Allosteric inhibition

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Allosteric inhibition

  1. 1. Allosteric Inhibition
  2. 2. Conformational change The enzyme switches back and forth between the two forms. They are in equilibrium. Inactive form Active form Allosteric Regulation of Enzymes
  3. 3. Conformational change Inactive form Active form Allosteric Regulation of Enzymes Allosteric site Active site
  4. 4. When the enzyme is in its inactive form, the allosteric sites on the regulatory subunits can accept inhibitor. Allosteric regulation Inactive form Allosteric Regulation of Enzymes Catalytic subunit Regulatory subunit
  5. 5. Allosteric regulation Inactive form Allosteric Regulation of Enzymes Catalytic subunit Regulatory subunit Allosteric inhibitor
  6. 6. When the enzyme is in its active form, the active sites on the catalytic subunits can accept substrate. Allosteric regulation Active form Allosteric Regulation of Enzymes
  7. 7. Once a site is filled with a substrate or inhibitor, binding at a second site of the same type is favored. Cooperativity Allosteric Regulation of Enzymes Substrate
  8. 8. Cooperativity Allosteric Regulation of Enzymes No product formation Product formation
  9. 9. <ul><li>Allosteric control involves binding at a site other than the active site </li></ul><ul><li>Allosteric control of aspartate transcarbamoylase (ATCase) from E. coli. </li></ul><ul><li>First and unique step in pyrimidine synthesis </li></ul><ul><li>Both substrates bind cooperatively to enzyme </li></ul>
  10. 10. The feedback inhibition of ATCase regulates pyrimidine synthesis <ul><li>Allosteric inhibition of ATCase by cytidine triphosphate (CTP), a pyrimidine (feedback inhibition) </li></ul><ul><li>Allosteric activation by adenosine triphosphate (ATP), a purine nucleotide </li></ul>
  11. 11. Kinetics of the ACTase reaction <ul><li>Sigmoidal rather than hyperbolic curve -> cooperative substrate binding </li></ul><ul><li>Allosteric effectors shift curve to the right or left </li></ul>
  12. 12. Allosteric changes alter ACTase’s substrate-binding site <ul><li>ACTase (300 kD), subunit composition c6r6, catalytic and regulatory subunits </li></ul><ul><li>Catayltic subunits arranged as two sets of trimers (c3) in complex with three sets of regulatory dimers (r2) </li></ul><ul><li>Each regulatory dimer contacts two catalytic subunits in different C3 trimers </li></ul><ul><li>Isolated catalytic trimer are active and not affected by allosteric regulators (ATP or CTP) </li></ul><ul><li>Regulatory subunits reduce activity of cat. subunits </li></ul>
  13. 13. Structure of ACTase
  14. 14. Allosteric changes alter ACTase’s substrate-binding site <ul><li>ATP preferentially binds to ATCase’s active (R or high substrate affinity) state -> increasing substrate af. </li></ul><ul><li>CTP as inhibitor binds to the inactive (T or low substrate affinity) state -> decreasing substrate af. </li></ul><ul><li>Similarly, the unreactive bisubstrate analog N-(phosphonacetyl)-L-aspartate (PALA) binds tightlx to the R-state but not to T-state </li></ul><ul><li>Large conformational changes, mostly quarternary shifts associated with T -> R transition </li></ul><ul><li>Substrate binding to one subunit thus increases affinity in the other subunits </li></ul>
  15. 16. Tertiary and quarternary conformational changes in ACTase No substrate Low affinity T state Substrate binding induces T -> R transition
  16. 17. DRUG DESIGN
  17. 18. DRUG DESIGN <ul><li>Most of the drugs in todays use were developed over the last 30 years, exceptions: digitalis (heart stimulants), quinine (malaria), mercury (syphilis) </li></ul><ul><li>How are new drugs discovered ? </li></ul><ul><ul><li>Mostly by screening of compound libraries, using an in vitro assay with the purified enzyme - > determining KI </li></ul></ul><ul><ul><li>Results in lead compound (good candidate has a dissociation constant of 1 µM) </li></ul></ul><ul><ul><li>Chemical modification of lead compound to improve its pharmacological properties (5-10000)! </li></ul></ul>
  18. 19. Quinine and chloroquine <ul><li>Antimalaria drugs, share quinoline ring system </li></ul><ul><li>Pass through cell membranes and inhibit heme crystallization/storage in parasite (Plasmodium) </li></ul>
  19. 20. TRADITIONAL DRUG DESIGN Lead generation: Natural ligand / Screening Biological Testing Synthesis of New Compounds Drug Design Cycle If promising Pre-Clinical Studies
  20. 21. Structure-based drug design accelerates drug discovery <ul><li>Since mid 1980s, rational drug design, based on protein structure </li></ul><ul><li>Model hydrogen bonding donors and acceptors, cavities etc. </li></ul><ul><li>Used to develop analgesics (pain relievers) Celebrex and Vioxx, HIV inhibitors! </li></ul>
  21. 22. Structure-based Drug Design (SBDD) Molecular Biology & Protein Chemistry 3D Structure Determination of Target and Target-Ligand Complex Modelling Structure Analysis and Compound Design Biological Testing Synthesis of New Compounds If promising Pre-Clinical Studies Drug Design Cycle Natural ligand / Screening
  22. 23. Combinatorial chemistry and high-throughput screening are useful drug discovery tools <ul><li>Rapid and cheap synthesis of large numbers of related compound that can then be used for robotic high throughput screening </li></ul><ul><li>The combinatorial synthesis of arylidene diamides: </li></ul><ul><li>if ten different variants of each R group are used in the synthesis, then 1000 different derivatives will be synthesized! </li></ul>
  23. 24. A drug’s bioavailability depends on how it is absorbed and transported in the body <ul><li>The in vitro assay to uncover lead compound is only the first step in the drug discovery process </li></ul><ul><li>Besides causing the desired response in its isolated target protein, a useful drug must be delivered in sufficiently high concentration to this protein where it resides in the human body </li></ul>
  24. 25. Bioavailability / Pharmacokinetics <ul><li>For example, orally delivered drugs: </li></ul><ul><li>pass through the stomach, must be chemically stable </li></ul><ul><li>must be absorbed from gatrointestinal tract, from bloodstream must bass through cell membrane etc. </li></ul><ul><li>Should not bind to other substances, lipohilic substances for example are absorbed by plasma proteins and fat tissue </li></ul><ul><li>Must survive detoxification reactions for xenobiotics by liver enzymes </li></ul><ul><li>Avoid rapid excretion by the kidney </li></ul><ul><li>Must pass from capillaries to target tissue </li></ul><ul><li>May cross blood-brain barrier </li></ul><ul><li>Pass through the plasma membrane </li></ul><ul><li>These are Pharmacokinetic parameters that define bioavailability </li></ul>
  25. 26. Clinical trials test for efficacy and safety <ul><li>Successful drug must be safe and efficacious in humans </li></ul><ul><li>Test first in animals </li></ul><ul><li>Test in humans through clinical trials, monitored by FDA (food and drug administration, USA) </li></ul><ul><li>Three increasingly detailed and expensive phases: </li></ul><ul><ul><li>Phase I, designed to test safety of drug candidate and dosage range, method, and frequency 20-100 volunteers, or volunteer patients with target disease! </li></ul></ul>
  26. 27. Clinical trials test for efficacy and safety <ul><li>Phase II, test drug efficacy against target disease, 100-500 volunteer patients, refine dosage, check for side effects single bind tests against placebo </li></ul><ul><li>Phase III, monitors adverse reactions from long-term use and confirms efficacy, 1000-5000 patients double blind test </li></ul><ul><li>Only 5 out of 5000 drug candidates that enter preclinical trials (3 years) reach clinical trials (7-10 years), of these, only 1 will be approved (cost 300 Mio $), majority fail in Phase II. </li></ul><ul><li>Phase IV, post-marketing surveillance, if 1 in 10’000 individuals show life-threatening side effects, it will be taken from market (e.g. Vioxx in 2004, etc.)! </li></ul>
  27. 28. Cytochromes P450 are often implicated in adverse drug reactions <ul><li>Why can a drug be tolerated well by many but show dangerous effects in few ? </li></ul><ul><li>Individual differences in drug tolerance due to differences in disease stage, other drugs taken, age, sex and environmental factors. </li></ul><ul><li>Cytochromes P450 function in large part to detoxify xenobiotics, participate in metabolic clearance of majority of drugs </li></ul><ul><li>Cytochromes P450 constitute a superfamily of heme-containing enzymes present in nearly all organisms </li></ul><ul><li>57 isoenzymes in human genome, eg. CYP2D6 -> + polymorphic variants Monooxygenases embedded in the ER </li></ul><ul><ul><li>RH + O2 + 2H+ + 2e- -> ROH + H2O (R: steroids, PCB, drugs, tobacco smoke, broiled meat etc…) </li></ul></ul><ul><li>Cytochromes P450 can also convert non-harmful drugs into highly toxic compounds Example acetaminophen (antipyretic /fever reducer) at low dose (1.2g/day) but at > 10g/day is highly toxic! </li></ul>

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