suicide inhibitors

SUICIDEINHIBITORS

Guided by

Dr. V. M. Motghare

Dr. Swarnank Parmar

JR-1

Dept. Of Pharmacology

GMC Nagpur

Overview

• Introduction• Types of Inhibition• Suicide inhibitors• Clinical examples• Conclusion• References

Introduction

• “Suicide inhibition, also known as suicide inactivation, is a form of irreversible enzyme inhibition that occurs when an enzyme binds a substrate analogue and forms an irreversible complex with it through a covalent bond during the "normal" catalysis reaction”

• Inhibitor binds to active site where it is modified by the enzyme to produce a reactive group that reacts irreversibly to form a stable inhibitor-enzyme complex

• Inhibitor has a functional group, usually a leaving group, that is replaced by a nucleophile in the enzyme active site

• Reaction with suicide inhibitor removes active enzyme from the system; this removal is measured as inhibition

ReversibleReversible

IrreversibleIrreversible

Type of Type of InhibitorsInhibitors

CompetitiveCompetitive

UncompetitiveUncompetitive

Non- CompetitiveNon- Competitive

Active Site Active Site DirectedDirected

SuicideSuicideInhibitorsInhibitors

• A special group of irreversible inhibitors is known as suicide inhibitors

• A suicide inhibitor is a relatively inert molecule that is transformed by an enzyme at its active site into a reactive compound that irreversibly inactivates the enzyme

• Relatively unreactive until they bind to the active site of the enzyme

Suicide Inhibitors

• First few steps of the reaction it functions like a normal substrate, but then it is converted into a very reactive compound that combines with the enzyme to block its activity

• They use the normal enzyme reaction mechanism to inactivate the enzyme, they are also known as mechanism-based inhibitors or transition state analogs

• Suicide inhibitors that exploit the transition state-stabilizing effect of the enzyme result in higher enzyme binding affinity than do substrate-based inhibitor designs

• Designing drugs that precisely mimic the transition state is a real challenge because of the unstable, poorly characterized structure of the transition state

• Prodrugs undergo one or more initial reactions to form an overall electrostatic and three-dimensional intermediate transition state complex form with close similarity to that of the substrate

• Serves as guidelines to further develop transition state molecules with modifications

• Such inhibitors are called suicide inhibitors because the Such inhibitors are called suicide inhibitors because the enzyme appears to commit suicideenzyme appears to commit suicide

• A common example of a suicide inhibitor is Allopurinol, the anti-gout drug that inhibits xanthine oxidase activity. The enzyme commits suicide by initially activating Allopurinol into Oxypurinol (a transition state analog) that binds very tightly to the molybdenum-sulfide (Mo-S) complex at the active site of xanthine oxidase

• Acyclovir is one of the most commonly used antiviral agents with very low toxicity. It is selectively converted into acyclo-guanosine monophosphate (acyclo-GMP) by viral thymidine kinase

• Acyclo-GMP is further phosphorylated into the active triphosphate form, acyclo-GTP, by cellular kinases

• Acyclo-GTP is a very potent inhibitor of viral DNA polymerase with over 100-fold greater affinity for viral than cellular polymerase.

• It is incorporated into viral DNA, resulting in chain termination

• The suicide inhibitor removes enzyme and reduces the formation of ES complex. The Vmax value is reduced and inhibition cannot be overcome by adding extra substrate. In this regard, suicide inhibition resembles non-competitive inhibition

Some clinical examples of suicide inhibitors

• 5-fluorouracil acts as a suicide inhibitor of thymidylate synthase during the synthesis of thymine from uridine

• Reaction is crucial for proliferation of cells, particularly those that are rapidly proliferating (such as fast-growing cancer tumors)

During thymidylate synthesis, NDuring thymidylate synthesis, N55,N,N1010- methyleneTHF is converted - methyleneTHF is converted to 7,8-dihydrofolate; methyleneTHF is regenerated in two stepsto 7,8-dihydrofolate; methyleneTHF is regenerated in two steps

• Inhibition causes cell death from lack of thymine to create more DNA

• This is often used in combination with Methotrexate, a potent inhibitor of dihydrofolate reductase enzyme

• Aspirin, which inhibits cyclooxygenase- Aspirin irreversibly inhibits COX-1 and modifies the enzymatic activity of COX-2

• Low doses of aspirin (75mg-81mg/day) sufficient to irreversibly acetylate serine530 of COX-1

• Inhibits platelets generation of TXA2 – antithrombin effect

• Intermediate doses (650mg-4g/day) inhibit COX-1 & COX-2 blocking PG production

• Penicillin, which inhibits DD-transpeptidase from building bacterial cell walls- Penicillin blocks the formation of the cross-link between the N-acetylmuramic acid and N-acetylglucosamine

• Benzothiazinones (BTZs) - antituberculosis drug, are suicide substrates of the FAD-dependent decaprenylphosphoryl-β-D-ribofuranose 2'-oxidase DprE1, an enzyme involved in cell-wall biogenesis

• Serine proteases are attractive targets for the design of enzyme inhibitors

• Within the class of serine proteases, Human Leucocyte Elastase(HLE) is one of the most destructive enzymes in the body

• Numerous inhibitors of serine proteases reported during the past three decades

• Coumarin-type molecules displayed high inhibitory potency towards various serine proteases

• Halomethyl dihydrocoumarins - shown to behave as the first general suicide inhibitors of serine protease

• Inhibit several proteases - human leucocyte elastase, porcine pancreatic elastase, thrombin, urokinase and human plasmin

• Series of mechanism-based inhibitors of Phospholipase A2 (SIBLINKS) were synthesized

• Phospholipid analogues that contain a para-substituted phenyl 3,3-dimethylglutaryl group in the place of the sn-2 acyl chain

• Effect of the phenyl leaving group on inhibitory activity was studied by varying electron-withdrawing ability of the para-substituted group

• Strong correlation observed between leaving group potential of suicide inhibitor and inhibitory activity of the derivative toward cobra venom phospholipase A2.

• TCAs act primarily as serotonin-norepinephrine reuptake inhibitors (SNRIs)

• Blocks serotonin transporter (SERT) and nor-epinephrine transporter (NET)-elevation of the synaptic concentrations of neurotransmitters

• Clavulanic acid is a suicide inhibitor, covalently bonding to a serine residue in the active site of the β-Lactamase

• Restructures clavulanic acid molecule, creating a more reactive species - attacked by another amino acid in the active site, permanently inactivating the enzyme

• This inhibition restores the antimicrobial activity of β-lactam antibiotics against lactamase-secreting resistant bacteria

• AZT (zidovudine) and other chain-terminating nucleoside analogues used to selectively inhibit HIV-1 reverse transcriptase in the treatment of HIV/AIDS

• Exemestane is an irreversible, steroidal aromatase inactivator, structurally related to the natural substrate androstenedione binds irreversibly to the active site of the enzyme causing its inactivation, an effect also known as suicide inhibition

• Sulbactum is an irreversible inhibitor of ß–lactamase, which prohibits penicillin-resistant strains of bacteria from metabolizing penicillin

• Sarin (Organophosphorus compound) is a potent suicide inhibitor of acetylcholinesterase degrading neurotransmitter Acetylcholine after its release

• Benzotriazole esters(used in SARS) reported as potent nonpeptidic suicide inhibitors of the enzyme proteinase, acts at active-site cysteine, acylated by benzotriazole esters

Selegiline (selective irreversible MAO-B inhibitor) although in the attached reference the compound is called a 'suicide inactivator' (not inhibitor)

• Serpins are a group of proteins with similar structures that were first identified as a set of proteins able to inhibit proteases

• Serpins are suicide inhibitors

• Serpin1 of Arabidopsis thaliana is a Suicide Inhibitor for Metacaspase 9

• Serpins are referred to as suicide inhibitors because they are cleaved by their target proteases

• Vigabatrin, an anticonvulsant is a irreversible suicide inhibitor of GABA-Transaminase (responsible for catabolism of GABA) increasing level of GABA in brain

• ß-haloamines represent a new series of suicide inhibitors of lysyl oxidase which can inactivate the enzyme faster than BAPN (ß-aminoproprionitrile) and may have antifibrotic potential

• Plasminogen activator inhibitor-1(PAI-1), representative of the serpin superfamily is the major regulator of tissue-type (tPA) and urokinase-type (uPA) plasminogen activators in vivo

• PAI-1 plays a role in the control of normal fibrinolysis as well as a number of physiological processes that are dependent on plasminogen activation

• PAI-1 acts as a suicide inhibitor that inactivates target proteinases by trapping a stabilized acyl enzyme intermediate

• Significant structural changes accompanying the reaction result in efficient and essentially irreversible impairment of the proteinase mechanism

• Eflornithine, one of the drugs used to treat sleeping sickness, is a suicide inhibitor of Ornithine Decarboxylase(ODC) preventing the natural substrate Ornithine from accessing the active site

• DFMO (difluoromethylornithine) and DFMA (difluoromethylarginine), irreversible suicide inhibitors of ornithine and arginine decarboxylase activities (ODC and ADC) respectively

• Inhibit the growth of six species of Microsporum and six species of Trichophyton

Hit and Run drugs

• Are the drugs whose effect lasts much longer than the drug itself

• Because, drug with a relatively short t½ still able to produce effect long after it has been eliminated - its intracellular action and partial binding to the receptor long after most of the extracellular drug has been eliminated

• Eg. Reserpine, Omeprazole, MAO inhibitors, Methyldopa, etc.

Name of the drug

Category Mechanism of action

T1/2

(hrs.)

Uses

Reserpine Adrenergic neuron blocking agent

Blocks the ability of aminergic transmitter vesicles to take up & store biogenic amines

24-48 Antihypertensive(rarely used)

Omeprazole Proton pump inhibitor

Blocks final common pathway of acid secretion in parietal cells

0.5-1.5 Peptic, gastric, duodenal ulcers & GERD

Methyldopa Centrally acting sympathetic drug

Activates α2 adrenoceptors

2 Hypertension

Conclusion

• Special group of irreversible inhibitors relatively unreactive until they bind to active site of enzyme

• Knowledge is highly useful in developing pharmaceutical agents with minimal side effects

• Designing drugs that precisely mimic the transition state - a real challenge because of unstable, poorly characterized structure of transition state

• Main goal of this approach - to create substrates that are unreactive and non-toxic

• Become active within that enzyme's active site and at the same time being highly specific for that enzyme

• Drugs based on this approach have the advantage of very few resulting side effects

• Suicide inhibitors are used in what is called "rational drug design" where the aim is to create a novel substrate, based on already known mechanisms and substrates

References • Journal of chemistry & biology 2008.04.011 -A ‘Structural View of the Inactivation of the SARS

Coronavirus Main Proteinas by Benzotriazole Esters’• ‘Development and evolution of therapies targeted to

the estrogen receptor for the treatment and prevention of breast cancer’ 2007 January;72(1): 7–25. doi:10.1016/j.steroids.2006.10.009.

• Bioorg Med Chem Lett. 1998 Aug 18;8(16): 2129-32. ‘The structure-activity relationships of a series of suicide inhibitors of phospholipase A2’

• ‘Benzothiazinones are suicide inhibitors of mycobacterial decaprenylphosphoryl-β-D-ribofuranose 2'-oxidase DprE1’ 2011 American Chemical Society

• Journal of American Chemical Society -2009 Mar 4;131(8):3057-62. doi: 10.1021/ja809683v

• Journal of American Chemical Society -2012 Jan 18;134(2):912-5. doi: 10.1021/ja211042r. Epub 2011 Dec 21