1 Carbonic anhydrases: One target for multiple diseases RNDr. Eva Havránková, PhD. Department of the Chemical Drugs, Masaryk University Brno Med Res Rev. 2020;40:2485–25652 What are carbonic anhydrases (CA) • CA, EC 4.2.1.1 • Metaloenzymes Biochem. J. (2016) 473, 2023–2032 3 Structure A B C D E Med Res Rev. 2020;40:2485–2565; Biochem. J. (2016) 473, 2023–20324 CA families α-CAs vertebrates, protozoa, algae, cytoplasm of green plants, Gram- bacteria β‐CAs Gram- bacteria, Gram+ bacteria, algae, chloroplasts of mono- and dicotyledons, fungi, Archaea γ‐CAs Archaea, cyanobacteria and most types of bacteria δ‐CAs marine diatoms ζ‐CAs marine diatoms η‐CAs protozoa θ‐Cas marine diatoms ι‐CAs marine diatoms Med Res Rev. 2020;40:2485–25655 CA presence in the cells Cytosol • CA I • CA II • CA III • CA VII • CA VIII • CA X • CA XI • CA XIII Membrane- bound • CA IV Trans- membrane • CA IX • CA XII • CA XIV Mitochondria • CA VA • CA VB Saliva and milk • CA VI Med Res Rev. 2020;40:2485–25656 CA presence in organs/tissue CA I Erythrocytes, gastrointestinal tract (GIT), and eye CA II Erythrocytes, eye, GIT, bone osteoclasts, kidney, lung, testis, and brain CA III Skeletal muscle and adipocytes CA IV Kidney, lung, pancreas, brain capillaries, colon, heart muscle, and eye CA V Liver, Heart and skeletal muscle, pancreas, kidney, spinal cord, and GIT CA VI Salivary and mammary gland CA VII Central nervous system (CNS) CA VIII Central nervous system (CNS) CA IX Tumors and gastrointestinal mucosa CA X Central nervous system (CNS) CA XI Central nervous system (CNS) CA XII Kidney, intestine, reproductive epithelia, eye, tumors, and CNS CA XIII Kidney, brain, lung, gut, and reproductive tract CA XIV Kidney, brain, liver, and eye Med Res Rev. 2020;40:2485–25657 hCAs are involved in….. • Respiration • Transportation of CO2 and bicarbonate • pH and CO2 homeostasis • Electrolyte secretion in various tissues • Biosynthetic reactions • Gluconeogenesis • Adipogenesis • Ureagenesis • Bone resorption • Calcification 8 In which form is most of the carbon dioxide transported through the bloodstream? • dissolved in plasma • Bonded to haemaglobine in form of carbaminohaemaglobine • Converted into hydrogencarbonate ions and then transported in plasma https://drawittoknowit.com/course/physiology/glossary/physiological-process/carbon-dioxide-transport9 CA in the breathing cycle CA II CA I J. Enzyme Inhib. Med. Chem.2016;31: 345-36010 CA Mechanism of Action How CA can be used in therapy? 11 12 Inhibitors of CAs Activators of CAs OverexpressionofCAs DeficitofCAs 13 Overexpression/deficit of CAs Med Res Rev. 2020;40:2485–2565 How many mechanism of inhibition of carbonic anhydrases do we know? 14 CA Inhibitors Mechanism of Action J. Enzyme Inhib. Med. Chem.2016;31: 345-36015 The Zinc Binders acetazolamide CA Inhibitors Mechanism of Action J. Enzyme Inhib. Med. Chem.2016;31: 345-36016 CAIs anchoring to the zinc-coordinated water/hydroxide ion CA Inhibitors Mechanism of Action J. Enzyme Inhib. Med. Chem.2016;31: 345-36017 CA inhibition by occlusion of the active site entrance CA Inhibitors Mechanism of Action J. Enzyme Inhib. Med. Chem.2016;31: 345-36018 Out of the active site binding as a CA inhibition mechanism CA Mechanism of Action J. Enzyme Inhib. Med. Chem.2016;31: 345-36019 Compounds acting as CAIs with an unknown mechanism of action Med Res Rev. 2020;40:2485–256520 CA I CA II CA VII CA IX CA III CA IV CA VI CA XII CA XIII CA XIV 21 How to discover new selective inhibitors ? 22 How to discover new selective inhibitors ? Knowledge of the structre Screening of anions Screening of drugs Combination of approaches Steal other people's work FEBS Lett. 2004;577:439–445; Metabolites. 2017;7:4823 Knowledge of the structure - X-ray crystal structure of CA IX - NMR - other J. Inorg. Biochem. 2012; 111:117-12924 Screening of anions J. Med. Chem. 2020; (63)13: 7422–744425 Screening of known drugs Compound KI* (nM) hCA I hCA II hCA IX AAZ 250 12.1 25.8 BRZ NT 3 37 DCP 1200 38 50 DZA 50 000 9 52 EZA 25 8 34 IND 31 15 24 MZA 50 14 27 26 How to treat cancer – Inhibition of hCA IX ? J. Med. Chem. 2020; (63)13: 7422–744427 Which isoenzyme is involved in the cancer? Metabolites. 2017;7:48; Cancer Metastasis Rev. 2019;38:65–77; Natl. Cancer Inst. 2001;93;266–276.; Cancer Cell. 2008;13:472–482; Nat. Rev. Drug Discov. 2008;7:168–181; Nat. Rev. Drug Discov. 2011;10:767–777. 28 Hypoxia → HIF-1 → aggressive tumor 29 hCA IX – How it helps the tumor Catalytic hydration of CO2 Maintaining pHe and pHi Up-regulation of metabolism Increased adhesion ability Migration Invasion Metastasis Interaction with signal cascades Support of angiogenesis Cancer Metastasis Rev. 2019;38:65–7730 What the hCA IX inhibitors do? • No cytotoxicity (cytostatic effect) • Inhibit all of the previously mentioned processes • Prevents the formation and spread of metastasis • Multiply increases sensitivity to existing treatments Eur. J. Med. Chem. 2022;228:114008; Eur. J. Med. Chem. 2021;216:113283 31 Inhibitors of CA IX 32 What about bacterial CAs ? Expert Opin. Ther. Pat. 2018; 28:745-75433 Bacterial CA Inhibition of CA: • disruption of essentials metabolic pathways • growth retardation • growth defects • volnurable to host defense mechanism Expert Opin. Ther. Pat. 2018; 28:745-75434 Bacterial CA 35 Does it have potential? • Wide range of disseases • Effective treatment • Great potential • But……… 36 Thank you for your attention.