The Pharmacophore X-Ray Structure of Abl-Tk/STI-571 (1IEP)

The functional groups (ionization considered) of a drug and the bioactive conformation they must
adopt to sustain high-affinity and specific non-covalent interactions with the molecular target.
N
Drug-MT binding forces are the same that stabilize protein tertiary structure: H H
N N N NCH3
• Hydrogen bonds
N O
• Hydrophobic interactions H H3C

O H
• Electrostatic interactions
HO NH3
• Ion-dipole interactions •• N Bioactive conformation of STI-571
from X-ray coordinates (1IEP)
• Dipole-dipole interactions O STI-571(a.k.a. imatinib or Gleevec®) Via Protein Data Bank
• Charge-transfer complexes H (http://www.rcsb.org/pdb/)
Bioactive conformation
• ʌ-cation

Solvation and intramolecular binding forces:

• Energy penalty of desolvation of drug and protein MT
• Hydrophobic collapse
• Intramolecular hydrogen bonds
--------------
Aldrich catalog:
(R)-(-)-Norepinephrine; Į-(aminomethyl)-3,4-dihydroxybenzyl alcohol; (HO)2C6H3CH(CH2NH2)OH
Wentland-SC-9

Wentland-SC-9k

CHEM-4330 - Spring, 2004 - Module 2 1 CHEM-4330 - Spring, 2004 - Module 2 2

Protein Structure
Nearly all Natural Amino Acids have a Center of Chirality
O P3 O P1 O P2' O P4'
H H H H
Primary (1o) structure: N
N
N
N
N
N
N
N 20 Natural AAs have (S)- or L- absolute
[Amino acid sequence] P4
H
O P2
H
O P1'
H
O P3'
H
O
R H
configuration except Cys (R-) and Gly
+ O-
H3N L- Ÿ L-glyceraldehyde (Fischer notation of
O absolute configuration)
Secondary (2o) structure: (R)- or (S)- Ÿ Cahn-Ingold-Prelog notation
[Conformation of segments of
of absolute configuration)
backbone (e.g., D-helix, E-sheet)]

Tertiary (3o) structure:

[3D arrangement of all atoms HO The tripeptide, H-Ser-Ala-Phe-OH,
In a protein (e.g., Abl-TK)] O drawn in the standard "zig-zag"/
H
H N OH N- to C-terminus representation.
N N
H H
O CH3 O
Quaternary (4o) structure:
[3D structure of proteins having
more than one peptide chain
(e.g., homodimeric HIV protease)] Wentland-SC-9v

Wentland-SC-9m

CHEM-4330 - Spring, 2004 - Module 2 3 CHEM-4330 - Spring, 2004 - Module 2 4

 Interactions that Stabilize the Secondary Structure of Proteins Amino Acid/Peptide Primer
R H

+ O- 20 Natural AAs have (S)- or L- absolute R H H O R'' H

H3N configuration except Cys (R)- and Gly N
O N N
H3C O O
H H R' H
•
•
•
•
•
•
Amino Acid R= 3 Letter Name 1 Letter Name
CH2 CH •• CO2H
• Glycine H Gly G
H3C Primary peptide structure (transoid form)
Alanine CH3 Ala A
•
•
•
•
•
•

Valine CH(CH3)2 Val V

Hydrophobic
•• Leucine CH2CH(CH3)2 Leu L
•
•
•
•
•
•

• Isoleucine (S)-CH(CH3)CH2CH3 Ile I O O

Serine CH2OH Ser S
•
•
•
•
•
•

..
N N N+
Threonine (R)-CH(OH)CH3 Thr T
CH2-S-S-CH2 •• •
H
•• Cysteine CH2SH Cys C H H
•
•
•
•
•
•

Disulfide • C O Methionine Met

CH2CH2SCH3 M
H-bond Restricted rotation due to amide resonance
Phenylalanine CH2C6H5 Phe F
•
•
•
•
•
•

Tyrosine CH2-4-C6H4OH Tyr Y

•• O
• Tryptophan CH2-3-indolyl Trp W
•
•
•
•
•
•

Histidine CH2-4-imidazolyl His H 1.23Å

Arginine (CH2)3NHC(=NH)NH2 Arg R 121.1o 123.2o

•
•
•
•
•
•

C C
O H2N Lysine (CH2)4NH2 Lys K 2Å 121.9o 1.4
5Å

D-Helix CH2 N
1.5
1.3
3
Aspartic Acid CH2CO2H Asp D C 115.6o
N
H
•
•
•
•
•
•

O H2N Glutamic Acid CH2CH2CO2H Glu E 119.5o 118.2o

Asparagine CH2CONH2 Asn N 1.0Å

Electrostatic H
Biochim. Biophys. Acta.
H2 N Glutamine CH2CH2CONH2 Gln Q 1974, 359, 298.

Proline Pro P
E-Pleated sheet +
-
N CO2
H H Wentland-SC-10a
Wentland-SC-10

CHEM-4330 - Spring, 2004 - Module 2 5 CHEM-4330 - Spring, 2004 - Module 2 6

H
Thr

H R O CH3
H R R
O H O H O
H
N N N N
N N N N
R RO O H O O
R H R R H
O
H O R H O R H O R H
H
N N N
N
N Glu N N N

O R R H R H
O-
O O O
R H

H R R R
H O H O H O
O
N N N
N N N N
O
R H O H O H O
O R R R
R

Wentland-SC-12c
Wentland-SC-13c

CHEM-4330 - Spring, 2004 - Module 2 7 CHEM-4330 - Spring, 2004 - Module 2 8

 Abl-Tk/STI-571 Non-covalent Interactions from 1IEP Enzyme-Ligand Non-Covalent Interactions: Competitive Inhibition

E H O+ S H O E·S [E · S]‡ E·P P + E

2 2
NCC (non-covalent complexes)

Inhibitor Substrate

X-H Inhibitor X-H X-H Substrate

glu286

thr315

glu286 koff
[E] [I] koff
thr315
CH2CH2
E·I E + I Ki = =
NCC kon [E · I] kon
CH3 CH H
O O O

H H N
N N N NCH3 "Slow tight-binding" inhibitors are characterized by:
- Slow (relative to diffusion control) "on rate"
N O
H3C - Very slow "off rate"
IC50 = 38 nM - Displacement of a structured H 2O from active site
N Wentland-SC-13d - Transition state analogue Wentland-SC-13f

CHEM-4330 - Spring, 2004 - Module 2 9 CHEM-4330 - Spring, 2004 - Module 2 10

Lineweaver-Burk Plots for Determination of Ki of a Competitive Inhibitor IC50 Value and Dose Response Curves

0.9 100
90

Enzyme inhibition (%)

0.8
80
70
PM

0.7 IC50 = 1.0 PM IC50 = 10 PM

3X

60 IC50 = [Inhibitor] that reduces

PM
=

0.6
2X
[I]

50 product formation by 50%

=
1 [I] M
(min/mM)
0.5 XP 40
[v] =1
[I] Both inhibitors are equally active;
0.4 30
0 PM
[I] = 20
one is 10-fold more potent
0.3
10
0.2

0.1 0.10 0.30 1.0 3.0 10 30 100

Inhibitor concentration - PM
{

1 0 1 2 3 4 5 6 7 8 9 10
Km 1 [S]
(mM-1) IC50 = Ki 1 + When [S] is 10-fold or more below its Km, then IC50 ~ Ki
1 [S] Km
1
Kmapp Km (1 + [I]/Ki)
Wentland-SC-14b

Wentland-SC-14a

CHEM-4330 - Spring, 2004 - Module 2 11 CHEM-4330 - Spring, 2004 - Module 2 12

Enzyme-Ligand Non-Covalent Interactions: Free Energy of Binding Drug-Protein Non-Covalent Interactions: Noncompetitive Inhibition
Inhibitor/drug binds to enzyme at a different site than substrate
'G = Gproducts- Greactants
o o o

X-H X-H

'Go = 'Ho - T'So

• Enthalpic (H) effects: H-bonds, (de)solvation, electrostatics, VDW, etc.

• Entropic (S) effects:
- Unbound ligand Ÿn S (translational and rotational energies) Point to Ponder - Complex kinetics may
substrate hinder quantification of activity; e.g., drug
- Bound ligand Ÿp S (fewer degrees of freedom) E · S · I can still be catalytically active
- Water release (ordered to disordered) Ÿn S

drug drug

Attributes of a competitve inhibitor:

X-H substrate X-H substrate X-H
- Active-site directed
- High affinity and specific non-covalent interactions with MT
- Does not act as alternate substrate
- "Drug-like" E·S E·S·I E·I
Wentland-SC-14c Wentland-SC-14d

CHEM-4330 - Spring, 2004 - Module 2 13 CHEM-4330 - Spring, 2004 - Module 2 14

Drug-Protein Interactions: Covalent Bonding of Drug to Enzyme

Drug-Protein Binding Forces
• Hydrogen bond - linear non-covalent bond between a donor H (O-H or N-H) and an acceptor O, N or F.
E + I* E • I* E I*
- Stabilization: 'Go = 'Ho - T'So ~ - 0.5 to -7 kcal/mol with 2.4-3.0 Å optimal
NCC
Alkylation: CH3

O
N H N ..
N H O

..
Br O H N
H
X-H drug* X drug* X
irreversible O X

+ acceptor (drug) donor (protein) donor (drug) acceptor (protein)

- Desolvation Penalty

O
S1 H H
S3 .. ..
O O

H2N
NH2 .. . . H .. .. . . + H2O
N H O O H O N H O N

..
+ N
H H
H H
HN
O X X
H2N O
Solvated drug and protein - unbound Drug - protein NCC
N
N
H Cl Enthalpic and entropic benefit in establishing H-bond contacts with MT may be offset by an
O uncompensatable desolvation penalty. Then why do this? SELECTIVITY and SOLUBLITY !!
S2
Wentland-SC-17
PPACK: inhibitor of human thrombin Wentland-SC-14f

CHEM-4330 - Spring, 2004 - Module 2 15 CHEM-4330 - Spring, 2004 - Module 2 16

 Drug-Protein Binding Forces Drug-Protein Binding Forces
o
• Electrostatic interactions ('G ~ -5 to -10 kcal/mol)
• Hydrophobic interactions ('Go ~ - 0.5 to -1 kcal/mol)
H
CH3 O O H NH2(CH2)4-Lys O H N • Enthalpic considerations - Van der Waals contacts
Drug N H CH2 Glu Drug Drug NH(CH2)3-Arg
CH3 O O O H N H2C O
H

HC O O

o
• Ion-dipole interactions ('G ~ -3 to -5 kcal/mol) • Dipole-dipole interactions ('Go ~ -1 to -3 kcal/mol)
H2C O O

CH3 O
-G H
Drug N H O +G Drug N·· +G
N
CH3 N H O
-G

+G
• Charge-transfer complexes ('Go ~ -1 to -7 kcal/mol) •S-Cation complexes ('Go ~ -0.5 to -1.5 kcal/mol) C
-G
OCOCH3 H H
CN CH2 +G
Drug Drug
H H
N O H3N CH2
(CH3)3N
Trp-84
C
HO CH2 Tyr CH2-Phe -G
N
H

S-cation interaction between

ACh and acetylcholine esterase
• Entropic considerations
Wentland-SC-17g
Wentland-SC-17d

CHEM-4330 - Spring, 2004 - Module 2 17 CHEM-4330 - Spring, 2004 - Module 2 18

Hydrophobic Interactions - Entropic Considerations

Hydrophobic Collapse
Ordered water molecules surrounding hydrophobic surfaces
• Change in conformation of a molecule bought about by dissolution in water relative to that
conformation observed in an organic environment.
+ N
H
• Energy in the form of decreased binding affinity may be required to adopt the bioactive
O conformation when that drug exists in a different, but stable conformation in water due to
X
Water release also stabilizes: intramolecular hydrophobic interactions, or conversely;

water release = nS Phe

Drug Drug Trp • If the hydrophobically-collapsed conformation is very similar to the bioactive conformation,
CH2
CH2 then the molecule is "preorganized" for binding resulting in ehanced binding affinity, e.g., Taxol:

N H
N AcO NOE's observed between the 4-acetyl methyl,
N H
PhCONH O 10 O
H OH 2-benzoyloxy phenyl and 3'-phenyl groups in
SS Stacking Edge-to-face
X O
3' O 13 DMSO-water solution.
OH 2
4
HO Vander Velde, D.G.; Georg, G.I.; Grunewald,
O O O
G.L.; Gunn, C.W.; Mitscher, L.A. J. Amer.
O O Chem. Soc. 1993, 115, 11650-11651.
2 H3C
• The larger the surface area the greater the effect (~ 28 cal/mole/Å )
Wentland-SC-18a
• H2O solvation of unbound ligand may have an uncompensatable enthalpic advantage
Wentland-SC-18

CHEM-4330 - Spring, 2004 - Module 2 19 CHEM-4330 - Spring, 2004 - Module 2 20

 Enzyme-Ligand Binding: A Closer Look
Factors Contributing to High Affinity Binding
From: Davis, A. M.; Teague, S. J. “Hydrogen Bonding, Hydrophobic Interactions, and Failure of
Lock and key (Fisher, 1894): the Rigid Receptor Hypothesis” Angew. Chem. Int. Ed. 1999, 38, 736-749.

L1 High affinity binding is generally achieved via induced fit of MT around a ligand having optimized:

+ L1 • Specific hydrophobic interactions

• Polar interactions
- Contribution of an HB is unpredictable
- Neutral-neutral HB contributes 0- to 15-fold in binding affinity
- Charge reinforced HB contributes up to 3000-fold in binding affinity
Induced fit (Koshland, 1958):

L2
L2 How do you achieve high affinity binding? “Stay tuned”
+ L2

Wentland-SC-18e

Teague, S. J. "Implications of Protein Flexibility for Drug Discovery." Nature Rev. - Drug Disc. 2003, 2, 527-541.

Wentland-SC-18c
CHEM-4330 - Spring, 2004 - Module 2 21 CHEM-4330 - Spring, 2004 - Module 2 22

The Power of Non-Covalent Interactions: ELISA-Based Colorimetric TK Assay

Biotin-Streptavidin Non-Covalent Interactions • Biotinylated peptide substrate "immobilized" to streptavidin-coated 96-well ELISA microtiter plate
koff (ELISA = Enzyme-Linked ImmunoSorbant Assay)
SA • B SA + B
kon
O • Add test compounds in varying concentrations and positive/negative controls
H H
[ SA ] [ B ] N OH • Add a Tyrosine Kinase and ATP to each well, incubate, and wash
Kd = = 4 x 10-14 M O S
• Add anti-phosphotyrosine antibody, incubate, and wash
[ SA • B ] N Biotin
H H
o o o MW = 244.2 • Add horse radish peroxidase (HRP)-conjugated anti-mouse IgG, incubate and wash
'G = 'H - T'S = - 2.303RT logKeq = - 18.3 kcal/mol
• Develop by adding HRP substrate reagent to each well
Every 10-fold increase in potency (K) Ÿ - 1.36 kcal/mol
• OD (optical density) measured by ELISA auto-reader (absorbance at 415 nm)
O Asn-49 • IC50 obtained is [drug] resulting in 50% inhibition
Binding interactions from 1STP.pdb:
Ser(-45)CH2 N

OH H O NH2
Ser(-27)CH2 O Protein ATP N
O N HRP
H H O H substrate
H R' N N
N O N H O O
O
Tyr-43 O H O S H CH2Ser-88 O
O -
CH2 OH O P O P O P O
N O
H O- O- O-
H
H
H
Hydrophobic pocket formed
H N +
HN O OH OH
by Trp-79, -92, -108 H
O O R
Asn(-23) O N H anti-phosphotyrosine
TK antibody
Asp-128

O
H O H O H O H OPO3-2
H H H
N O N O N O R'
N H O
O S O S O S
O
signal Y
N N N CH2 O P O- B
-
biotinylated peptide
H H H H O
H H H H N SA
Binding stabilizes dipolar resonance contributors O
H
R
Weber, P.C.; Ohlendorf, D.H.; Wendoloski, J.J.; Salemme, F.R. Science 1989, 243, 85. Wentland-SC-20a N H

CHEM-4330 - Spring, 2004 - Module 2 23 CHEM-4330 - Spring, 2004 - Module 2 24 Wentland-SC-20d