substrate and being a respect, it is worth noting that, a significantly better substrate for the enzyme MAO-B than higher MAO-B activity was found in as a mechanism based inhibitor for all the hippocampi from epileptic patients as species studied (7). Moreover from our compared with non-epileptic control subjects, previous studies, the compound 2- after correction for age-related changes (15). (benzylamino)acetamide, another glycine Higher brain monoamine levels have been derivative acts as substrates for MAO-B and as shown to reduce seizure susceptibility (16).It inhibitors for MAO-A & MAO-B and has been reported that the synthesis and displaying lower anticonvulsant activity (8). It release of monoamines, dopamine in seems apparent that, the 'delivery of glycine to particular, is increased in seizure foci the brain' as aprerequisite for anticonvulsant compared with no focus regions in the human activity remains an imperfect hypothesis. brain (17). This may represent defense Since the amino acid alanine,unlike glycine, response to seizure susceptibility. does not possess an anticonvulsant activity, an Administration of the MAO-B inhibitor investigation of the mechanism of action of selegiline to Parkinson's disease patients was this compound with MAO may help in shown to enhance brain dopamine understanding the anticonvulsant behavior of concentrations (18). Therefore, it is possible this group of compounds. Introduction of a that, The MAO-B inhibitory property of FCE benzyloxy group in the para position of a 26743 might contribute to its anti-epileptic phenyl ring in MAO substrates and inhibitors activity by increasing brain dopamine levels has been shown to transform the substrates during long-term treatment. In the present into inhibitors (9) and to increase the work the behavior of FCE 26743 as a MAO-A inhibitory potency of the inhibitors (10,11). and –B substrate and inhibitor was examined FCE 26743 showed potent protective in human and rat liver mitochondria. properties in a number of models of epilepsy Furthermore the kinetic parameters of in rodents, such as bicuculline-induced inhibition of rat liver mitochondrial MAO-A convulsions and lethality, maximal and -B, and human liver mitochondrial MAO- electroshock and kainic acid-induced seizures A and MAO-B by FCE 26743 were and status epilepticus (8,12). determined in vitro. In vitro studies with rat brain homogenates (13) showed FCE 26743 to be a potent 2. Experimental procedures inhibitor of MAO-B [IC50 = 0.17 ± 0.01 µM, 2.1. Materials. 5-Hydroxytryptamine (5-HT) [side chain-2- were determined. FCE 26743 was examined as 14C] creatine sulphate and phenylethylamine - a substrate of MAO by the direct spectrophotometric assay at 283 nm. The (PEA)(ethyl-114C) hydrochloride were effects of FCE 26743 on the activity of MAO- obtained from Amersham International or New A and -B were determined using the England Nuclear. Benzylamine HCL and 5- radiochemical assay by the method of (25) as Hydroxytryptamine creatine sulphate (5-HT) modified by (26) using 5- were obtained from Sigma Co. FCE 26743 and Hydroxytryptamine(5-HT) and its aldehyde derivative were kindly given by phenylethylamine(PEA), as the selective Pharmacia-Farmitalia Carlo Erba. All other substrates for MAO-A and -B, respectively. chemicals were standard laboratory chemicals IC values (concentration of the inhibitor and were of analytical reagent grade whenever 50 possible. giving 50% inhibition) were determined at zero-time and after 30 min preincubation of 2.2. Methods. the enzyme and inhibitor using the computer program Kaleidagraph . Rat liver mitochondria and human liver The kinetic behavior of FCE 26743 on mitochondria were prepared by the method of MAO-A and-B from both species were (19). The mitochondrial pellet obtained was determined (a) spectrophotometrically, by suspended in a small volume of 0.1M coupled or direct assays by varying the potassium phosphate buffer, pH 7.2 and stored substrate concentration (5-HT and o benzylamine, respectively), in the presence of at -20 C until use for MAO-B or MAO-A several fixed concentrations of the inhibitor. activity. Human liver was obtained within 12 (b) Radiochemically, using 5-HT and PEA as hours of death and transferred to the laboratory substrates in the presence of several fixed on ice. concentrations of the inhibitor. The Ki values The aldehyde dehydrogenase (ALDH) was were calculated by fitting the data of the partly purified from ox liver by a modification apparent K /V values (slopes)versus the (20) of the method of (21) and IU of activity is m max defined as the amount that catalyzes the inhibitor concentrations using the computer o program Mac-CurveFit. Extended time courses formation of 1µ mol product / min at 37 C in of inhibition of human and rat liver + the presence of 500 µM NAD and 3mM mitochondrial MAO-A and -B were acetaldehyde. The molar extinction coefficient determined spectrophotometrically by ( ) of NADH at 340 nm was taken to be monitoring the product formation by either 6.22x103 M-1.cm-1 (22).
Load more

Recommended publications
  • The Serpintine Solution
    & Experim l e ca n i t in a l l C C f a Journal of Clinical & Experimental o r d l i a o n l o r g u Lucas et al., J Clin Exp Cardiolog 2017, 8:1 y o J Cardiology ISSN: 2155-9880 DOI: 10.4172/2155-9880.1000e150 Editorial Open Access The Serpintine Solution Alexandra Lucas, MD, FRCP(C)1,2,*, Sriram Ambadapadi, PhD1, Brian Mahon, PhD3, Kasinath Viswanathan, PhD4, Hao Chen, MD, PhD5, Liying Liu, MD6, Erbin Dai, MD6, Ganesh Munuswami-Ramanujam, PhD7, Jacek M. Kwiecien, DVM, MSc, PhD8, Jordan R Yaron, PhD1, Purushottam Shivaji Narute, BVSc & AH, MVSc, PhD1,9, Robert McKenna, PhD10, Shahar Keinan, PhD11, Westley Reeves, MD, PhD12, Mark Brantly, MD, PhD13, Carl Pepine, MD, FACC14 and Grant McFadden, PhD1 1Center for Personalized Diagnostics, Biodesign Institute, Arizona State University, Tempe AZ, USA 2Saint Josephs Hospital, Dignity Health Phoenix, Phoenix, AZ, USA 3NIH/ NIDDK, Bethesda MD, USA 4Zydus Research Centre, Ahmedabad, India 5Department of tumor surgery, The Second Hospital of Lanzhou University, Lanzhou, Gansu, P.R.China 6Beth Israel Deaconess Medical Center, Harvard, Boston, MA, USA 7Interdisciplinary Institute of Indian system of Medicine (IIISM), SRM University, Chennai, Tamil Nadu, India 8MacMaster University, Hamilton, ON, Canada 9Critical Care Medicine Department, Clinical Center, National Institutes of Health, Bethesda MD, USA 10Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL, USA 11Cloud Pharmaceuticals, Durham, North Carolina, USA 12Division of Rheumatology, University of Florida,
    [Show full text]
  • Understanding Drug-Drug Interactions Due to Mechanism-Based Inhibition in Clinical Practice
    pharmaceutics Review Mechanisms of CYP450 Inhibition: Understanding Drug-Drug Interactions Due to Mechanism-Based Inhibition in Clinical Practice Malavika Deodhar 1, Sweilem B Al Rihani 1 , Meghan J. Arwood 1, Lucy Darakjian 1, Pamela Dow 1 , Jacques Turgeon 1,2 and Veronique Michaud 1,2,* 1 Tabula Rasa HealthCare Precision Pharmacotherapy Research and Development Institute, Orlando, FL 32827, USA; [email protected] (M.D.); [email protected] (S.B.A.R.); [email protected] (M.J.A.); [email protected] (L.D.); [email protected] (P.D.); [email protected] (J.T.) 2 Faculty of Pharmacy, Université de Montréal, Montreal, QC H3C 3J7, Canada * Correspondence: [email protected]; Tel.: +1-856-938-8697 Received: 5 August 2020; Accepted: 31 August 2020; Published: 4 September 2020 Abstract: In an ageing society, polypharmacy has become a major public health and economic issue. Overuse of medications, especially in patients with chronic diseases, carries major health risks. One common consequence of polypharmacy is the increased emergence of adverse drug events, mainly from drug–drug interactions. The majority of currently available drugs are metabolized by CYP450 enzymes. Interactions due to shared CYP450-mediated metabolic pathways for two or more drugs are frequent, especially through reversible or irreversible CYP450 inhibition. The magnitude of these interactions depends on several factors, including varying affinity and concentration of substrates, time delay between the administration of the drugs, and mechanisms of CYP450 inhibition. Various types of CYP450 inhibition (competitive, non-competitive, mechanism-based) have been observed clinically, and interactions of these types require a distinct clinical management strategy. This review focuses on mechanism-based inhibition, which occurs when a substrate forms a reactive intermediate, creating a stable enzyme–intermediate complex that irreversibly reduces enzyme activity.
    [Show full text]
  • Insights Into the Role of Tick Salivary Protease Inhibitors During Ectoparasite–Host Crosstalk
    International Journal of Molecular Sciences Review Insights into the Role of Tick Salivary Protease Inhibitors during Ectoparasite–Host Crosstalk Mohamed Amine Jmel 1,† , Hajer Aounallah 2,3,† , Chaima Bensaoud 1, Imen Mekki 1,4, JindˇrichChmelaˇr 4, Fernanda Faria 3 , Youmna M’ghirbi 2 and Michalis Kotsyfakis 1,* 1 Laboratory of Genomics and Proteomics of Disease Vectors, Biology Centre CAS, Institute of Parasitology, Branišovská 1160/31, 37005 Ceskˇ é Budˇejovice,Czech Republic; [email protected] (M.A.J.); [email protected] (C.B.); [email protected] (I.M.) 2 Institut Pasteur de Tunis, Université de Tunis El Manar, LR19IPTX, Service d’Entomologie Médicale, Tunis 1002, Tunisia; [email protected] (H.A.); [email protected] (Y.M.) 3 Innovation and Development Laboratory, Innovation and Development Center, Instituto Butantan, São Paulo 05503-900, Brazil; [email protected] 4 Faculty of Science, University of South Bohemia in Ceskˇ é Budˇejovice, 37005 Ceskˇ é Budˇejovice, Czech Republic; [email protected] * Correspondence: [email protected] † These authors contributed equally. Abstract: Protease inhibitors (PIs) are ubiquitous regulatory proteins present in all kingdoms. They play crucial tasks in controlling biological processes directed by proteases which, if not tightly regulated, can damage the host organism. PIs can be classified according to their targeted proteases or their mechanism of action. The functions of many PIs have now been characterized and are showing clinical relevance for the treatment of human diseases such as arthritis, hepatitis, cancer, AIDS, and cardiovascular diseases, amongst others. Other PIs have potential use in agriculture as insecticides, anti-fungal, and antibacterial agents.
    [Show full text]
  • Competitive Inhibition That Barrier, As We Shall See
    Chapter 4 Catalysis In living systems, speed is everything. Providing the reaction speeds necessary to support life are the catalysts, mostly in the form of enzymes. Catalysis Introduction Introduction Activation Energy General Mechanisms of Action If there is a magical component to life, an argument can surely be made for it Substrate Binding being catalysis. Thanks to catalysis, reactions that could take hundreds of years Enzyme Flexibility to complete in the “real world,” occur in seconds in the presence of a catalyst. Active Site Chymotrypsin Chemical catalysts, like platinum, speed reactions, but enzymes (which are simply Enzyme Parameters super-catalysts with a twist) put chemical catalysts to shame. To understand VMAX & KCAT enzymatic catalysis, we must first understand energy. In Chapter 2, we noted the KM tendency for processes to move in the direction of lower energy. Chemical Perfect Enzymes Lineweaver-Burk Plots reactions follow this universal trend, but they often have a barrier in place that Enzyme Inhibition must be overcome. The secret to catalytic action is reducing the magnitude of Competitive Inhibition that barrier, as we shall see. No Effect On VMAX Increased KM Non-Competitive Inhibition Uncompetitive Inhibition Suicide Inhibition Control of Enzymes Allosterism Covalent Control of Enzymes Other Controls of Enzymes Ribozymes Enzyme Rate Enhancements 81 Activation Energy Another feature to note about catalyzed reactions is Click HERE, HERE, and HERE for The figure below schematically depicts the energy changes that the reduced energy barrier Kevin’s Catalytic Mechanism occur during the progression of a simple reaction. In the figure, lectures on YouTube (also called the activation the energy differences during the reaction are compared for a energy or free energy of catalyzed (plot on the right) and an uncatalyzed reaction (plot on activation) to reach the transition state of the catalyzed reaction.
    [Show full text]
  • Optimizing Defence, Counter-Defence and Counter-Counter Defence in Parasitic and Trophic Interactions – a Modelling Study
    1 Optimizing defence, counter-defence and counter-counter defence in parasitic and trophic interactions – A modelling study Stefan Schuster1*, Jan Ewald1,2, Thomas Dandekar2, Sybille Dühring1 1Dept. of Bioinformatics, Friedrich Schiller University of Jena Ernst-Abbe-Platz 2, 07743 Jena, Germany 2Dept. of Bioinformatics, Biocenter Julius Maximilian University of Würzburg Am Hubland, 97074 Würzburg *Corresponding author, e-mail: [email protected] Abstract In host-pathogen interactions, often the host (attacked organism) defends itself by some toxic compound and the parasite, in turn, responds by producing an enzyme that inactivates that compound. In some cases, the host can respond by producing an inhibitor of that enzyme, which can be considered as a counter-counter defence. An example is provided by cephalosporins, β-lactamases and clavulanic acid (an inhibitor of β-lactamases). Here, we tackle the question under which conditions it pays, during evolution, to establish a counter- counter defence rather than to intensify or widen the defence mechanisms. We establish a mathematical model describing this phenomenon, based on enzyme kinetics for competitive inhibition. We use an objective function based on Haber’s rule, which says that the toxic effect is proportional to the time integral of toxin concentration. The optimal allocation of defence and counter-counter defence can be calculated in an analytical way despite the nonlinearity in the underlying differential equation. The calculation provides a threshold value for the dissociation constant of the inhibitor. Only if the inhibition constant is below that threshold, that is, in the case of strong binding of the inhibitor, it pays to have a counter-counter defence.
    [Show full text]
  • Bsc Chemistry
    Subject Chemistry Paper No and Title 16, Bio-organic and Biophysical Chemistry Module No and Title 30, Enzymes as targets or drug design Module Tag CHE_P16_M30 Chemistry PAPER No. : 16, Bioorganic and biophysical chemistry MODULE No. : 30, Enzymes as targets of drug design TABLE OF CONTENTS 1. Learning outcomes 2. Why Enzymes as Drug Targets? 3. Enzyme structure and Catalysis 4. Concept of Enzyme Inhibition 5. Types of Enzyme Inhibition 5.1. Nonspecific Inhibitors 5.2. Specific Inhibitors 6. Summary Chemistry PAPER No. : 16, Bioorganic and biophysical chemistry MODULE No. : 30, Enzymes as targets of drug design 1. Learning outcomes After studying this module you shall be able to: Know about enzymes as drug targets Learn about enzyme structure and catalysis Know the concept of enzyme inhibition Understand the different types of enzyme inhibition 2. Introduction 2.1 Why Enzymes as Drug Targets? Medicine in twenty first century has become a science in which drug molecules are directed against the macromolecules. Enzymes hold a prominent position among the biological macromolecules that can be used as drug targets. Some features of enzyme structures and reaction pathways which make them attractive and primary target for drug action are as follows They play an essential role in biological life processes and pathophysiology The structures of active sites of enzymes and the ligand binding pockets are highly amenable for high affinity interactions with small drug like molecules Presence of potential allosteric sites Conformational variations in the binding sites Can be directly used as a therapeutic agent 3. Enzyme structure and catalysis Enzymes are biological catalysts as well as can act as receptors by acting with the substrates.
    [Show full text]
  • Imexon and Gemcitabine: Mechanisms of Synergy Against Human Pancreatic Cancer
    Imexon and Gemcitabine: Mechanisms of Synergy against Human Pancreatic Cancer Item Type text; Electronic Dissertation Authors Roman, Nicholas Publisher The University of Arizona. Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 02/10/2021 18:39:06 Link to Item http://hdl.handle.net/10150/194494 IMEXON AND GEMCITABINE: MECHANISMS OF SYNERGY AGAINST HUMAN PANCREATIC CANCER by NICHOLAS ORDUÑO ROMÁN _________________________ A Dissertation Submitted to the Faculty of the COMMITTEE ON PHARMACOLOGY AND TOXICOLOGY (GRADUATE) In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY In the Graduate College THE UNIVERSITY OF ARIZONA 2 0 0 5 2 THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE As members of the Dissertation Committee, we certify that we have read the disser tation prepared by Nicholas Ordu ño Rom án entitled Imexon and Gemcitabine: Mechanisms of Synergy against Human Pancreatic Cancer and recommend that it be accepted as fulfilling the dissertation requirement for the Degree of Doctor of Philosophy _____________________________________ ___________________________ Date:_______________ Robert T. Dorr, Ph.D. 4/12/05 _________ _______________________________________________________ Date:_______________ Terrence J. Monks. Ph.D. 4/12/05 _____ ___________________________________________________________ Date:_______________ Nathan J. Cherrington, Ph.D. 4/12/05 __________________________________________ ______________________ Date:_______________ Garth Powis, D. Phil. 4/12/05 ________ __ ______________________________________________________ Date:_______________ Terry H. Landowski, Ph.D. 4/12/05 Final approval and acceptance of this dissertation is contingent upon the candidate’s submission of the final copies of the dissertatio n to the Graduate College.
    [Show full text]
  • Identification of Regulatory Mechanisms of the Hepatic
    Available online at www.sciencedirect.com Computers and Chemical Engineering 32 (2008) 356–369 Identification of regulatory mechanisms of the hepatic response to thermal injury E. Yang a, T.J. Maguire a, M.L. Yarmush a, F. Berthiaume b, I.P. Androulakis a,∗ a Biomedical Engineering Department, Rutgers University, Piscataway, NJ, USA b Center for Engineering in Medicine/Surgical Services, Massachusetts General Hospital, Harvard Medical School and the Shriners Hospitals for Children, Boston, MA, USA Received 30 October 2006; received in revised form 11 February 2007; accepted 13 February 2007 Available online 25 February 2007 Abstract The purpose of this paper is to evaluate the hypothesis that a systems biology approach can be developed such that a set of transcription factors, relevant to burn-induced inflammatory response can be identified and modulated to control the host response. We explore a novel method for identifying coherent and informative expression motifs and we subsequently determine conserved transcription factor binding sites for the sub-sets of co-expressed genes. The responses are rationalized in the context of burn-induced inflammation and the putative transcription factors are rationalized in the context of intervention targets for controlling gene expression. © 2007 Published by Elsevier Ltd. Keywords: Gene expression; Burn; Inflammation; Transcritpion factors 1. Introduction to thermal injury predispose to the later hypermetabolic state would make it possible to define points of intervention by which Deep thermal injury over greater than 20 percent of the total such outcomes can be avoided. body surface area is one of the most severe forms of trauma. The response to burn injury and trauma results from a com- Following the early acute phase response dealing with the initial plex interplay between inflammation caused by the initial injury injury and shock (Carter, Tompkins, Yarmush, Walker, & Burke, and hypermetabolism.
    [Show full text]
  • Reversible and Mechanism-Based Irreversible Inhibitor Studies on Human Steroid Sulfatase and Protein Tyrosine Phosphatase 1B
    Reversible and Mechanism-Based Irreversible Inhibitor Studies on Human Steroid Sulfatase and Protein Tyrosine Phosphatase 1B by F. Vanessa Ahmed A thesis presented to the University of Waterloo in fulfilment of the thesis requirement for the degree of Doctor of Philosophy in Chemistry Waterloo, Ontario, Canada, 2009 © F. Vanessa Ahmed 2009 Author’s Declaration I hereby declare that I am the sole author of this thesis. This is a true copy of the thesis, including any required final revisions, as accepted by my examiners. I understand that my thesis may be made electronically available to the public. ii Abstract The development of reversible and irreversible inhibitors of steroid sulfatase (STS) and protein tyrosine phosphatase 1B (PTP1B) is reported herein. STS belongs to to the aryl sulfatase family of enzymes that have roles in diverse processes such as hormone regulation, cellular degradation, bone and cartilage development, intracellular communication, and signalling pathways. STS catalyzes the desulfation of sulfated steroids which are the storage forms of many steroids such as the female hormone estrone. Its crucial role in the regulation of estrogen levels has made it a therapeutic target for the treatment of estrogen-dependent cancers. Estrone sulfate derivatives bearing 2- and 4- mono- and difluoromethyl substitutions were examined as quinone methide-generating suicide inhibitors of STS with the goal of developing these small molecules as activity- based probes for proteomic profiling of sulfatases. Kinetic studies suggest that inhibition by the monofluoro derivatives is a result of a quinone methide intermediate that reacts with active-site nucleophiles. However, the main inhibition pathway of the 4- difluoromethyl derivative involved an unexpected process in which initially formed quinone methide diffuses from the active site and decomposes to an aldehyde in solution which then acts as a potent, almost irreversible STS inhibitor.
    [Show full text]
  • Cefoxitin, N-Formimidoyl Thienamycin, Clavulanic
    1594 THE JOURNAL OF ANTIBIOTICS NOV. 1982 CEFOXITIN, N-FORMIMIDOYL THIENAMYCIN, CLAVULANIC ACID, AND PENICILLANIC ACID SULFONE AS SUICIDE INHIBITORS FOR DIFFERENT TYPES OF ~-LACTAMASES PRODUCED BY GRAM-NEGATIVE BACTERIA TETSUO SAWAI and KIKUO TSUKAMOTO Faculty of Pharmaceutical Sciences, Chiba University, Chiba, Japan (Received for publication July 30, 1982) Cefoxitin, N-formimidoyl thienamycin (MK0787), clavulanic acid, and penicillanic acid sulfone (CP45,899) were studied to determine their potency as suicide inhibitors of three very different kinds of Vii-lactamases produced by Gram-negative bacteria: type Ib penicillinase (TEM-2 type), a typical cephalosporinase (the enzyme of Proteus morganii), and a cephalo- sporinase with broad substrate specificity (the enzyme of Proteus vulgaris). All these ~-lactams were confirmed to be quite stable to the three 19-lactamases. The absolute values of the turn- over numbers (kcat) for these enzyme-catalyzed hydrolyses were determined, the values ranged from 0.25 minute-1 to 660 minute-1. All the j3-lactams studied, except cefoxitin, acted as suicide inhibitors of the typical cephalosporinase. Although cefoxitin did not exhibit such an effect, it was a powerful competitive inhibitor of this enzyme. Although the four j3-lactams acted as suicide inhibitors of the P. vulgaris cephalosporinase, the inactivated enzyme partially regained its activity after removing the effect of the free inhibitor. Type lb penicillinase was also inactivated by the four p-lactams, and the activity of the inactivated enzyme, except in the case of cefoxitin, was partially restored. The rate constants for enzyme inactivation or reacti- vation were calculated and are presented. The information obtained from this study suggests that the catalytic center of the P.
    [Show full text]
  • Chapter 8. Enzymes: Basic Concepts and Kinetics
    CHAPTER 8. ENZYMES: BASIC CONCEPTS AND KINETICS coelenterazine The activity of an enzyme is responsible for the glow of the luminescent jellyfish at left. The enzyme aequorin catalyzes the oxidation of a compound by oxygen in the presence of calcium to release CO2 and light CHAPTER 8. ENZYMES: BASIC CONCEPTS AND KINETICS Introduction . Enzymes are large biological molecules that catalyze chemical transformations . The molecule also mediates the transformation of one form of energy into another . 25% of the genes in the human genome encode enzymes . Catalytic power and specificity – important characteristics . Active site – the place where enzymatic reactions take place CHAPTER 8. ENZYMES: BASIC CONCEPTS AND KINETICS Introduction . Nearly all known enzymes are proteins • RNA can function as a catalyst . Proximity effect – enzymes bring substrates closer together in an optimal orientation . They catalyze reactions by stabilizing transition states 8.1 CATALYTIC POWER AND SPECIFICITY Rate enhancement by enzymes . Enzymes accelerate reactions by factors of as much as a million or more 8.1 CATALYTIC POWER AND SPECIFICITY Substrate specificity . A substrate is a molecule upon which an enzyme acts . Some proteases cleaves specific amide bonds in a peptide . The specificity of an enzyme is due to the precise interaction of the substrate with the enzyme . This precision is a result of the intricate 3D structure of the enzyme protein Fig 8.1 Enzyme specificity. (A) Trypsin cleaves on the carboxyl side of arginine and lysine residues, whereas (B) thrombin cleaves Arg-Gly bonds in particular sequences only 8.1 CATALYTIC POWER AND SPECIFICITY Cofactors . The catalytic activity of enzymes depends on the presence of small molecules termed cofactors .
    [Show full text]
  • Crossroads of Antibiotic Resistance and Biosynthesis
    Review Crossroads of Antibiotic Resistance and Biosynthesis Timothy A. Wencewicz Department of Chemistry, Washington University in St. Louis, One Brookings Drive, St. Louis, MO 63130, USA Correspondence to : [email protected] https://doi.org/10.1016/j.jmb.2019.06.033 Abstract The biosynthesis of antibiotics and self-protection mechanisms employed by antibiotic producers are an integral part of the growing antibiotic resistance threat. The origins of clinically relevant antibiotic resistance genes found in human pathogens have been traced to ancient microbial producers of antibiotics in natural environments. Widespread and frequent antibiotic use amplifies environmental pools of antibiotic resistance genes and increases the likelihood for the selection of a resistance event in human pathogens. This perspective will provide an overview of the origins of antibiotic resistance to highlight the crossroads of antibiotic biosynthesis and producer self-protection that result in clinically relevant resistance mechanisms. Some case studies of synergistic antibiotic combinations, adjuvants, and hybrid antibiotics will also be presented to show how native antibiotic producers manage the emergence of antibiotic resistance. © 2019 Elsevier Ltd. All rights reserved. Introduction panel of isogenic Escherichia coli strains. Plazomicin is a semisynthetic aminoglycoside derived from Antibiotics continue to be the most important sisomicin and is designed to chemically block the resource in the global management of infectious sites of chemical modification by aminoglycoside diseases [1,2]. The increased occurrence of antibi- inactivating enzymes, including N-acetyltransferases otic resistance in human pathogens has raised (AACs), O-nucleotidylyltransferases, and O- global concern as antibiotics steadily lose efficacy in phosphotransferases (APHs) [9,10]. Indeed, plazomi- clinical and community settings.
    [Show full text]