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4-7 ; 5-2,5; 6-3,4
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
Nu
N
H
H2N
O
H CO2H
N
H
OH
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 color 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.
5-2) This question should refer to Figure 5.3 and not Figure 5.2.
The possible binding interactions for the functional groups in each molecule are
shown as HBD (hydrogen bond donor), HBA (hydrogen bond acceptor), ionic and
vdw (van der Waals interactions). It should also be noted that van der Waals
interactions involving alkyl groups or alkyl chains are possible.
The following amino acids have side chains which could interact by hydrogen
bonding: Ser, Thr, Tyr, Asn, Gln.
The following amino acids have side chains which could interact by ionic
interactions: Asp, Glu, His, Lys, Arg.
The following amino acids have aromatic or heteroaromatic groups in their side
chains which could interact by van der Waals interactions; Phe, Tyr, Try.
The following amino acids contain alkyl side chains which could interact by van
der Waals interactions; Val, Leu. Ile. Met, Pro.
In addition, the peptide links between amino acids in the binding site can interact
with ligands by hydrogen bonding.
Acetylcholine
HBA
O
H3C
O
HBA
Ionic
NMe3
It is also known that three of the four methyl groups fit into hydrophobic pockets
and participate in van der Waals interactions (see sections 19.7 and 15.14.1).
Noradrenaline and adrenaline
HBD H
HBD H
HBA
O
O
HBA
HBD
H HBA
O H HBD
H
N
R
HBA
vdw
HBD H
HBDH
HBA
O
HBD
H
HBA HBD
O H
H
H HBD
N
R
vdw
Ionic
O
HBA
The amino group of both nordrenaline and adrenaline can exist as the free base
or as the protonated, ionized form. Note that the nitrogen can act as a HBA in the
free base but not when it is ionized. Further details on the binding interactions of
noradrenaline and adrenaline can be found in sections 15.14.1, 20.8 and 20.9
Dopamine
HBD H
HBD H
HBD
H
HBA
O
N
HBD
H
HBA
vdw
HBD H
HBD H
O
HBA
HBD
H
HBA
O
N
vdw
O
HBA
H
HBD
H HBD
Ionic
The amino group of dopamine can exist as the free base or as the protonated,
ionized form. Note that the nitrogen can act as a HBA in the free base but not
when it is ionized. Further details on the binding interactions of dopamine can be
found in section 15.14.1.
Glycine
Glycine is an amino acid which is more likely to exist as the zwitterion with both
the amino and carboxylic acid groups being ionized.
HBD
H
HBD H
HBA
O
N
C
HBA
HBD
H
O
HBA
HBD
HBD H H
N
H Ionic
HBD
HBA
O
Ionic
O
HBA
Serotonin
HBD HBD
H H
HBD
N H
HBD
HBD
H
N H
HBA
HBA
O
H
HBD
Ionic
HBA
O
vdw
N
H HBD
H
HBD
vdw
N
H HBD
The amino group of serotonin can exist as the free base or as the protonated,
ionized form. Note that the nitrogen can act as a HBA in the free base but not
when it is ionized. Note also that the heterocyclic nitrogen is unlikely to be a good
HBA since its lone pair interacts with the ring's  system. Further details on the
binding interactions of serotonin can be found in section 15.14.1.
-Aminobutyric acid
HBD
H
HBD H
HBA
O
N
HBD
HBD
H
O
HBA
HBA
HBA
O
HBD H
H
N
H
HBD Ionic
Ionic
O
HBA
Glutamic acid
HBA
O
HBD
H
O
HBA
HBA
O
H
HBA N
H
H
HBD HBD
Ionic
HBD
H
HBA
O
O
HBA
O
HBA
HBA
O
H
Ionic
Ionic
O
HBA
N
H
H
HBD
HBD H
HBD
5-5) The inactive metabolite has a methyl ether rather than a phenol group. This
indicates that the phenol group is an important binding group when isoprenaline
interacts with the adrenergic receptor. For example, the hydrogen atom of the
phenol group may act as a hydrogen bond donor to a corresponding hydrogen
bond acceptor in the binding site. This interaction is no longer possible for the
inactive metabolite. Another possibility is that the phenolic oxygen acts as a
hydrogen bond acceptor and that the methyl group in the metabolite prevents this
interaction due to its size and bulk (see also sections 11.2.6 and 20.10.3).
HO
H
OHH
N
H
CH3
CH3
HO
Isoprenaline
MeO
OHH
N
HO
CH3
CH3
Inactive metabolite
6-3) It is worth considering the interactions of estradiol and raloxifene with the
estrogen receptor (Box 6.2) in order to answer this question.
His 524
Me OH
H
H
H
N
Side
chain
H
Glu353
Asp351
H
H
His 524
O
Glu353
O
OH
O
Hydrophic skeleton
H2O
H
O
S
Arg394
Oestradiol
Arg394
Raloxifene
Both estradiol and raloxifene contain functional groups that can interact through
hydrogen bonding to the amino acids Glu353, Arg394 and His524. Both molecule
have hydrophobic skeletons that position these groups correctly and match the
hydrophobic nature of the binding site. Estradiol is an agonist whereas raloxifene
is an antagonist. This is due to the extra interaction with Asp351 that is possible
for raloxifene.
Turning now to tamoxifen, this molecule is also hydrophobic and of a similar size
to the above, allowing it to fit the hydrophoic binding site. It does not have the
phenol or alcohol functional groups present in estradiol or raloxifene, but it does
have a group that can interact with Asp351 in the same way as raloxifene,
resulting in it acting as an antagonist.
Asp351
H
Me2N
O
Tamoxifen
4) Although tamoxifen itself is an antagonist, its metabolite is an agonist. This is
because it has lost the group that is so crucial for antagonist activity (the side
chain containing the amine). It also contains a phenol group which can mimic the
phenolic group of estradiol (see above question 3).
Asp351
Asp351
O
HO
H
Me2N
Glu353
Glu353
Tamoxifen
Arg394
H
Arg394
O
Metabolite