Enzymes As Drug Targets PDF

Enzymes as Drug Targets Chapter 7 CH456 Medicinal Chemistry © 2025 James H. Gerlach Enzymes As Drug Targets 7.1 Inhibitors Acting at the Active Site of an Enzyme 7.2 Inhibitors Acting at Allosteric Binding Sites 7.3 Uncompetitive and Non-competitive Inhibitors 7...

Enzymes as Drug Targets Chapter 7 CH456 Medicinal Chemistry © 2025 James H. Gerlach Enzymes As Drug Targets 7.1 Inhibitors Acting at the Active Site of an Enzyme 7.2 Inhibitors Acting at Allosteric Binding Sites 7.3 Uncompetitive and Non-competitive Inhibitors 7.4 Transition-State Analogues: Renin Inhibitors 7.5 Suicide Substrates 7.6 Isozyme Selectivity of Inhibitors 7.7 Medicinal Uses of Enzyme Inhibitors 7.8 Enzyme Kinetics (not covered) 7.1.1 Reversible Inhibitors Substrate binding at the active site of an enzymes if the substrate binds too weakly it will not remain in the active site long enough for the reaction to occur. If substrate binds too strongly, product is not released from the active site. Therefore, most enzyme substrates have similar binding affinities. FIGURE 7.1 Example of an enzyme being ‘clogged up’ if the product remains bound. Competitive Inhibitors Similar in structure to the natural substrate or product but usually binds to the active site more strongly. Drug is competing with for the enzyme binding site (competitive inhibition). The longer the drug remains in the binding site, the greater the inhibition of the enzyme. FIGURE 7.2 Competitive inhibition. Dihydrofolate reductase is inhibited by methotrexate, which prevents binding of its substrate, folic acid. Binding site in blue Inhibitor in green Substrate in black BOX 7.1 A cure for antifreeze poisoning Ethylene glycol Pure ethylene glycol freezes at about −12 °C but a mixture of 60% ethylene glycol and 40% water freezes at −45 °C. Moderately toxic with an oral LDLo = 786 mg/kg for humans. LDLo (lowest lethal dose) is the least amount of drug that can produce death. The major danger is due to its sweet taste. Oxidized in a series of enzymatic reaction to oxalic acid. First step is oxidation by alcohol dehydrogenase (ADH), so ethylene glycol is acting as a competitive inhibitor of the natural substrate. Treatment is large doses of the natural substrate – ethanol.  Methanol CH3OH Ethanol CH3CH2OH Fomepizole* Ethylene glycol (CH2OH)2 *Fomepizole (4-methylpyrazole) is an inhibitor of alcohol dehydrogenase with an affinity for the enzyme 8,000× higher 7.1.2 Irreversible Inhibitors Irreversible inhibitors They can form a covalent bond with a key amino acid in the active site. Most effective irreversible inhibitors contain an electrophilic functional group capable of reacting with a nucleophilic group that is present on an amino acid side chain. Most common amino acids are serine (Ser, S) or cysteine (Cys, C) because these contain nucleophilic groups (OH or SH). Electrophilic groups commonly found in irreversible inhibitors include alkyl halides, epoxides, α,β-unsaturated ketones or strained lactones and lactams. Highly toxic nerve agents contain electrophilic fluorophosphonate groups (e.g., diisopropyl fluorophosphate is an irreversible anti- cholinesterase). Irreversible Inhibition of an Enzyme With an Alkylating Agent FIGURE 7.3 Irreversible inhibition of an enzyme with an alkylating agent. (X = halogen leaving group). FIGURE 7.4 Examples of electrophilic functional groups. Enzymes As Drug Targets 7.1 Inhibitors Acting at the Active Site of an Enzyme 7.2 Inhibitors Acting at Allosteric Binding Sites 7.3 Uncompetitive and Non-competitive Inhibitors 7.4 Transition-State Analogues: Renin Inhibitors 7.5 Suicide Substrates 7.6 Isozyme Selectivity of Inhibitors 7.7 Medicinal Uses of Enzyme Inhibitors 7.8 Enzyme Kinetics (not covered) Allosteric Binding Sites Regulation of enzymes by natural allosteric inhibitors is common. An inhibitor binding at an allosteric site on an enzyme can alter the conformation of the enzyme's active site. Drugs can be designed to mimic this activity. If drug contains a reactive group that allows formation of a covalent bond, then the inhibition is irreversible. 6-Mercaptopurine is an allosteric inhibitor. It inhibits the first enzyme involved in synthesis of purines. It is used to treat leukemias (e.g., acute lymphocytic leukemia) and autoimmune diseases (e.g., Crohn's disease). FIGURE 7.5 6- Mercaptopurine. Allosteric Inhibitors of BCR-ABL (1 of 3) BCR-ABL BCR-ABL is a fusion protein created by a chromosomal translocation between chromosomes 9 and 22, forming the Philadelphia chromosome. It is a constitutively active tyrosine kinase associated with certain cancers, particularly chronic myeloid leukemia (CML) and some cases of acute lymphoblastic leukemia (ALL). The constant activation of this kinase promotes uncontrolled cell proliferation and survival, leading to cancer progression. Allosteric Inhibitors of BCR-ABL (2 of 3) Allosteric inhibitors of BCR–ABL Unlike traditional inhibitors that target the ATP-binding site (active site) of the kinase, allosteric inhibitors bind to a different site on the protein, called the allosteric site, which is remote from the catalytic site. These inhibitors bind to a regulatory pocket of the BCR-ABL protein such as the myristoyl binding site, which controls the protein's structural configuration and function. Allosteric inhibitors are highly selective for BCR-ABL, which minimizes damage to normal cells and reduces side effects. Allosteric inhibitors can be combined with ATP-competitive inhibitors to achieve dual inhibition, providing a synergistic effect for greater efficacy. Allosteric Inhibitors of BCR-ABL (3 of 3) Type 1 and type 2 kinase-inhibitor complexes The terms type 1 and type 2 kinase-inhibitor complexes describe the mode of binding of small-molecule inhibitors to kinase enzymes, including BCR- ABL. Type 1 inhibitors bind to the active conformation of the kinase where the ATP-binding site is accessible and are competitive enzyme inhibitors. Type 1 inhibitors may cause off-target effects due to binding to other kinases. Type 1 inhibitors are often first-line therapies for CML. Type 2 inhibitors bind to the inactive conformation of the kinase at an allosteric site. This stabilizes the inactive state, preventing kinase activation. Type 2 inhibitors are more selective, as the inactive state is unique to each kinase. Type 2 inhibitors are used when resistance or intolerance to type 1 inhibitors develops. “Growing Arsenal of ATP-Competitive and Allosteric Inhibitors of BCR–ABL” Cancer Research 2012 Type 1 inhibitors target the active conformation of the kinase domain. Type 2 inhibitors target the inactive conformation of the kinase domain. BCR-ABL Fusion Gene (Philadelphia Chromosome) ABL Kinase Structure SH3 = Negative Regulatory Domain Autoregulation of BCR-ABL ABL Kinase Domain (blue) in Complex With 2nd Generation Tyrosine Kinase Inhibitor (TKI) Nilotinib (red) Enzymes As Drug Targets 7.1 Inhibitors Acting at the Active Site of an Enzyme 7.2 Inhibitors Acting at Allosteric Binding Sites 7.3 Uncompetitive and Non-competitive Inhibitors 7.4 Transition-State Analogues: Renin Inhibitors 7.5 Suicide Substrates 7.6 Isozyme Selectivity of Inhibitors 7.7 Medicinal Uses of Enzyme Inhibitors 7.8 Enzyme Kinetics (not covered) Uncompetitive and Non-competitive Inhibitors Uncompetitive inhibitors Bind reversibly to an enzyme, but only when the substrate is already bound to the active site. i.e., The inhibitor binds to the enzyme–substrate complex. Inhibition is dependent on sufficient substrate being present to form the enzyme–substrate complex, therefore an uncommon type of drug. Uncompetitive and Non-competitive Inhibitors Non-competitive inhibitors Binds to an allosteric binding site and inhibits the enzyme- catalyzed reaction without affecting the strength of substrate binding. Binding of the allosteric inhibitor distorts the active site sufficiently to prevent the catalytic mechanism but has no effect on the substrate binding. In practice this ideal situation is extremely rare and most drugs that are non‑competitive inhibitors also affect substrate binding. Enzymes As Drug Targets 7.1 Inhibitors Acting at the Active Site of an Enzyme 7.2 Inhibitors Acting at Allosteric Binding Sites 7.3 Uncompetitive and Non-competitive Inhibitors 7.4 Transition-State Analogues: Renin Inhibitors 7.5 Suicide Substrates 7.6 Isozyme Selectivity of Inhibitors 7.7 Medicinal Uses of Enzyme Inhibitors 7.8 Enzyme Kinetics (not covered) Renin and Hypertension Renin Renin is a protease that converts angiotensinogen to angiotensin I, which is further converted to the active peptide hormone angiotensin II by the angiotensin-converting enzyme (ACE). Angiotensin II causes an increase in blood pressure (hypertension). FIGURE 7.6 Inhibition of renin to block the synthesis of angiotensin I and angiotensin II. Mechanism of Renin-Catalyzed Hydrolysis Renin is an aspartic acid protease secreted by the kidneys that participates in the body's renin– angiotensin–aldosterone system (RAAS). The active site of renin contains two aspartate residues and a bridging water molecule that are crucial to the mechanism by which a peptide bond in the substrate is hydrolysed. It is not possible to isolate the high-energy state (i.e., the tetrahedral intermediate). Mechanism of Renin-Catalyzed Hydrolysis FIGURE 7.7 Mechanism of renin-catalyzed hydrolysis. Renin Transition-State Analogues Renin transition-state analogues mimic the transition state. Design of these drugs is based on the reaction intermediate. Therefore, they should bind more strongly that either substrate or product. Since the intermediate is less stable than the substrate, it is presumed that it is closer in character to the transition state. Implies that the transition state is more tetrahedral in character than planar. Therefore, drugs based on the structure of the tetrahedral intermediate are more likely to mimic the transition state. Renin Transition-State Analogues FIGURE 7.8 Aliskiren. Purine Nucleoside Phosphorylase: Substrate, Transition State and Immucillin-H Transition-State Analogue Substrate, Transition State and Transition State Analogue Inhibitors for P. falciparum PNP Enzymes As Drug Targets 7.1 Inhibitors Acting at the Active Site of an Enzyme 7.2 Inhibitors Acting at Allosteric Binding Sites 7.3 Uncompetitive and Non-competitive Inhibitors 7.4 Transition-State Analogues: Renin Inhibitors 7.5 Suicide Substrates 7.6 Isozyme Selectivity of Inhibitors 7.7 Medicinal Uses of Enzyme Inhibitors 7.8 Enzyme Kinetics (not covered) Suicide Inhibitors Designed to undergo an enzyme-catalyzed transformation. Converted to a highly reactive species that can form a covalent bond in the active site of the target enzyme. e.g., Clavulanic acid – inhibits bacterial β-lactamases that are responsible for penicillin resistance. FIGURE 7.9 Reaction catalyzed by bacterial β- lactamase enzymes. β-Lactam Drugs Penicillin G Clavulanic acid Clavulanic Acid Acting as a Suicide Substrate FIGURE 7.10 Clavulanic acid acting as a suicide substrate. Clavulanic Acid Acting as a Suicide Substrate (Better) Rise of Resistance – Decrease in New Antibiotics Enzymes As Drug Targets 7.1 Inhibitors Acting at the Active Site of an Enzyme 7.2 Inhibitors Acting at Allosteric Binding Sites 7.3 Uncompetitive and Non-competitive Inhibitors 7.4 Transition-State Analogues: Renin Inhibitors 7.5 Suicide Substrates 7.6 Isozyme Selectivity of Inhibitors 7.7 Medicinal Uses of Enzyme Inhibitors 7.8 Enzyme Kinetics (not covered) Tissue-Specific Isozymes Allows for Tissue- Selective Drugs Identification of tissue-specific isozymes allows for development of tissue-selective drugs. e.g., Inflammatory diseases such as rheumatoid arthritis Indometacin (aka indomethacin) inhibits the enzyme cyclooxygenase involved in the synthesis of prostaglandins but also inhibits synthesis of beneficial prostaglandins. Cyclooxygenase isozymes: COX-1 and COX-2 (active in rheumatoid arthritis) Drugs have been developed to be selective for the COX-2 isozyme. e.g., Valdecoxib, rofecoxib and celecoxib Tissue-Specific Isozymes Allows for Tissue- Selective Drugs FIGURE 7.11 Cyclooxygenase inhibitors. Enzymes As Drug Targets 7.1 Inhibitors Acting at the Active Site of an Enzyme 7.2 Inhibitors Acting at Allosteric Binding Sites 7.3 Uncompetitive and Non-competitive Inhibitors 7.4 Transition-State Analogues: Renin Inhibitors 7.5 Suicide Substrates 7.6 Isozyme Selectivity of Inhibitors 7.7 Medicinal Uses of Enzyme Inhibitors 7.8 Enzyme Kinetics (not covered) Enzyme Inhibitors Used Against Microorganisms The goal is to identify crucial enzymes in microorganisms that are not present in humans or enzymes that are sufficiently different to allow selective targeting. Natural product screening Many fungal strains produce metabolites that act as inhibitors of bacterial enzymes but have no effect on fungal enzymes. e.g., Penicillin and cephalosporin C Enzyme Inhibitors Used Against Viruses Enzyme inhibitors are important in the battle against viral infections. e.g., Herpes virus and HIV/AIDS Successful antiviral drugs include Aciclovir (aka acyclovir) for herpes Zidovudine (aka azidothymidine, AZT) and saquinavir for HIV/AIDS Aciclovir Zidovudi Saquinavir ne Enzyme Inhibitors Used Against the Body’s Own Enzymes Search continues for new enzyme inhibitors, especially those that are selective for a specific isozyme, or act against recently discovered enzymes. Some current research projects include: Investigations into inhibitors of the COX-2 isozyme (section 7.6). Matrix metalloproteinases (anti-arthritic and anticancer drugs; section 21.7.1) Aromatases (anticancer agents; section 21.4.5) Caspases implicated in the processes leading to cell death. Inhibitors of caspases may have potential in the treatment of stroke victims. Vast amount of research is also taking place on kinase inhibitors. Kinase enzymes catalyse the phosphorylation of proteins and play an important role in signalling pathways within cells (see also Chapter 5 and section 21.6.2). Enzyme Modulators Enzyme modulators are agents that bind to the allosteric binding site of an enzyme and modulate its activity by making it more sensitive to low levels of substrate. Alternatively, they convert the enzyme from an inactive conformation to an active conformation. Compared to enzyme inhibitors, there are far fewer drugs acting as enzyme modulators. e.g., Riociguat is a vasodilator that stimulates an enzyme called soluble guanylate cyclase (section 26.5.1). Table 7.1 Drugs That Are Enzyme Inhibitors (1 of 2) Drug Target enzyme Field of therapy Relevant Section Aspirin Cyclooxygenase Anti-inflammatory 13.1.9 Captopril and enalapril Angiotensin-converting Antihypertension 28.3.3 and Case enzyme (ACE) study 2 Simvastatin HMG-CoA reductase Lowering cholesterol Case study 1 levels Phenelzine Monoamine oxidase Antidepressant 25.12.5 Clorgiline, Moclobemide Monoamine oxidase-A Antidepressant Box 7.4, 25.12.5 Selegiline Monoamine oxidase-B Parkinson’s disease Box 7.4 Methotrexate, Dihydrofolate reductase Anticancer 21.3.1 pemetrexed, pralatrexate 5-Fluorouracil, raltitrexed Thymidylate synthase Anticancer 21.3.2 Gefitinib, imatinib, etc. Tyrosine kinases Anticancer 21.6.2 Sildenafil Phosphodiesterase enzyme Treatment of male 12.4.4.2 (PDE5) erectile dysfunction. 28.5.2 Vasodilator in cardiovascular medicine. Allopurinol Xanthine oxidase Treatment of gout Hydroxycarbamide Ribonucleotide reductase Anticancer 21.3.3 Pentostatin Adenosine deaminase Antileukaemia 21.3.4 Cytarabine, DNA polymerases Anticancer 21.3.5 gemcitabine, fludarabine Table 7.1 Drugs That Are Enzyme Inhibitors (2 of 2) Drug Target enzyme Field of therapy Relevant Section Physostigmine, Acetylcholinesterase Myasthenia gravis, 24.12–24.15 donepezil, tacrine, glaucoma, Alzheimer’s organophosphates disease Various structures Matrix metalloproteinase Potential anticancer agents 21.7.1 Racecadotril Enkephalinase Treatment of diarrhoea 26.8.4 Zileuton 5-Lipoxygenase Anti-asthmatic Bortezomib Proteasome Anticancer 21.7.2 Vorinostat Histone deacetylase Anticancer 21.7.3 Lonafarnib Farnesyl transferase Anticancer 21.6.1 Sacubitril Neprilysin Vasodilator 28.5.3 Aliskiren Renin Antihypertensive 28.3.2 and Case study 9 Apixaban Factor Xa Anticoagulant 28.9.1.3 and Case study 10 Key Points (1 of 2) Enzyme inhibition is reversible if the drug binds through intermolecular interactions. Irreversible inhibition results if the drug reacts with the enzyme and forms a covalent bond. Competitive inhibitors bind to the active site and compete with either the substrate or the cofactor. Allosteric inhibitors bind to an allosteric binding site, which is different from the active site. They alter the shape of the enzyme such that the active site is no longer recognizable. Transition-state analogues are enzyme inhibitors designed to mimic the transition state of an enzyme-catalyzed reaction mechanism. They bind more strongly than either the substrate or the product. Key Points (2 of 2) Suicide substrates are molecules that act as substrates for a target enzyme, but which are converted into highly reactive species as a result of the enzyme-catalyzed reaction mechanism. These species react with amino acid residues present in the active site to form covalent bonds and act as irreversible inhibitors. Drugs that selectively inhibit isozymes are less likely to have side effects and will be more selective in their effect. Enzyme inhibitors are used in a wide variety of medicinal applications.