| Enzyme Inhibitor |
Article Index for Enzyme |
Shopping Inhibitor |
Website Links For Enzyme |
Information AboutEnzyme Inhibitor |
| CATEGORIES ABOUT ENZYME INHIBITOR | |
| medicinal chemistry | |
| enzymes | |
| metabolism | |
| inhibitors | |
|
Enzyme inhibitors are Molecule s that bind to Enzyme s and decrease their Activity . Since blocking an enzyme's activity can kill a Pathogen or correct a Metabolic imbalance, many drugs are enzyme inhibitors. They are also used as Herbicide s and Pesticide s. Not all molecules that bind to enzymes are inhibitors; '' Enzyme Activator s'' bind to enzymes and increase their Enzymatic Activity . The binding of an inhibitor can stop a Substrate from entering the enzyme's Active Site and/or hinder the enzyme from Catalysing its reaction. Inhibitor binding is either Reversible or irreversible. Irreversible inhibitors usually react with the enzyme and change it chemically. These inhibitors modify key Amino Acid residues needed for enzymatic activity. In contrast, reversible inhibitors bind Non-covalently and different types of inhibition are produced depending on whether these inhibitors bind the Enzyme , the enzyme-substrate complex, or both. Many Drug Molecules are enzyme inhibitors, so their discovery and improvement is an active area of research in Biochemistry and Pharmacology . A medicinal enzyme inhibitor is often judged by its Specificity (its lack of binding to other proteins) and its potency (its Dissociation Constant , which indicates the concentration needed to inhibit the enzyme). A high specificity and potency ensure that a drug will have few Side Effects and thus low Toxicity . Enzyme inhibitors also occur naturally and are involved in the regulation of metabolism. For example, enzymes in a Metabolic Pathway can be inhibited by downstream products. This type of Negative Feedback slows flux through a pathway when the products begin to build up and is an important way to maintain Homeostasis in a Cell . Other cellular enzyme inhibitors are Protein s that specifically bind to and inhibit an enzyme target. This can help control enzymes that may be damaging to a cell, such as Protease s or Nuclease s; a well-characterised example is the Ribonuclease Inhibitor , which binds to Ribonuclease s in one of the tightest known Protein–protein Interaction s.Shapiro R, Vallee BL. ''Interaction of human placental ribonuclease with placental ribonuclease inhibitor.'' Biochemistry. 1991 Feb 26;30(8):2246–55. PMID 1998683 Natural enzyme inhibitors can also be poisons and are used as defenses against predators or as ways of killing prey. REVERSIBLE INHIBITORS Types of reversible inhibitor Reversible inhibitors bind to enzymes with non-covalent interactions such as Hydrogen Bond s, Hydrophobic Interaction s and Ionic Bond s. Multiple weak bonds between the inhibitor and the active site combine to produce strong and specific binding. In contrast to Substrate s and irreversible inhibitors, reversible inhibitors generally do not undergo chemical reactions when bound to the enzyme and can be easily removed by dilution or dialysis. There are three kinds of reversible enzyme inhibitors. They are classified according to the effect of varying the concentration of the enzyme's substrate on the inhibitor.Berg J., Tymoczko J. and Stryer L. (2002) ''Biochemistry.'' W. H. Freeman and Company ISBN 0-7167-4955-6
Quantitative description of reversible inhibition Reversible inhibition can be described quantitatively in terms of the inhibitor's Binding to the enzyme and to the enzyme–substrate complex, and its effects on the Kinetic Constants of the enzyme. In the classic Michaelis–Menten Scheme below, an enzyme (E) binds to its substrate (S) to form the enzyme–substrate complex ES. Upon catalysis, this complex breaks down to release product P and free enzyme. The inhibitor (I) can bind to either E or ES with the Dissociation Constant s ''K''i or ''K''i', respectively.
Measuring the dissociation constants of a reversible inhibitor As noted above, an enzyme inhibitor is characterized by its two : where the modifying factors α and α' are defined by the inhibitor concentration and its two dissociation constants : : Thus, in the presence of the inhibitor, the enzyme's effective ''K''m and ''V''max become (α/α')''K''m and (1/α')''V''max, respectively. However, the modified Michaelis-Menten equation assumes that binding of the inhibitor to the enzyme has reached equilibrium, which may be a very slow process for inhibitors with sub-nanomolar dissociation constants. In these cases, it is usually more practical to treat the tight-binding inhibitor as an irreversible inhibitor (see below); however, it can still be possible to estimate ''K''i' kinetically if ''K''i is measured independently. The effects of different types of reversible enzyme inhibitors on enzymatic activity can be visualized using graphical representations of the Michaelis–Menten equation, such as methods, as described above. Special cases
Examples of reversible inhibitors ]] As enzymes have evolved to bind their substrates tightly, and most reversible inhibitors bind in the active site of enzymes, it is unsurprising that some of these inhibitors are strikingly similar in structure to the substrates of their targets. An example of these substrate mimics are the , a protease inhibitor based on a peptide and containing three Peptide Bond s, is shown on the right. As this drug resembles the protein that is the substrate of the HIV protease, it competes with this substrate in the enzyme's active site. Enzyme inhibitors are often designed to mimic the Transition State or intermediate of an enzyme-catalysed reaction. This ensures that the inhibitor exploits the transition state stabilising effect of the enzyme, resulting in a better binding affinity (lower ''K''i) than substrate-based designs. An example of such a transition state inhibitor is the antiviral drug Oseltamivir ; this drug mimics the planar nature of the ring Oxonium Ion in the reaction of the viral enzyme Neuraminidase . ]] However, not all inhibitors are based on the structures of substrates. For example, the structure of another HIV protease inhibitor Tipranavir is shown on the left. This molecule is not based on a peptide and has no obvious structural similarity to a protein substrate. These non-peptide inhibitors can be more stable than inhibitors containing peptide bonds, because they will not be substrates for Peptidase s and are less likely to be degraded in the cell. In drug design it is important to consider the concentrations of substrates to which the target enzymes are exposed. For example, some Protein Kinase inhibitors have chemical structures that are similar to Adenosine Triphosphate , one of the substrates of these enzymes. However, drugs that are simple competitive inhibitors will have to compete with the high concentrations of ATP in the cell. Protein kinases can also be inhibited by competition at the binding sites where the kinases interact with their substrate proteins, and most proteins are present inside cells at concentrations much lower than the concentration of ATP. As a consequence, if two protein kinase inhibitors both bind in the active site with similar affinity, but only one has to compete with ATP, then the competitive inhibitor at the protein-binding site will inhibit the enzyme more effectively.Bogoyevitch MA, Barr RK, Ketterman AJ. ''Peptide inhibitors of protein kinases—discovery, characterisation and use.'' Biochim Biophys Acta. 2005 Dec 30;1754(1–2):79–99. PMID 16182621 IRREVERSIBLE INHIBITORS Types of irreversible inhibition Irreversible inhibitors usually Covalent ly modify an enzyme, and inhibition cannot therefore be reversed. Irreversible inhibitors often contain reactive functional groups such as Nitrogen Mustard s, Aldehyde s, Haloalkane s or Alkene s. These Electrophilic groups react with amino acid side chains to form covalent adducts. The residues modified are those with side chains containing Nucleophile s such as Hydroxyl or Sulfhydryl groups; these include the amino acids Serine (as in DFP , right), Cysteine , Threonine or Tyrosine .Lundblad R. L. ''Chemical Reagents for Protein Modification'' CRC Press Inc (2004) ISBN 0-8493-1983-8 Irreversible inhibition is different from irreversible enzyme inactivation. Irreversible inhibitors are generally specific for one class of enzyme and do not inactivate all proteins; they do not function by destroying will hydrolyse the Peptide Bond s holding proteins together, releasing free amino acids.N. Price, B. Hames, D. Rickwood (Ed.) ''Proteins LabFax'' Academic Press (1996) ISBN 0-12-564710-7 Analysis of irreversible inhibition
The binding and inactivation steps of this reaction are investigated by incubating the enzyme with inhibitor and assaying the amount of activity remaining over time. The activity will be decrease in a time-dependent manner, usually following Exponential Decay . Fitting these data to a Rate Equation gives the rate of inactivation at this concentration of inhibitor. This is done at several different concentrations of inhibitor. If a reversible EI complex is involved the inactivation rate will be saturable and fitting this curve will give ''k''inact and ''K''i.Maurer T, Fung HL. ''Comparison of Methods for Analyzing Kinetic Data From Mechanism-Based Enzyme Inactivation: Application to Nitric Oxide Synthase.'' AAPS PharmSci. (2000) 2(1)E8. PMID 11741224 Another method that is widely used in these analyses is Mass Spectrometry . Here, accurate measurement of the mass of the unmodified native enzyme and the inactivated enzyme gives the increase in mass caused by reaction with the inhibitor and shows the stoichiometry of the reaction. This is usually done using a MALDI-TOF mass spectrometer. In a complementary technique, Peptide Mass Fingerprinting involves digestion of the native and modified protein with a Protease such as Trypsin . This will produce a set of Peptide s that can be analysed using a mass spectrometer. The peptide that changes in mass after reaction with the inhibitor will be the one that contains the site of modification. Special cases
Examples of irreversible inhibitors with the lower molecule of an inhibitor bound irreversibly and the upper one reversibly. Created from PDB 1GXF .]] Diisopropylfluorophosphate (DFP) is shown as an example of an irreversible protease inhibitor in the figure Above Right . The enzyme hydrolyses the phosphorus–fluorine bond, but the phosphate residue remains bound to the serine in the Active Site , deactivating it.J. A. Cohen, R. A. Oosterbaan and F. Berends ''Organophosphorus compounds'' Meth. Enzymol. (1967) 11, 686 Similarly, DFP also reacts with the active site of Acetylcholine Esterase in the Synapses of neurons, and consequently is a potent neurotoxin, with a lethal dose of less than 100 mg.Brenner, G. M. (2000): ''Pharmacology.'' Philadelphia, PA: W.B. Saunders Company. ISBN 0-7216-7757-6 Suicide Inhibition is a unusual type of irreversible inhibition where the enzyme converts the inhibitor into a reactive form in its active site. An example is the inhibitor of Polyamine biosynthesis, α-difluoromethylornithine or DFMO, which is an analogue of the amino acid Ornithine , and is used to treat African Trypanosomiasis (sleeping sickness). Ornithine Decarboxylase can catalyse the decarboxylation of DFMO instead of ornithine, as shown above. However, this decarboxylation reaction is followed by the elimination of a fluorine atom, which converts this catalytic intermediate into a conjugated Imine , a highly electrophilic species. This reactive form of DFMO then reacts with either a cysteine or lysine residue in the active site to irreversibly inactivate the enzyme. Since irreversible inhibition often involves the initial formation of a non-covalent EI complex, it is sometimes possible for an inhibitor to bind to an enzyme in more than one way. For example, in the figure showing Trypanothione Reductase from the human protozoan parasite '' Trypanosoma Cruzi '', two molecules of an inhibitor called ''quinacrine mustard'' are bound in its active site. The top molecule is bound reversibly, but the lower one is bound covalently as it has reacted with an amino acid residue through its Nitrogen Mustard group.Saravanamuthu A, Vickers TJ, Bond CS, Peterson MR, Hunter WN, Fairlamb AH. ''Two interacting binding sites for quinacrine derivatives in the active site of trypanothione reductase: a template for drug design.'' J Biol Chem. 2004 Jul 9;279(28):29493–500. PMID 15102853 DISCOVERY AND DESIGN OF INHIBITORS New drugs are the products of a long approaches that quickly produce large numbers of novel compounds and High-throughput Screening technology to rapidly screen these huge chemical libraries for useful inhibitors. More recently, an alternative approach has been applied: Rational Drug Design uses the Three-dimensional Structure of an enzyme's active site to predict which molecules might be inhibitors. These predictions are then tested and one of these tested compounds may be a novel inhibitor. This new inhibitor is then used to try to obtain a structure of the enzyme in an inhibitor/enzyme complex to show how the molecule is binding to the active site. This structure is then inspected and changes made to the inhibitor to try to optimise binding. This test and improve cycle is then repeated until a sufficiently potent inhibitor is produced. Typically, this process aims to produce an inhibitor with a dissociation constant of <10-9 M .Hunter WN. ''Rational drug design: a multidisciplinary approach.'' Mol Med Today. 1995 Apr;1(1):31, 34. PMID 9415135 USES OF INHIBITORS Enzyme inhibitors are found in nature and are also designed and produced as part of Pharmacology and Biochemistry . Natural Poison s are often enzyme inhibitors that have evolved to defend a plant or animal against Predators . These natural toxins include some of the most poisonous compounds known today. Artificial inhibitors are often used as drugs, but can also be Insecticide s such as Malathion , Herbicide s such as Glyphosate , or Disinfectants such as Triclosan . Chemotherapy The most common uses for enzyme inhibitors are as drugs to treat disease. Many of these inhibitors target a human enzyme and aim to correct a pathological condition. However, not all drugs are enzyme inhibitors. Some, such as Anti-epileptic Drugs , alter enzyme activity by causing more or less of the enzyme to be produced. These effects are called Enzyme Induction And Inhibition and are alterations in Gene Expression , which is unrelated to the type of enzyme inhibition discussed here. Other drugs interact with cellular targets that are not enzymes, such as Ion Channel s or Membrane Receptors . An interesting example of a medicinal enzyme inhibitor is , which causes an erection. Since the drug decreases the activity of the enzyme that halts the signal, it makes this signal last for a longer period of time. Another example of the structural similarity of some inhibitors to the substrates of the enzymes they target is seen in the figure comparing the drug Methotrexate to Folic Acid . Folic acid is the oxidised form of the substrate of Dihydrofolate Reductase , an enzyme that is potently inhibited by methotrexate. Methotrexate blocks the action of dihydrofolate reductase and thereby halts Thymidine biosynthesis. This block of Nucleotide biosynthesis is selectively toxic to rapidly growing cells, therefore methotrexate is often used in cancer Chemotherapy .McGuire JJ. ''Anticancer antifolates: current status and future directions.'' Curr Pharm Des. 2003;9(31):2593–613. PMID 14529544 Drugs also are used to inhibit enzymes needed for the survival of from the bacteria ''Streptomyces'' R61 (the protein is shown as a Ribbon-diagram ). Drug Design is facilitated when an enzyme that is essential to the pathogen's survival is absent or very different in humans. In the example above, humans do not make peptidoglycan, therefore inhibitors of this process are selectively toxic to bacteria. Selective toxicity is also produced in antibiotics by exploiting differences in the structure of the Ribosome s in bacteria, or how they make Fatty Acid s. Metabolic control Enzyme inhibitors are also important in metabolic control. Many Metabolic Pathway s in the cell are inhibited by Metabolite s that control enzyme activity through Allosteric Regulation or substrate inhibition. A good example is the allosteric regulation of the Glycolytic Pathway . This Catabolic pathway consumes Glucose and produces ATP , NADH and Pyruvate . A key step for the regulation of glycolysis is an early reaction in the pathway catalysed by Phosphofructokinase-1 (PFK1). When ATP levels rise, ATP binds an allosteric site in PFK1 to decrease the rate of the enzyme reaction; glycolysis is inhibited and ATP production falls. This Negative Feedback control helps maintain a steady concentration of ATP in the cell. However, metabolic pathways are not just regulated through inhibition since enzyme activation is equally important. With respect to PFK1, Fructose 2,6-bisphosphate and ADP are examples of metabolites that are allosteric activators.Okar DA, Lange AJ. ''Fructose-2,6-bisphosphate and control of carbohydrate metabolism in eukaryotes.'' Biofactors. 1999;10(1):1–14. Physiological enzyme inhibition can also be produced by specific protein inhibitors. This mechanism occurs in the s.Oliver CJ, Shenolikar S. ''Physiologic importance of protein phosphatase inhibitors.'' Front Biosci. 1998 Sep 1;3:D961–72. PMID 9727084 Acetylcholinesterase inhibitors Acetylcholinesterase (AChE) is an enzyme found in animals from insects to humans. It is essential to nerve cell function through its mechanism of breaking down the neurotransmitter Acetylcholine into its constituents, Acetate and Choline . This is somewhat unique among neurotransmitters as most, including Serotonin , Dopamine , and Norepinephrine , are absorbed from the Synaptic Cleft rather than cleaved. A large number of AChE inhibitors are used in both medicine and agriculture. Reversible competitive inhibitors, such as Edrophonium , Physostigmine , and Neostigmine , are used in the treatment of Myasthenia Gravis and in anaesthesia. The Carbamate pesticides are also examples of reversible AChE inhibitors. The Organophosphate insecticides such as Malathion , Parathion , and Chlorpyrifos irreversibly inhibit acetylcholinesterase. Natural poisons s, pulses contain Trypsin Inhibitor s that interfere with digestion.]] Animals and plants have evolved to synthesize a vast array of poisonous products including (taxol), an organic molecule found in the Pacific Yew Tree , binds tightly to Tubulin dimers and inhibits their assembly into Microtubule s in the Cytoskeleton .Abal M, Andreu JM, Barasoain I. ''Taxanes: microtubule and centrosome targets, and cell cycle dependent mechanisms of action.'' Curr Cancer Drug Targets. 2003 Jun;3(3):193–203. PMID 12769688 Many natural poisons act as s, from the plant species in the '' Solanaceae '' family (includes Potato , Tomato and Eggplant ), that are Acetylcholinesterase inhibitors. Inhibition of this enzyme causes an uncontrolled increase in the acetylcholine neurotransmitter, muscular paralysis and then death. Neurotoxicity can also result from the inhibition of receptors; for example, Atropine from deadly nightshade ('' Atropa Belladonna '') that functions as a Competitive Antagonist of the Muscarinic Acetylcholine Receptors .DeFrates LJ, Hoehns JD, Sakornbut EL, Glascock DG, Tew AR. ''Antimuscarinic intoxication resulting from the ingestion of moonflower seeds.'' Ann Pharmacother. 2005 Jan;39(1):173-6. PMID 15572604 Although many natural toxins are secondary metabolites, these poisons also include peptides and proteins. An example of a toxic peptide is s and is a known carcinogen that can also cause acute liver hemorrhage and death at higher doses.Bischoff K. ''The toxicology of microcystin-LR: occurrence, toxicokinetics, toxicodynamics, diagnosis and treatment.'' Vet Hum Toxicol. 2001 Oct;43(5):294-7. PMID 11577938 Proteins can also be natural poisons, such as the , an extremely potent protein toxin found in Castor Oil Beans . This enzyme is a glycosidase that inactivates ribosomes. Since ricin is a catalytic irreversible inhibitor, this allows just a single molecule of ricin to kill a cell.Hartley MR, Lord JM. ''Cytotoxic ribosome-inactivating lectins from plants.'' Biochim Biophys Acta. 2004 Sep 1;1701(1–2):1–14. PMID 15450171 SEE ALSO
REFERENCES EXTERNAL LINKS
|
|
|