CHEM3407 Part 2 Stu

1/23/2015
CHEM3407/3410 & BPHM3015
Hongzhe SUN
Chemistry & Pharmacy
HKU
2nd Semester, 2015
Targets and Mechanisms of Drugs Action

Overview
TARGET
Enzyme
Receptor
Ion Channel
DNA, RNA
MECHANISM
specific & irreversible inhibition
binding with agonist and antagonist
blocking the entry to cell
alkylation (N-coordination), intercalation
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3. Structural Protein as Drug Target

Hardly use as drug targets
Tubulin a heterodimer (- and -subunits), (weight ~55 kDa) is a
structural protein that self-polymerizes to form actin microtubules in the
presence of GTP (but depolymerizes if tubulin dimers bind GDP),
the microtubules serve as structural components of cells and involve
in controlling mitosis, cytokinesis and vesicular transport.
GTP
Polymerization
Depolymerization
GDP
Tubulin (, -subunit)
(soluble globular protein)
GTP: Guanosine triphosphate
Actin Microtubule
(insoluble filaments)
GDP: Guanosine diphosphate (energy source)
3. Structural Protein as Drug Target
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CHEM3407 Medicinal Chemistry
Anticancer drug Taxol
discovered in 1971 and clinically used in 1992

(9 yrs to isolate since discovery)
1977- clinical tested by NCI
(National Cancer Institute)
1980- pre-clinical toxicity tested
1992- July 22nd, drug for ovarian cancer
binds to the -subunit of tubulin, and accelerates
polymerization and stabilizes the resulting microtubules, inhibits
depolymerization (Nature, 1979, 277, 665-667)
Taxol arrests (stops) cell division through irreversible and out of
control microtubule formation, thus deplete free tubulin needed for
cell division and the accumulated microtubule initiates apoptosis
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Binding site in drug-target complex
- taxol binds the M-loop region of -tubulin since the peptide residues
are highly acidic and divergent, might induce stronger GDP capture
-tubulin
GTP
Taxol
GDP
-tubulin
PNAS 2003, 100, 6394; JMB 2001,
313, 1045 (PDB ID: 1JFF)
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Multi-step synthesis of Taxol
Other Mechanism of Action

- anticancer drugs e.g. Tesetaxel,
Vincristine, Vinblastine also targets
tubulin as antimicrotubule agents,
that inhibit tubulin polymerization
during early stages in mitosis of cancer
cells as the mitotic spindles used in
separating chromosomes are made of
stabilized microtubules
Vincristine
Tesetaxel
Vinblastine
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4 Proteins as Drug Targets: Enzymes

General Aspects of Enzyme
- enables reaction(s) took place in the biological space (biosynthesis)
- speeds up the reaction but does not affect the equilibrium
- provide a reaction surface (the active site) including suitable
(chemically and physically) environment (hydrophobic region)
- bring reactants together and position reactants correctly via
intermolecular forces in the active site (hollow or cleft region of
enzyme), weaken some related chemical bonds in the reactants
- sometimes serve as acid or base for the enzymatic reaction, or as a
nucleophile (as a ligand for metal)
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4 Proteins as Drug Targets: Enzymes

- substrate is bound to the enzyme to form an enzyme-substrate
complex (ES), which later undergoes a reaction to form the enzymebound product (EP), followed by the release of this product
P
Product
S
Release
ES
EP
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Enzyme-substrate interactions
1. Lock-and-key model
Active
site +
2. Induced fit model

Active
site
+
transition state
conformation
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How to regulate enzymatic activity?

- gene expression regulated by the amount of substrate (lac operon)*
- organelle compartmentalization (proteases in lysosome)
- change in conformation (e.g. allosteric site) i.e. enzyme is activated
or de-activated by +ve/-ve allosteric binding
enzyme is
activated by
an allosteric
binding
10% active
100% active
- activates by protein phosphorylation (Tyr-OH, Ser-OH, The-OH
Res-OPO3)
* The lac operon is set of structural genes required for the transport and metabolism of lactose in
Escherichia Coli and enteric bacteria. It is regulated by the availability of glucose and of lactose.
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Competitive inhibition
Non-competitive inhibition
- both substrate and inhibitor

compete the same active site
- inhibitor binds at a different region

(-ve allosteric or inhibitor site)
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Enzyme Inhibition: reversible or irreversible?

Both competitive and non-competitive inhibitions of enzyme can be
reversible or irreversible, depends on the extent of the +ve or -ve
allosteric binding between inhibitor and enzyme
- allosteric binding site (binding site differ from the active site) controls
the activity of enzyme by binding of a small molecule (inhibitor or
activator) to allosteric binding site that alters not only this site but also
the active site. Inhibitor disables the induced fit mechanism by -ve
allosteric binding, making original active site unrecognizable to substrate
Induced
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Enzyme catalyzed reactions

Substrate-active site interaction
Mostly, side-chains (and sometimes backbone C=O & N-H) of the
amino acids in the active site interact with the substrate via
- H-bond
- Ionic bonding
- v.d.w. interaction or dipole-dipole interaction
- Hydrophobic interaction
Catalytic role:
Amino acids in the active site act as nucleophiles
(serine and cysteine) or acid/base catalyst (histidine)
Cofactor (e.g. metals) is required sometimes (Zn2+ in
carbonic anhydrase)
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Enzyme-catalyzed reactions
- binding of pyruvic acid at the active site of lactate dehydrogenase
Imidazole (base)
Imidazolium (acid)
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Enzyme catalyzed reactions

Chymotrypsin (digestive enzyme) undergoes proteolysis by
cleavage of peptide bond where the carboxyl side of the peptide bond
belongs to Tyr, Trp or Phe. These 3 a.a. have aromatic rings well-fit to
the hydrophobic pocket of this enzyme. Both hydrophobic and shape
complementarity between such 3 a.a. in substrate and the active site in
this enzyme accounts for high substrate specificity
Active site contains catalytically
active side groups of Ser195, His57
and Asp102 residues
Leucine (Leu) and methionine
(Met), why?
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the cleavage
of a peptide bond of a protein (top) by
the enzyme (bottom), Ser, His and Asp are
the catalytic residues used for proteolysis
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e.g. Lysozyme
- catalyzes the glycosidic bond cleavage of polysaccharide chain
S + E
ES
EP
E + P
D-glucose
Before glucose binding
After glucose binding
Proteases (Digestive Enzymes)
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Four major classes of proteases (aspartic, serine, cysteine,

and metallo-based proteases) selectively catalyze the hydrolysis of
peptide bonds in polypeptide chains, depend on the type of residues
mainly involved in their active sites. exopeptidase cleaves terminal
a.a. residue (carboxypeptidase A);endopeptidase for internal a.a.
(trypsin, chymotrypsin, pepsin)
Fastest switching on and off regulatory mechanism on
physiological processes: digestion, blood clotting, wound healing,
cell signaling/migration, immunological defense, apoptosis, etc.
Ideal drug target (i.e. secreted bacterial exotoxin or metabolic types)
Protease-based inhibitors have been clinically used, i.e. Dr. David
Ho, an AIDS researcher who is famous for pioneering the use of
protease inhibitors in treating HIV-infected patients since 1996.
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a cocktail of drugs for AIDS
HIV Protease Enzyme
HIV protease is essential in the life cycle (replication & release of

mature viral particles from infected cells) of HIV (Human
immunodeficiency virus), a retrovirus causes AIDs in human being
A family of aspartyl protease specifically catalyzing the cleavage of
the phenylalanine-Proline or Phe-Pro peptide bond
One aspartic acid (Asp25) is crucial to the catalytic mechanism
A small, dimeric (99 a.a. /monomer), its active site has C2 symmetry
HIV protease (wild type)
- HIV protease inhibitor ?

symmetric? hydrophobic, 1 peptide
inhibitor-HIV protease complex
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Any other advantages of using aspartyl protease?
1. Small enzyme which could be either synthesized or

prepared by recombinant DNA technology
2. Structural determination could be determined by Xray or NMR with and without the inhibitor
3. Convenient for structurally-based drug design
Strategy for development?
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HIV Protease Enzyme
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Clinically Used HIV-1 Protease Inhibitors

- specifically & effectively target at the active site
Saquinavir (IC50< 50 nM, Roche)
Ritonavir (Abbott)
Nelfinavir (Lilly)
Amprenavir (Vetex)
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Active Site of HIV protease enzyme

- active site: Asp25-Thr26-Gly27
- a broad range of substrate, particularly contains a peptide bond
between the Pro and Phe residues (other aromatic is possible)
Asp25
Asp25
Asp25
Asp25
Thr26
Thr26
Two Asp25 residues are

almost coplanar!
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Active site
Protein
substrate
Phe
Pro
Catalytic mechanism
unstable
Acid
Nu:
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Transition-state inhibitor
- a compound mimics the transition-state of enzyme-catalyzed reaction

- why bother? transition-state is likely bound to the active site stronger
than either the substrate or the product (advantage!)
- any inhibitor resembling the transition-state, is also likely to have
stronger binding to the active site of enzyme
Unstable (acetal)
hydroxyethylamine
N
statines
OH
O
N
OH
R
OH
N
OH
dihydroxyethylene amide
- transition-state isostere to mimic the tetrahedral transition-state

Isoteres - molecules or ions with the same number of atoms and the same number of valence electrons
(e.g. Na+ & H+; benzene & pyridine; thiophene & furan, etc)
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- binding sub-sites (S1 to S3) & (S1 to S3) in the enzyme are located on
either side of the catalytic region
S3
S2
S1
CONH2
Ph
Ph
H
N
O
N
N
H
H
N
N
H
NH
CONH2
S1
S3
S2
- without strong inhibitor, HIV protease catalyzes the cleavage of the

peptide bond between phenylalanine and proline residues (Phe-Pro)
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Inhibitor-bound HIV-1 Protease (PDB ID: 1HXB)

- HIV-1 protease binds with anti-viral drug Saquinavir, its binding
site was later confirmed by X-ray structure
S3
S1
S3
O
O
H H
N
O
D25
D25
S2
H
N
H H
NH
N
H
OH
CONH2
Hydrogen bonded to D25/D25
S1
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HIV-1 Protease Inhibitor: Structure-based drug

design starting from Phe-Propeptide
CO2H
H
Z
N
H H
N
H2N
H
O
CO2tBu
OH
Compound I
(IC50 = 6500 nM)
L-Phe-L-Pro
H H
N
N
H H
CO2tBu
N
OH
CONH2
Asparagine
Compound II
(IC50 = 140 nM)
O
O
H
N
O
H
N
H
NH
H
N
H
N
H
H
OH
C ONH2
Saquinavir (highly potent) (IC50< 0.4 nM)
CO2tBu
H
OH
CONH2
Compound III
(IC50 = 23 nM)
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tBu=
tertiary butyl (-C(CH3)3

O
Z= benzyloxycarbonyl
O
Please read Chap 20 on antiviral drug design (Medicinal Chemistry)
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How Saquinavir binds?
- substitutents of Saquinavir occupy 5 sub-sites S1-S3 & S1, S3

- H-bond between OH group of drug and the CO2 group of Asp25
- the two C=O of the transition isostere of the drug acts as HBA to one
bridging H2O molecule that serves as HBD to two ILE50 residues
S1
S3
S3
O
N
H H
N
O
H
N
H H
CONH2
NH
N
H
OH
H
S2
S1
Some Remarks on Saquinavir

- Saquinavir entered into clinical trial in 1991 and market in 1995
- however 45% patients developed drug resistance
- low oral bioavailability (~ 4%) due to high affinity to the proteins
in blood (i.e. 98% to plasma proteins)
- new potent inhibitors with better bioavailability are now sought!
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Development of amprenavir (Vertex) and darunavir

Less peptide analogue to avoid protein binding
Darunavir binding to HIV-1 protease like a molecular

crab
(PDB: 2ien)
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HIV-1 protease with cyclic sulfamide inhibitor

strategy: binding of inhibitor toward the active site prevents selfassembly of HIV-1 protease, resulting in non-infective virions (virus)
7-mr
CYCLIC SULFAMIDE INHIBITOR, AHA047
(PDB ID: 1g2k)
surface
tube
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Other Possibility? Cyclic urea inhibitor

- cyclic urea inhibitor (1) has symmetric binding to HIV-1 protease,
whereas non-symmetric binding for cyclic sulfamide inhibitors (2, 3)
1
1, Ki= 12 nM
symmetric binding
Ki= 23 nM
Ki= 3.4 nM
non-symmetric binding
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Design and synthesis of symmetric cyclic

sulfamide as HIV-1 Protease Inhibitors
O
S
- both S2/S2 subsites are hydrophilic,

compounds 3 and 25 binds stronger and
spatially fit well, than the others
PhO
OPh
OH
HO
pKi
observed Ki
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Non-symmetric cyclic sulfamide

O
R1
O
S
R2
PhO
OPh
HO
OH
(JMC 2001, 44, 155)
- non-symmetric cases are

less tightly bound, than the
symmetric analogs
(based on Ki) i.e.
both molecular shape &
dipole moment may be
important in binding!
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Cyclic Sulfamide Inhibitors (Model Fitting)

O
R1
R1
O
S
R2
Ki (nM)
R2
PhO
OPh
HO
OH
rotate?
HBA
or
HBD
HIV-1 protease bound with drug #16
Arg
HIV-1 protease bound with drug #21
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Intermolecular Forces ?
- all these non-covalent interactions (H-bonds, C-H, C-HO=S)
are co-operative that enable highly specific and efficient binding
Inhibitor (16) in the binding site

of HIV-1 protease (H-bond networks)
Binding potential surface of the binding site showing

hydrophobic and H-bonds interactions around
the inhibitor (16) (Red: negative, O atoms ;
Gray: neutral, C atoms; Blue: positively charged)
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TARGETING HIV REPLICATION

http://www.youtube.com/watch?v=7CLFTrZOeEg&feature=related
References on HIV protease inhibitors:

Ghosh AK et al Enhancing protein backbone bindinga fruitful
concept for combating drug-resistant HIV Angew Chem 2012, 51,
1778 1802. (development of darunavir (Prezista))
Development strategy of HIV protease inbibitors:

1. Mimic the substrate (-Pro-Phe-): competitive inhibitors
2. Based on symmetric dimer: symmetric molecules
3. Enhancing protein backbone binding: overcoming drugresistance
5 Lead Optimization in Drug Design

- to optimise the binding interactions, so that more efficient + specific!
why needed?
- to increase activity and reduce dose levels
- to increase selectivity and reduce side effects
what strategies?
- 1. vary alkyl substituents
- 2. vary aryl substituents
- 3. extension of structure
- 4. chain extensions / contractions
- 5. ring expansions / contractions
- 6. ring variation
- 7. isosteres and (bio)isosteres
- 8. simplification of structure
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1 Vary Alkyl Substituents
Rationale:
- alkyl group in lead compound may interact with hydrophobic
region in binding site
- vary length and bulkiness of alkyl group to optimise interaction
ANALOGUE
LEAD COMPOUND
CH3
H3C
CH3
CH3
van der Waals

interactions
Hydrophobic
pocket
1 Vary Alkyl Substituents
Rationale:
- vary length of n-alkyl group to introduce selectivity
N
CH3
N
Fit
CH3 Fit
No Fit
Fit
Receptor 1
N
CH3
CH3
S teric
Block
Receptor 2
Binding region for N
Less selective
More selective
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1. Vary Alkyl Substituents

H
HO
OH
H
N
Adrenaline
(hormone & HO
neurotransmitter)
H
HOCH2
OH
H
N
CH3
CH3
CH3
CH3
HO
Ventolin
(asthma medicine)
CH3
H
O
Propranolol
(-Blocker),
treat hypertension
N
H
CH3
OH
-Adrenoceptor
+
Adrenaline
H-Bonding
region
H-Bonding
region
Van der Waals

bonding region
Ionic
bonding
region
H-Bonding
region
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-Adrenoceptor
Methyl
group is
good to fit!
ADRENOCEPTOR-ADRENALINE COMPLEX
2 Vary Aryl Substituents

Rationale:
- vary the identity of substituent of aryl group
- vary position of substituents of aryl group
Weak
H-Bond
Strong
H-Bond
(increased
activity)
H
O
Binding
site
Y
para Substitution
Binding Region
(H-Bond)
Binding
site
Binding Region
(for Y)
meta Substitution
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- vary position of a given substituent of the aryl group to enhance

binding interactions, therefore the drugs activity
O
MeO2SHN
6
7
NR
Benzopyrans
- anti-arrhythmic () activity of this drug is found to be the
best when the -NHSO2CH3 substituent is at the 7-position of the ring
- (another possibility) vary the position of substituent to increase

binding strength indirectly via the electronic effect
..
..
NH2
NH2
NH2
O
N
O
meta-position
(inductive electron
withdrawing effect)
N
O
para-position (more electron withdrawing

due to both resonance and inductive effects)
- binding strength of NH2 (as a HBD) depend on the position of NO2

i.e. stronger HBD when NO2 is at the para position
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TARGETING HIV REPLICATION

http://www.youtube.com/watch?v=7CLFTrZOeEg&feature=related
References on HIV protease inhibitors:

Ghosh AK et al Enhancing protein backbone bindinga fruitful
concept for combating drug-resistant HIV Angew Chem 2012, 51,
1778 1802.
3 Extension - Extra Functional Groups

Rationale:
to explore target binding site for more binding regions to achieve
additional binding interactions
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3 Extension - Extra Functional Groups

- e.g. inhibitor of angiotensin () converting enzyme (ACE)
4 Chain Extension / Contraction

Rationale :
- useful if a chain is present connecting two binding groups, A & B
- vary the length of that chain to optimise interactions
Weak
interaction
Strong
interaction
Chain
extension
RECEPTOR
Binding regions
A&B
RECEPTOR
HO
Binding groups
Optimized chain length n = 2

O
N
N-Phenethylmorphine
(CH2)n
H
HO
Binding
group
Binding
group
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5 Ring Expansion / Contraction

Rationale : to improve -overlap of binding groups with binding regions
6 MR to
7 MR
R
hydrophobic regions
N
O-
N
H2
CH3
N
O-
O-
N
H2
O-
O
O
Eanlaprilat
Good activity
N
N
H2
O-
O
O
O-
Cliazaprilat
Good activity
Poor activity
6 Ring Variations
Rationale :
- replace aromatic or heterocyclic rings with other rings of similar sizes
- often attempt this strategy to resolve patent problems (avoids conflicts
or fulfils some requirements for new patent application)
F
SO2CH3
General structure
for NSAIDS
SO2CH3
N
N
X
X
CF3
Br
DuP697
F
SC-58125
SO2CH3
SO2CH3
Core
scaffold
SC-57666
NSAIDS - Nonsteroidal anti-inflammatory drugs ()
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6 Ring Variations
- sometimes results in better medicinal properties after this strategy
Example:
N
N
N
N
OH
Cl
OH
Cl
Ring variation
Structure I
Antifungal agent
UK - 46245
improved selectivity
vs. fungal enzyme
6 Ring Variations
Example: Nevirapine (anti-viral agent)
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6 Ring Variations
Example : Pronethalol (-blocker)
H
HO
OH
H
NHR
OH H
N
Me
Me
HO
R= CH3, Adrenaline
Pronethalol
R= H, Noradrenaline
Selective for
-receptors
over -receptors
7 Isosteres and Bio-isosteres
Rationale (isosteres):
- replace a functional group with a group of same valency (isostere)
e.g. OH replaced by SH, NH2, CH3 or O replaced by S, NH, CH2
- enables more controllable changes in steric & electronic properties
- might affect the extents of binding and/or stability
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- useful for correlating the structure-activity relationship (SAR) of drug
Me
Me
NH
H
OH
Propranolol (b-blocker)
- replace OCH2 with CH=CH, SCH2, CH2CH2 eliminates activity
- replace OCH2 with NHCH2 retains activity
- implies O must involve in drug-target binding and O is HBA
Rationale (bio-isosteres):
- replace a functional group with another group which retains the
same biological activity, both do not necessarily have the same valency
Example:
Antipsychotics
()
O
N
Et
N
Et
N
H
OMe
OMe
EtO2S
EtO2S
Sultopride
pyrrole is the bio-isostere

for the amide
DU 122290
- gave an improved selectivity for D3 receptor over D2 receptor !

- implies hydrophobic interaction is better than dipole/H-bond
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8. Simplification of Structure
Why bother?
- lead compounds isolated from natural sources are often structurally
complicated and difficult to be synthesized without total synthesis
- simplify the lead molecule and make synthesis of its analogues more
easier, quicker and cheaper (attractive reason!)
- simpler structures may fit binding site easier and increase activity that
allows faster SAR development for less expensive manufacturing
processes of drugs with comparable activity
- simpler structures may (or may not) be more selective and less toxic if
those existing functional groups are removed (trial-&-error?)
Rationale:
- retain the basic pharmacophore(s) (i.e. key chemical/structural moiety
of the lead compound that likely relate to observed medicinal activity)
- remove unnecessary functional groups, e.g. chiral centers, chiral rings
8. Simplification of Structure
- the key idea is not pruning groups off the lead compound, the
simplified structure could be made by rationalized total synthesis
Me
N
CO2Me
Et2NCH2CH2
O
O
O
COCAINE H
NH2
Pharmacophore
COCAINE
(anaesthetic, stimulant)
PROCAINE
(local anaesthetic)
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Structure-based drug design (case study 1)

Anti-hypertensives () ACE inhibitor
- Angiotensin Converting Enzyme (ACE)
membrane-bound zinc(II) metalloprotein that catalyzes:
Angiotensin I
ACE
Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu
Asp-Arg-Val-Tyr-Ile-His-Pro-Phe
Angiotensin II
His-Leu
- Angiotensin II stimulates blood vessel constriction, blood pressure

- ACE inhibitor becomes an useful antihypertensive agent
Problem: - ACE is difficult to be crystallized as crystals for X-ray
Solution: - study on Zn protein analog e.g. carboxypeptidase (CP),
Peptide~~~~aa3-aa2-aa1
CP
Peptide~~~~aa3-aa2 + aa1
help to identify the binding site for ACE inhibitor. The solved X-ray
structure of CP as well as ease of protein preparation favors drug design
Peptide~~~~aa3-aa2-aa1
CP
Peptide~~~~aa3-aa2 + aa1
Normal activity
CP
CP + polypeptide
OH
OH
O
Inhibition
L-Benzylsuccinic acid
- the lead compound for ACE inhibitor
L-benzylsuccinic acid ???
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Drug Design structural variations of the Lead
1st non-peptide ACE inhibitor
proposed binding site in ACE
Epidermal Growth Factor Receptor (EGFR)

- acts both as a receptor (binding domain - binds EGF) and an enzyme
(kinase or catalytic domain - catalyzes protein phosphorylation, i.e.
tyrosine OH residues via ATP hydrolysis)
- as cellular signaling process via EGF-EGFR binding is normally tightly
controlled, over-expression of EGFR triggers the formation of epithelial
cancer, thus EGFR becomes a target for cancer therapy
Protein
HN
HO
EGF
Protein
CH2
ATP
Protein
ADP
EGFR
(PDB id: 1ivo)
HN
CH2
Protein
O
P
-
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Strategy
- the kinase portion of EGFR was produced by recombinant DNA
technology and was used in screening compounds for competing ATP
binding site & inhibiting protein phosphorylation
ATP (adenosine triphosphate)
kinase portion
of EGFR
- at least 2 H-bonds, 1 hydrophobic interaction between EGFR & ATP
EGFR Inhibitor Design - Mimicking ATP?

- random screening: pyrazolopyrimidine is the lead compound
Hydrophobic pocket
CGP_59326
ribose pocket
i.e. 2 H-bonds, 2 hydrophobic interaction between EGFR & CGP_59326
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EGFR Inhibitor Design Optimization

- random screening: pyrazolopyrimidine becomes the lead compound
Cl
Positioning of aryl group

NH2
NH2
H2N
NH2
Lead Compound
IC50= 0.22 M
IC50= 0.8 M
N
H
N
N
IC50= 2.7 M

Cl
Cl
NH2
N
H2N
N
H
IC50= 0.22 M
H
NH
N
N
IC50= 0.16 M
Cl
X
N
N
N
N
NH
NH
N
N
NH
NH
N
X = OH, IC50= 0.001 M
Cl
IC50= 0.033 M
Cl
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X
chain extension
NH
NH
N
Cl
chain contraction
NH
N
Y
H
X= OH
IC50=0.001 M
X= Cl
IC50=0.033 M
X= OCH3 IC50=0.008 M
X= NHC(O)C(CH3)3, inactive
NH
X = OH, IC50= 0.001 M
vary aryl substituents
N
N
Cl
X= Cl
IC50=0.026 M
X= OCH3 IC50=0.008 M
N
N
NH
N
Cl
X= OH, Y=H IC50=0.026 M

X= H, Y=OH IC50=0.006 M

Simplifying Lead Compound (staurosporine )
- a metabolite from bacterium (Streptomyces Staurosporeus)
which is a highly potent protein kinase inhibitor by competing the
binding site with ATP in kinase portion of EGFR
- anti-fungal, anti-hypertensive properties, high affinity to kinases but
low selectivity (i.e. inhibits all serine, threonine and tyrosine kinases)
H
N
N
O
H3 C
H 3C
*
*
O
H3 C
NH
staurosporine
enzymes
bio-synthesis route
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EGFR complexation with 1,2,3,4-Tetrahydrogen Staurosporine
77
(PDB: 2itw)
Strategy: easier to synthesize in lab and also retain its biological

activity; i.e. asymmetric center (avoid complication due to different
stereoisomers) & use available fragment (maleimide, rather latacm)
H
Acryriaflavin A
N
O
O
(from marine species)
selectively inhibits PKC
Indole ring
Maleimide ring
H
N
N
H
N
H
simplification
bis-indolylmaleimide
N
H
(still PKC selective)

N
H
H
N
Phthalimide ring
1
aniline ring
PKC (protein kinase C controls the function of other
proteins via phosphorylation of OH groups in Ser, The
a.a in the protein substrate of PKC.
NH
HN
Dianilinophthalimide (CGP_52411)
39
1/23/2015
Simplified EGFR Inhibitor Optimization

H
N
H
N
Vary aryl subsitutent

HO
HN
NH
HBD & HBA
small-size &
electronegative
substituents
CGP_52411
H
N
NH
OH
H
N
HO
CGP_53353
HN
NH
HN
HN
NH
poor HBA
(hydrophobic)
- CGP_53353 has similar activity as CGP_52411 and is more stable

toward human metabolism, thus this drug enters to the clinical trials
Simplified EGFR Inhibitor Optimization

Ring extension: from 5-member imide ring to 6-member ring
maleic hydrazide ring
H
N
HN
pyridazinone ring
NH
HN
O
NH
HN
NH
HN
NH
CGP_52411
(IC50 0.7 M)
CGP_54690
(IC50 0.12 M)
(but lacking cellular activity
because it is cell impermeable)
HN
CGP_54690
(IC50 0.18 M)
- good activity both
in vitro and in vivo
Chain extension: increasing the distance between the

aniline rings and the phthalimide core give no activity
40
1/23/2015
(Qualitative) Structure Activity Relationship

QSAR established based on > 250 analogues
- CGP_52411 is highly selective for EGFR with an IC50 of 0.7 M
and is chosen for pre-clinical trials
4 key SAR being as a EGFR inhibitor
i) phthalimide N atom must be unsubstituted
(i.e. R = H), otherwise no activity
Phthalimide ring
-
ii) both C=O groups in phthalimide core

must be present, otherwise no activity
iii) aniline N atom must be unsubstituted
(i.e. R2 = H) otherwise too water
insoluble or bulky!
iv) the two aniline groups must be present
and their substitutent (R1) must be small,
otherwise no activity. If R1 = F, activity
R
N
aniline ring
R1
R1
NR2
R2N
(CGP_52411)
Binding Site Evidence Structural Validation!

- a competitive inhibitor for EGFR, ATP hydrolysis in epithelial cancer cells
Hydroprobic interactions
CGP_52411
- binding interactions of ATP and CGP_52411 in binding site shows a

close resemblance (or similar binding pattern, i.e. H-bond + hydrophobic)
41
1/23/2015
Clinically used inhibitor:

IRESSA (AstraZeneca): an inhibitor of the EGFR
IRESSA: is used to treat patients with locally-advanced or metastatic non
small cell lung cancer who have previously received chemotherapy or who are
not suitable for chemotherapy.
By inhibiting the activity of this enzyme, IRESSA slows tumor growth,
metastasis (spread of tumors from one part of the body to another) and
angiogenesis (formation of new blood vessels supporting growth of the tumor)
Q: Analysis the potential interaction of the anticancer drug with EGFR.
83
Clinically used inhibitor:

IRESSA (AstraZeneca): an inhibitor of the EGFR
(PDB: 2ito)
Mimic ATP?
Please watch the video at http://www.youtube.com/watch?v=OjLy04cEkB8
84
42
1/23/2015
Important open e-resource for published proteins

http://www.rcsb.org/ (highly recommended)
PDB id (4 letter)
useful details: sources

a.a. sequence, Mw, class
diseases, references, etc
Structural Determination Methods in Drug Design

1) NMR Spectroscopy
2) X-ray Crystallography
3) Modelling (Computer-aided Drug Design)
43
1/23/2015
Structural Determination of Biomolecules using

X-ray and NMR methods
solution structure
High-field NMR spectrometer
High-power X-ray diffractometer
crystal (solid-state) structure
Structural Determination by X-ray
Braggs Law = 2dsin
diffraction pattern
Fourier Transform (Ihkl (x,y,z)
place atoms
(solution)
improve fitting
(refinement)
X-ray crystal structure

Electron Density Map
Model building
44
1/23/2015
X-ray crystallography
Resolution and structural information

5.5 - see overall shape of the molecule - helices
show up as rods of strong intensity
3.5 - can see most of the main chain (some
ambiguity)
3.0 - side chains will be partially resolved
2.5 - side chains are well resolved. Can see the
plane of the peptide bond. Atoms located to 0.4
Electron
1.5 - Atoms located to 0.1
0.77 - Bond lengths in small molecules measured to 0.005
PDB: 2H1W
Resolution () 2.6
2AC4
2.1
Structural Determination by NMR
2H1W
1.2
density
Trp
89
45
1/23/2015
Nuclear Magnetic Resonance (NMR)
Chemical shift (0-10 ppm)
design pulse sequence

for 2D/3D NMR experiments
isotope-enriched
protein sample
spatial
correlations of
neighboring a.a.
2D
1H-15N
NMR: fingerprint of a protein
Recombinant DNA technology
solution structure
(ensembles)
NMR-based screening
SAR-by-NMR: a combinatorial chemistry concept
(Please read NMR in drug discovery Nat. Rev. Drug
Discov. 2002, 1, 211-219
92
46
1/23/2015
Inhibition of an influenza A virus proton (H+) channel M2
Furthering Reading
M2: pH-gated proton

channels in the viral lipid
envelope
97-residue single-pass
membrane protein
Homo-tetramer in its native
state, forming a channel
His37 is the sensor & Try41
is the gate
Rimantadine-binding pocket
(Schnell JR, Nature 2008, 451, 591-595;

93
Stouffer Nature 2008, 451, 596-599)
Comparison of two methods

X-ray crystallography & NMR are complementary techniques
NMR
1. Short time scale, protein folding
2. Solution, purity
3. <20 kDa, domain (max. 64 kDa)
4. Atomic nuclei, chemical bonds
5. Resolution limit at 2-3.5
6. Primary structure must be known
(aa or nucleotide sequence)
7. Functional active site
X-ray crystallography
long time scale, static structure
single crystal, purity
any size, domain, complex
electron density
resolution limit ~ 1-3.5
primary structure must be know
(except if resolution is 2 or below for
every single residue)
active or inactive
47
1/23/2015
Summary:
1. General introduction of drugs and drug discovery

2. Drug targets: Proteins (enzymes), nucleic acids, lipids &
carbohydrates
3. Proteins as drug targets: structural proteins, transporters,
ion channels etc
4. Proteins as drug targets: enzymes- discovery of anti-HIV
and anticancer drugs (proteinase)
5. Lead optimization: strategies- increase activity, selectivity
and reduce dosage & side-effects
Techniques:
Chemical techniques: synthesis & structural characterization (X-ray & NMR)
optimizations
Biochemical techniques: bioassays; structure-based drug design; recombinant
DNA technology; proteomics/metabollomics
95
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CHEM3407/3410 & BPHM3015

Targets and Mechanisms of Drugs Action

3. Structural Protein as Drug Target

3. Structural Protein as Drug Target

CHEM3407 Medicinal Chemistry

Anticancer drug Taxol

discovered in 1971 and clinically used in 1992

CHEM3407 Medicinal Chemistry

Binding site in drug-target complex

Multi-step synthesis of Taxol

CHEM3407 Medicinal Chemistry

CHEM3407 Medicinal Chemistry

Other Mechanism of Action

CHEM3407 Medicinal Chemistry

4 Proteins as Drug Targets: Enzymes

CHEM3407 Medicinal Chemistry

4 Proteins as Drug Targets: Enzymes

CHEM3407 Medicinal Chemistry

2. Induced fit model

CHEM3407 Medicinal Chemistry

How to regulate enzymatic activity?

- activates by protein phosphorylation (Tyr-OH, Ser-OH, The-OH

CHEM3407 Medicinal Chemistry

- both substrate and inhibitor

- inhibitor binds at a different region

CHEM3407 Medicinal Chemistry

Enzyme Inhibition: reversible or irreversible?

CHEM3407 Medicinal Chemistry

Enzyme catalyzed reactions

CHEM3407 Medicinal Chemistry

- binding of pyruvic acid at the active site of lactate dehydrogenase

CHEM3407 Medicinal Chemistry

Enzyme catalyzed reactions

CHEM3407 Medicinal Chemistry

CHEM3407 Medicinal Chemistry

Before glucose binding

After glucose binding

Proteases (Digestive Enzymes)

CHEM3407 Medicinal Chemistry

Four major classes of proteases (aspartic, serine, cysteine,

CHEM3407 Medicinal Chemistry

a cocktail of drugs for AIDS

HIV Protease Enzyme

CHEM3407 Medicinal Chemistry

HIV protease is essential in the life cycle (replication & release of

HIV protease (wild type)

- HIV protease inhibitor ?

inhibitor-HIV protease complex

CHEM3407 Medicinal Chemistry

Any other advantages of using aspartyl protease?

1. Small enzyme which could be either synthesized or

Strategy for development?

CHEM3407 Medicinal Chemistry

HIV Protease Enzyme

CHEM3407 Medicinal Chemistry

Clinically Used HIV-1 Protease Inhibitors

Saquinavir (IC50< 50 nM, Roche)

CHEM3407 Medicinal Chemistry

Active Site of HIV protease enzyme

Two Asp25 residues are

CHEM3407 Medicinal Chemistry

CHEM3407 Medicinal Chemistry

- a compound mimics the transition-state of enzyme-catalyzed reaction