Enzyme inhibitors C6215 - Lecture 9 Igor Kučera C 6215 Lecture 9 IgorKučera slope = KM vlim ( 1 + ) [I] Kis intercept y = 1 vlim ( 1 + ) [I] Kii Reversible inhibition C 6215 Lecture 9 IgorKučera Tuley and Fast, Biochemistry 2018, 57, 3326 C 6215 Lecture 9 IgorKučera Iwanami et al., Biosci. Biotech. Biochem. 1995, 59, 459 k = 4.2 × 10-3 s-1 K = 3.1 mM kobs values An example of an affinity labeling experiment C 6215 Lecture 9 IgorKučera Rask-Andersen et al., Nature Rev. 2011, 10, 579 Drug targets C 6215 Lecture 9 IgorKučera Inhibition of HMG-CoA reductase Reduction of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) to mevalonate, catalyzed by the HMG-CoA reductase, is the major point of regulation on the pathway to cholesterol Statins, originally discovered by a Japanese biochemist Akira Endo (*1933) in moulds, are effective in reducing the low-density cholesterol (LDL-C) by inhibiting HMG-CoA reductase. The first statin commercially marketed (Merck) was lovastatin isolated from the mould Aspergillus terreus. There are various other statins in the market. Statin side effects include: muscle pain (myalgia), muscle disease (myopathy) and liver abnormalities. Since statins block production of farnesyl diphosphate, an intermediate in the biosynthesis of coenzyme Q10, statin-induced CoQ10 deficiency may be instrumental in the pathogenesis of myopathy. Endo, Proc. Jpn. Acad., Ser. B, 2010, 86, 484 Ginter and Simko, Bratisl. Lek. Listy 2009, 110, 664 C 6215 Lecture 9 IgorKučera Inhibition of angiotensin-converting enzyme (ACE) https://www.ncbi.nlm.nih.gov/books/NBK82803 ACE is located in the capillaries of the lungs and in endothelial and kidney epithelial cells. It is a zinc metalloprotease that converts the inactive decapeptide angiotensin I to the octapeptide angiotensin II by removing the dipeptide His-Leu from the C-terminus. Angiotensin II binds to the type 1 angiotensin II receptor (AT1), which sets off a number of actions that result in vasoconstriction and increased blood pressure. ACE also inactivates bradykinin, a vasodilator nonapeptide. inhibitor Ki (nM) captopril 1.1 enalaprilate 0.78 lisinopril 0.27 Wang et al., J. Chem. Inf. Model 2011, 51, 1074 Hippuryl-His-Leu lisinopril C 6215 Lecture 9 IgorKučera Inhibition of xanthine oxidase/dehydrogenase allopurinol* febuxostat oxypurinol Pacher et al., Pharmacol. Rev. 2006, 58, 87 Gout is a disorder of purine metabolism. The final metabolite, uric acid, crystallizes in the form of monosodium urate, precipitating and forming deposits (tophi) in joints, on tendons, and in the surrounding tissues. This is accompanied by recurrent attacks of acute arthritic joint inflammation. Treatment with XO/XDH inhibitors results in an accumulation of xanthine and hypoxanthine – compounds more soluble than uric acid. *Allopurinol is converted to oxypurinol (alloxanthin) which binds tightly to Mo(IV) of the molybdopterin cofactor. C 6215 Lecture 9 IgorKučera Inhibition of gastric proton pump Hydrochloric acid secretion by the parietal cells Gastric juice: 155 mM HCl (pH 0.8) 15 mM KCl http://www.vivo.colostate.edu/hbooks/pathphys/digestion/stomach/parietal.html -SH -S-After acid transformation to sulfenamides the drugs bind covalently to sulfhydryl groups of cysteines of the proton pump. omeprazole Jain et al., Bioorg. Med. Chem. 2007, 15, 1181 C 6215 Lecture 9 IgorKučera The androgen hormone testosterone is secreted in the adrenal glands and ovaries in women and in the adrenal glands and testes in men. T is taken from circulation to cells and converted to dihydrotestosterone (DHT), the preferred ligand for androgen receptor (AR), by the enzyme 5 alpha-reductase (5αR). Finasteride is a synthetic 4-azasteroid and is the first 5α-RI approved for treatment of benign prostatic enlargement (BPE) and subsequently androgenic alopecia. testosterone dihydrotestosterone Azzouni et al., Adv. Urol. 2012, 530121 Inhibition of 5 alpha-reductase C 6215 Lecture 9 IgorKučera Inhibition of phosphodiesterase-5 Jeon et al., Cell. Mol. Life Sci. 2005, 62, 1198 Nitric oxide (NO) released from nerve endings in the penis and from endothelial cells activates soluble guanylate cyclase in smooth muscle cells. This results in increased levels of cGMP, activation of PKG, decreased Ca2+, muscle relaxation and penile erection. PDE5 degrades cGMP. Sildenafil fits into the same site of PDE5 as cGMP, thus inhibiting the enzyme. (Viagra) C 6215 Lecture 9 IgorKučera Prostaglandins have diverse effects, including the transmission of pain information to the brain, modulation of the hypothalamic thermostat, and inflammation. TXA2, which is formed from PGH2 via the enzyme thromboxane synthase, acts as a potent vasoconstrictor and inducer of platelet aggregation. Aspirin (acetylsalicylic acid ) is widely used to treat pain, fever, or inflammation and as a long term treatment to prevent heart attacks, ischaemic strokes, and blood clots in people at high risk. Aspirin irreversibly acetylates a serine residue of cyclooxygenase, thereby blocking synthesis of prostaglandin G2 and H2, and consequently thromboxane A2 generation. Inhibition of cyclooxygenases PGE2 Sweeny et al., Nature Rev. Cardiol. 2009, 6, 273 Hochgesang et al., J. Am. Chem. Soc. 2000, 122, 6514 C 6215 Lecture 9 IgorKučera Joshi S.N. et al. Cancer Treat. Res. Commun. 2021, 26, 100267 C 6215 Lecture 9 IgorKučera Tetrahydrofolate, a metabolically active form of the vitamin folic acid, is involved in onecarbon metabolism, which includes the synthesis of purines and thymidylate for DNA synthesis. Methotrexate is a slow and tight binding competitive inhibitor of dihydrofolate reductase (DHFR) (Ki=5 pM). Prevention of THF regeneration causes a toxic response on cells performing DNA replication, especially on rapidly proliferating cells. The drug is used in anticancer antimetabolite chemotherapy particularly for pediatric leukemia. Inhibition of dihydrofolate reductase C 6215 Lecture 9 IgorKučera hydrolysis self-destruction (minimized by attaching a suitable substituent R) https://www.futurelearn.com/courses /everyday-chemistry/0/steps/22314 Sauvage and Terrak, Antibiotics 2016, 5, 12 irreversible inactivation of transpeptidase Inhibition of bacterial transpeptidase by beta-lactam antibiotics meso-diaminopimelate side chain D-AlaTP: transpeptidase GT: glykosyltransferase C 6215 Lecture 9 IgorKučera Liao et al., J. Oral Microbiol. 2017, 9 Antimicrobial action of fluoride HF diffusion into cells and dissociation to H+ and FInhibition of enzymes: • enolase • F-ATPase • PPase • urease • arginine deiminase Fluoride metal complexes as phosphate mimics Enolase - Mg2F2Pi inhibitory complex Qin et al., Biochemistry 2006, 45, 793 C 6215 Lecture 9 IgorKučera Inhibition of viral reverse transcription emtricitabine tenofovir disoproxil tenofovir tenofovir phosphate tenofovir diphosphate an acyclic phosphonate nucleotide adenylate kinase NDP kinase a nucleoside emtricitabine triphosphate termination of DNA elongation C 6215 Lecture 9 IgorKučera https://medicalxpress.com/news/2013-02-flu-drug-virus-tracks.html sialic acid glycan transition state sialic acid oseltamivir transition state mimic https://basicmedicalkey.com/study-neuraminidase-inhibitors-and-the-influenza-virus/ Inhibition of viral neuraminidase (sialidase) Sialidase is the influenza virus surface enzyme recognizing the sialic acid N-acetylneuraminic acid (Neu5Ac) moiety, which is typically associated as α-linked terminal saccharidic unit of mammalian glycoconjugates. Its inhibition decreases the release of virus from infected cells. Neu5Ac zanamivir (Relenza) oseltamivir (Tamiflu) C 6215 Lecture 9 IgorKučera Pesticides are substances that are meant to control pests, including weeds. Pesticides Target organism: • herbicides • insecticides • rodenticides • ... Chemical structure: • organic • inorganic • biological • ... Target site of herbicides: • photosynthesis • pigment biosynthesis o protoporphyrinogen oxidase o 4-hydroxyphenylpyruvate dioxygenase • lipid biosynthesis • amino acid biosynthesis o glutamine synthetase o EPSP synthase o acetolactate synthase • … C 6215 Lecture 9 IgorKučera In the photosynthetic electron transport chain, plastoquinone (PQ) binds to the D1 protein in the PS II reaction center, accepts two electrons and two protons, and is released as plastoquinol (PQH2). Triazine (e.g., atrazine, simazine) and substituted urea (e.g., diuron, monuron) herbicides bind to the plastoquinone (PQ)-binding site on the D1 protein and prevents the binding of PQ. Excitation energy cannot be dissipated by normal electron flow and activated oxygen species are generated. Inhibition of photosystem II Cobb and Reade, Herbicides and Plant Physiology 2010 C 6215 Lecture 9 IgorKučera Inhibition of protoporphyrinogen oxidase PPO is the last common enzyme in the biosynthetic pathway leading to heme and chlorophyll. In plants, there are two isoforms of PPO; the plastidic PPO1 and the mitochondrial PPO2. PPO1 is located in the thylakoid and in the envelope membranes of chloroplasts; PPO2 is situated on the outer surface of the inner mitochondrial membrane. Inhibition of PPO causes accumulation of the protoporphyrinogen-IX substrate in the cytoplasm where it is nonenzymatically oxidized to the photosensitive protoporphyrin IX. With exposure to light, this protoporphyrin IX generates singlet oxygen molecules that cause lipid peroxidation and cell death. Therefore, PPO-inhibiting herbicides are also known as light-dependent bleaching herbicides. PPO catalyzes the six electron oxidation of protoporphyrinogen IX to the fully conjugated macrocyclic protoporphyrin IX. Hao et al., Chimia 2011, 65, 961 diphenylethers phenylpyrazoles, oxadiazoles, triazolinones, thiadiazoles, pyrimidindiones, oxazolidinedione, N-phenylphthalimides, and others C 6215 Lecture 9 IgorKučera Inhibition of the HPPD enzyme stops the catabolic degradation of tyrosine to plastoquinones and tocopherol (vitamin E). Plastoquinones are important for photosynthesis and carotenoid biosynthesis. Vitamin E protects biological membranes against oxidative stress and the photosynthetic apparatus against photo-inactivation. Inhibition of 4-hydroxyphenylpyruvate dioxygenase 2H C 6215 Lecture 9 IgorKučera Inhibition of acetyl coenzyme A carboxylase ACCase is a biotin-containing enzyme that catalyzes the ATP dependent carboxylation of acetyl-CoA to form malonyl-CoA. This reaction is the first step in the synthesis of fatty acids. Two forms of ACCase are found in higher plants. In dicotyledons a heteromeric form (termed the prokaryotic form) of ACCase is located in plastids. This form of ACCase is absent from the monocotyledon grasses due to the absence of the accD gene that encodes the β-CT polypeptide. A homomeric form of ACCase is present in both dicotyledons and grasses. Its inhibitors selectively injure grasses. aryloxyphenoxypropionates (Fops) cyclohexanediones (Dims) Cobb and Reade, Herbicides and Plant Physiology 2010 C 6215 Lecture 9 IgorKučera Herbicidal inhibitors of amino acid biosynthesis Tan, Amino Acids 2006, 30, 195 C 6215 Lecture 9 IgorKučera Inhibition of glutamine synthetase Glutamine synthetase catalyzes the conversion of L-glutamate to Lglutamine in the presence of ATP and ammonia. γ - glutamyl phosphate is first produced from ATP and L-glutamate, and then ammonia reacts with this complex to release Pi and L-glutamine. Glufosinate, as an L-glutamate analogue, can also be phosphorylated to produce an enzyme-glufosinatephosphate complex to which ammonia cannot bind and the enzyme is irreversibly inhibited. The result is a rapid build-up of a high ammonia level, depletion of glutamine and several other amino acids and halting photosynthesis. Glufosinate can inhibit bacterial, plant and mammalian glutamine synthetase in vitro. It is only moderately toxic to mammals, apparently because of its rapid clearance by the kidneys. C 6215 Lecture 9 IgorKučera Inhibition of EPSP synthase glyphosate Glyphosate (Roundup) is a nonselective total herbicide. Its target enzyme, EPSP synthase, is involved in the biosynthesis of the aromatic amino acids tryptophan, phenylalanine and tyrosine, and numerous secondary plant products (vitamins, lignins, alkaloids, and a wide array of phenolic compounds such as flavonoids). X-ray crystallographic studies show that glyphosate functions by occupying the binding site of the phosphoenolpyruvate, mimicking an intermediate state of the ternary enzyme–substrate complex. Schonbrunn et al., PNAS 2001, 98,1376 C 6215 Lecture 9 IgorKučera Inhibition of acetolactate synthase The imidazolinones, sulphonylureas triazolopyrimidines, sulfonylaminocarbonyltriazolines and pyrimidinyloxy-benzoates are chemically different, yet all share the same site of action, namely acetolactate synthase, a key enzyme in the biosynthesis of the branched-chain amino acids leucine, isoleucine and valine. IMs and SUs were shown to block a channel through which access to the active site is gained. The diminution of the branchedchain amino acid pool contributes to a cessation of protein synthesis and inhibition of cell division. C 6215 Lecture 9 IgorKučera Target Site Resistance Enhanced Metabolism Resistance Enhanced Compartmentalisation Resistance Susceptible (TS = Target Site) Mechanisms of herbicide resistance Cobb and Reade, Herbicides and Plant Physiology 2010 C 6215 Lecture 9 IgorKučera Genetically modified herbicide-tolerant crops The term Genetically Modified (GM) is used to denote the transfer of genes from one organism to another or the alteration of gene expression using genetic engineering techniques. Introduction of new genes into plants: Agrobacterium tumefaciens, particle bombardment, electroporation, microinjection … • Glyphosate-tolerant crops Insertion of the EPSP synthase CP4 gene (crops with the trade name Roundup-Ready) Insertion of the glyphosate oxidoreductase (gox) gene (z Ochrobactrum anthropi) + ½ O2 C 6215 Lecture 9 IgorKučera • Glufosinate-tolerant crops Created by insertion of the bar or pat genes from streptomyces. These genes encode the enzyme acetyl transferase that detoxifies glufosinate by acetylation. In 1995 the first glufosinate-resistant crop, canola, was brought to market, and it was followed by corn in 1997, cotton in 2004, and soybeans in 2011. C 6215 Lecture 9 IgorKučera Vlček and Pohanka, Chem. Listy 2011, 105, 908 Neurotoxic compounds, the mode of action of which is the inhibition of acetylcholinesterase; this inhibition blocks the hydrolysis of the AChE substrate acetylcholine, a neurotransmitter. The toxic action is a result of excess stimulation at the neuromuscular junction by an accumulation of acetylcholine. The active form is the “oxon” (P=O), the “thion” (P=S) being converted in vivo to the oxon by the cytochrome P450 monooxygenase system; the oxon then phosphorylates the serine residue in the active center of the enzyme. 1. Dimethoate activation by cyt P450 1b. Hydrolysis (preferred in vertebrates) 2. Phosphorylation of the serine hydroxyl group in the active site of acetylcholinesterase 3. Spontaneous dealkylation (“aging”), conversion of the inhibited enzyme into a nonreactivable form Organophosphorous insecticides C 6215 Lecture 9 IgorKučera C 6215 Lecture 9 IgorKučera Carbamate insecticides Kill insects by reversibly inactivating the enzyme acetylcholinesterase. An attack of the carbonyl function of the carbamate through Sergenerates a temporary (in the order of min to hr) carbamylated complex that is incapable of further substrate catalysis. Štěpánková and Komers, Curr. Enzyme Inhib. 2008, 4, 160 C 6215 Lecture 9 IgorKučera Carbofuran: illegal killing of animals and suicides https://www.iucnosgbull.org/Volume27/Polednikova_et_al_2010.html http://www.karbofuran.cz/index.php?m Many animals - mostly raptors and other carnivores - have been poisoned by carbofuran in the Czech Republic. The poisoning substance is now banned in the EU, however, it had been widely used in agriculture as insecticide and large supplies are still available. https://en.wikipedia.org/wiki/M%C4%83d%C4%83lina_Manole Suicidal ingestion cases are also known, e.g. Madalina Manole (1967-2010), a Romanian pop star C 6215 Lecture 9 IgorKučera GGCX: γ-glutamyl carboxylase VKOR: vitamin K epoxide reductase VKR: vitamin K reductase • Anticoagulants Act by blocking of the vitamin K cycle, resulting in inability to produce essential blood-clotting factors—mainly coagulation factors II (prothrombin) and VII (proconvertin). Designed to have high potency and long residence times in the body. Death occurs after a period of several days to two weeks, usually from internal hemorrhaging. bromadiolone 4-hydroxycoumarins diphenadione 1,3-indandiones Rodenticides C 6215 Lecture 9 IgorKučera • Metal phosphides React with the acid in the digestive system to generate the phosphine gas: Zn3P2 + 6 H+ → 3 Zn2+ + 2 PH3 PH3 slows down mitochondrial respiration by inhibiting Complex IV (cytochrome c oxidase). Partial inhibition of mitochondrial respiration enhances electron leakage from the transport chain, leading to increased reactive oxygen species (ROS) generation. Death occurs commonly within several hours after bait ingestion. The garlic-like odor of baits attracts rodents, but repels other mammals. Birds are not sensitive to the smell and can be poisoned. STUTOX 5 % Zn3P2 LD50 (rat): 250 mg LD50 (mouse): 20 mg C 6215 Lecture 9 IgorKučera