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Patrick: An Introduction to Medicinal Chemistry 3/e Chapter 4: Proteins as drug targets – enzymes Answers 1) The enzyme catalysed reduction of an aldehyde requires one equivalent of the cofactor NADH, which is oxidised to NAD+. However, if ethanol is added to the reaction, aldehyde dehydrogenase can catalyse its oxidation to acetaldehyde. In the process, NAD+ is converted back to NADH. As a result, only a catalytic quantity of NADH is required. It is important to appreciate that enzymes can catalyse reactions in either direction until an equilibrium is reached. In this case two separate reactions involving the same enzyme are coupled together to recycle the cofactor. Aldehyde dehydrogenase Aldehyde NAD+ NADH Acetaldehyde Alcohol Aldehyde dehydrogenase Ethanol 2) The structures of isoleucine and valine are as follows. H H2N H3C CO2H H2N CH3 H3C H CO2H CH3 Valine Isoleucine Isoleucine has a larger side chain than valine and so there is less space available in that region of the active site for COX-1 than there is in the corresponding region in COX-2. Drugs can be designed that take advantage of this difference such that they fit into the active site of COX-2 but cannot fit the active site of COX-1 3) Acetylcholine contains an ester functional group and a quaternary nitrogen which is charged. Thus hydrogen bonds and ionic interactions may be important. There are also methyl groups which might fit into hydrophobic pockets. The actual binding interactions for acetylcholine are discussed in section 19.16.3.1. HBA O H3C C O HBA H3C N CH3 CH3 ionic OXFORD © Oxford University Press, 2006. All rights reserved. Higher Education Patrick: An Introduction to Medicinal Chemistry 3/e Chapter 4: Proteins as drug targets – enzymes 4) A mechanism similar to that described for the hydrolysis of peptide bonds by chymotrypsin (section 4.5.3) would be feasible, involving a catalytic triad of serine, histidine and aspartate. Serine would serve as a nucleophile, histidine as an acid/base catalyst and aspartate as an activating and orientating group. The actual mechanism for the hydrolysis of acetylcholine is described in section 19.16.3.2. 5) In general, binding interactions will involve acetylcholine being bound in a particular conformation such that the binding interactions are maximised. As a result, certain bonds might be placed under strain with the result that they are broken more easily. For example, a hydrogen bond involving the carbonyl oxygen of acetylcholine may result in a weakening of the carbonyl bond such that it is more easily broken in the first stage of the enzyme catalysed mechanism 6) The reaction catalysed by acetylcholinesterase on acetylcholine is the hydrolysis of the ester to produce acetic acid and choline. With neostigmine, the corresponding reaction would be the following: Me2N O NMe3 O HO OH Me2N O NMe3 + Neostigmine In fact, this does not happen and a stable complex with the enzyme is formed. Following the mechanism through, serine acts as a nucleophile and a nucleophilic substitution reaction takes place on the urethane with loss of the phenol group. However, once this has occurred, serine is 'capped' with a stable urethane group which is resistant to hydrolysis. OXFORD © Oxford University Press, 2006. All rights reserved. Higher Education Patrick: An Introduction to Medicinal Chemistry 3/e Chapter 4: Proteins as drug targets – enzymes O MeHN H O Stage 2 H O :N NH MeHN :N C OAr NH :O: Ar : : : Stage 1 O Physostigmine -ArOH Stage 3 MeHN : : O C H N O Ar Stage 4 O NH MeHN :O: :N C O Ar NH H : : : O: +H 2O Hydrolysis VERY SLOW O O MeHN :N O H NH :N NH Stable carbamoyl intermediate Full details of this process are described in section 19.17.1.1 7) A transition state inhibitor is a drug which mimics the transition state for an enzyme catalysed reaction. Such a drug should bind more strongly than either the substrate or the product, and be a strong inhibitor as a result. The enzyme catalysed hydrolysis of the peptide bond between Phe or Pro should involve the enzyme using a nucleophilic group to form a bond to the carbonyl group, resulting in the intermediate shown. ENZ Nu ENZ H CO2H H2N N H O H CO2H Nu H2N N H OH OXFORD © Oxford University Press, 2006. All rights reserved. Higher Education Patrick: An Introduction to Medicinal Chemistry 3/e Chapter 4: Proteins as drug targets – enzymes The transition state for this stage should resemble the intermediate more than the starting material and so drugs mimicing the transition state should have characteristics also seen in the intermediate. The similarities in structure I to the intermediate are highlighted in colour below. extra methylene group O O H N H H H N OH O Me C Me O Me tetrahedral centre Note the presence of the tetrahedral centre bearing the alcohol group - also present in the intermediate. Note also that there is an extra methylene group between this tetrahedral centre and proline. This is necessary if the inhibitor is to be stable to the enzyme. Further details regarding the design of this inhibitor can be found in sections 17.7.4.2 and 17.7.4.3 An IC50 6500 nM means that the concentration of inhibitor required to inhibit the enzyme by 50% is 6500 nanomolar. 8) It is proposed that the binding interactions between an enzyme and a substrate are optimal during the transition state of the enzyme catalysed reaction. This is a reasonable proposition since the speed and effectiveness of a catalysed reaction is crucially dependent on how much the catalyst stablises the transition state. The more stable the transition state, the easier the reaction will occur and the more effective the enzyme will be as a catalyst. Therefore, it is more important that enzymes form their strongest interactions with 'guest molecules' at the transition state of the reaction, rather than to the substrate at the beginning of the process, or the product at the end. Indeed strong interactions with substrate or product are likely to be detrimental since it could result in a slow 'turnover' with substrate and product spending too much time in the active site. OXFORD © Oxford University Press, 2006. All rights reserved. Higher Education