Covalent Bonding Of Drug To Enzyme

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. Drug-MT binding forces are the same that stabilize protein tertiary structure:

H N

N

N

H N

NCH3

• Hydrogen bonds N

• Hydrophobic interactions

H O

• Electrostatic interactions

H NH3

HO ••

• Ion-dipole interactions • Dipole-dipole interactions

O

• Charge-transfer complexes

H

O

H3C

N

Bioactive conformation of STI-571 from X-ray coordinates (1IEP) Via Protein Data Bank (http://www.rcsb.org/pdb/)

STI-571(a.k.a. imatinib or Gleevec®) 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

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Protein Structure

Nearly all Natural Amino Acids have a Center of Chirality Primary (1o) structure: [Amino acid sequence]

O

H N P4

P3 N H

O

H N O

P2

P1 N H

O

P2'

O

H N P1'

N H

O

H N O

P3'

P4' N H

R

O

20 Natural AAs have (S)- or L- absolute configuration except Cys (R-) and Gly

H O-

+ H3N

L- Ÿ L-glyceraldehyde (Fischer notation of absolute configuration) (R)- or (S)- Ÿ Cahn-Ingold-Prelog notation of absolute configuration)

O

Secondary (2o) structure: [Conformation of segments of backbone (e.g., D-helix, E-sheet)]

Tertiary (3o) structure: [3D arrangement of all atoms In a protein (e.g., Abl-TK)]

HO

The tripeptide, H-Ser-Ala-Phe-OH, drawn in the standard "zig-zag"/ N- to C-terminus representation.

O H N

H N H

O

OH N H CH3

O

Quaternary (4o) structure: [3D structure of proteins having more than one peptide chain (e.g., homodimeric HIV protease)]

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Interactions that Stabilize the Secondary Structure of Proteins

Amino Acid/Peptide Primer R

H O-

+ H3N

20 Natural AAs have (S)- or L- absolute configuration except Cys (R)- and Gly

R

O

• • • • • •

H3C CH

CH2

H3C

• • • • • •

CO2H

•• •

Hydrophobic

• • • • • •

• • • • • •

•• • N

CH2-S-S-CH2

• • • • • •

•• •

Disulfide

C

O

• ••

H

• • • • • •

H-bond

• • • • • •

• • • • • •

•• • O

D-Helix

H2N

• • • • • •

CH2 O

H2N

N H

Electrostatic

H2 N E-Pleated sheet

Amino Acid Glycine Alanine Valine Leucine Isoleucine Serine Threonine Cysteine Methionine Phenylalanine Tyrosine Tryptophan Histidine Arginine Lysine Aspartic Acid Glutamic Acid Asparagine Glutamine Proline

H

H

N

3 Letter Name R= Gly H Ala CH3 Val CH(CH3)2 Leu CH2CH(CH3)2 Ile (S)-CH(CH3)CH2CH3 Ser CH2OH Thr (R)-CH(OH)CH3 Cys CH2SH Met CH2CH2SCH3 Phe CH2C6H5 Tyr CH2-4-C6H4OH Trp CH2-3-indolyl His CH2-4-imidazolyl Arg (CH2)3NHC(=NH)NH2 Lys (CH2)4NH2 Asp CH2CO2H Glu CH2CH2CO2H Asn CH2CONH2 Gln CH2CH2CONH2 Pro + N H

CO2

O

H

1 Letter Name G A V L I S T C M F Y W H R K D E N Q P

R'' H

O

N

N

H

O

H

R'

Primary peptide structure (transoid form)

O

O .. N

N+

H

H

Restricted rotation due to amide resonance O 1.23Å 121.1o

123.2o

C 2Å 1.5

121.9o 115.6o

C

5Å 1.4

C

1.3 3

N 119.5o

118.2o 1.0Å

Biochim. Biophys. Acta. 1974, 359, 298.

H

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H H

R H

H N

H

N

RO

N

R

O H

O

H O R

H N

Glu

R

H

O R

O

O-

O H

H

O

O R

N

R H

O

H R

N

R H

O R

H

H N

N

O

H

O

R

O

N

N

N

R

H

O

H

N

N R

N O

H

N

N

R

O

N

N

R

H

O

N

N R

CH3

O

R

O

Thr

N

N

N

O O

R

R

H

O

R

O

H

R

H

O

R

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Enzyme-Ligand Non-Covalent Interactions: Competitive Inhibition

Abl-Tk/STI-571 Non-covalent Interactions from 1IEP

E H O+ S H O 2

E·S

2

[E · S]‡

E·P

Substrate

Inhibitor

X-H

glu286

P + E

NCC (non-covalent complexes)

X-H

Inhibitor

X-H

Substrate

thr315

koff

glu286 thr315 CH3 CH

N N

E·I

CH2CH2 O

H

H

N

N

Ki =

koff [E] [I] = kon [E · I]

N

"Slow tight-binding" inhibitors are characterized by: - Slow (relative to diffusion control) "on rate" - Very slow "off rate" - Displacement of a structured H 2O from active site - Transition state analogue

NCH3 O

H3C

E + I

O

O

H

NCC

kon

IC50 = 38 nM N

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Lineweaver-Burk Plots for Determination of Ki of a Competitive Inhibitor

IC50 Value and Dose Response Curves 100 Enzyme inhibition (%)

0.9

PM

0.8

[I] =

3X

0.7 0.6

1 (min/mM) [v]

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10

[I]

0.5

=

2X

[I]

0.4

PM

M XP =1 0 PM [I] =

90 80 70

IC50 = 1.0 PM

IC50 = 10 PM IC50 = [Inhibitor] that reduces product formation by 50%

60 50 40

Both inhibitors are equally active; one is 10-fold more potent

30 20

0.3

10 0.2 0.10

0.1

0.30

1.0

3.0

10

30

100

{

Inhibitor concentration - PM 0

1 Km 1 Kmapp

1

2

3

4

5

6

7

8

9

10

1 (mM-1) [S]

1 Km (1 + [I]/Ki)

IC50 = Ki 1 +

[S] Km

When [S] is 10-fold or more below its Km, then IC50 ~ Ki

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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: Point to Ponder - Complex kinetics may hinder quantification of activity; e.g., E · S · I can still be catalytically active

- Unbound ligand Ÿn S (translational and rotational energies) substrate

- Bound ligand Ÿp S (fewer degrees of freedom)

drug

- Water release (ordered to disordered) Ÿn S drug

drug

Attributes of a competitve inhibitor: X-H

- Active-site directed

X-H

substrate

X-H

substrate

- High affinity and specific non-covalent interactions with MT - Does not act as alternate substrate - "Drug-like"

E·S

E·S·I

E·I

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Drug-Protein Interactions: Covalent Bonding of Drug to Enzyme E + I*

E • I*

E

14

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.

I*

- Stabilization: 'Go = 'Ho - T'So ~ - 0.5 to -7 kcal/mol with 2.4-3.0 Å optimal

NCC Alkylation:

CH3 O N

drug*

X-H

X

irreversible

X

drug*

+

N

O

N

H

O

acceptor (drug)

.. O

H

..

H

Br

N

H

X

donor (drug)

donor (protein)

acceptor (protein)

- Desolvation Penalty O

S1

S3 H2N

H

N

H

O

+

H

.. O

N

H

X

O O N H

PPACK: inhibitor of human thrombin 15

H

.. . . O

N

+ H2O H

Drug - protein NCC

Enthalpic and entropic benefit in establishing H-bond contacts with MT may be offset by an uncompensatable desolvation penalty. Then why do this? SELECTIVITY and SOLUBLITY !!

Cl O

S2

N H X

Solvated drug and protein - unbound

N

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O .. . . H O H

HN H2N

.. .. O

..

NH2

H

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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 Drug

N

O

O

H CH3

NH2(CH2)4-Lys

H

O

Drug

CH2 Glu

H

• Enthalpic considerations - Van der Waals contacts

N

Drug

NH(CH2)3-Arg

O

O

O

H

N H

• Dipole-dipole interactions ('Go ~ -1 to -3 kcal/mol)

o

• Ion-dipole interactions ('G ~ -3 to -5 kcal/mol)

CH3 Drug

N

H

-G O

O

HC

O

O

H2C

O

O O

H Drug

+G

CH3

N

+G

N··

H

N

O -G

• Charge-transfer complexes ('Go ~ -1 to -7 kcal/mol)

N

H

OCOCH3 CH2 O

H3N

Drug

(CH3)3N

H

-G H

CH2 Trp-84 N

+G

H

C -G

CH2-Phe

CH2 Tyr

HO

+G C

•S-Cation complexes ('Go ~ -0.5 to -1.5 kcal/mol)

CN Drug

H2C

H

S-cation interaction between ACh and acetylcholine esterase

• Entropic considerations Wentland-SC-17g

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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

O

X

Water release also stabilizes: Phe

water release = nS

Drug

Drug

Trp

CH2 CH2

N

N

H

N H

H

SS Stacking X

• Energy in the form of decreased binding affinity may be required to adopt the bioactive conformation when that drug exists in a different, but stable conformation in water due to intramolecular hydrophobic interactions, or conversely; • If the hydrophobically-collapsed conformation is very similar to the bioactive conformation, then the molecule is "preorganized" for binding resulting in ehanced binding affinity, e.g., Taxol:

AcO 10

O

PhCONH

OH

Edge-to-face 3'

O

NOE's observed between the 4-acetyl methyl, 2-benzoyloxy phenyl and 3'-phenyl groups in DMSO-water solution.

O

O

13 2

OH

4

HO O

O

Vander Velde, D.G.; Georg, G.I.; Grunewald, G.L.; Gunn, C.W.; Mitscher, L.A. J. Amer. Chem. Soc. 1993, 115, 11650-11651.

O O

O H3C

2

• The larger the surface area the greater the effect (~ 28 cal/mole/Å )

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• H2O solvation of unbound ligand may have an uncompensatable enthalpic advantage Wentland-SC-18

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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 the Rigid Receptor Hypothesis” Angew. Chem. Int. Ed. 1999, 38, 736-749.

Lock and key (Fisher, 1894):

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

L1

• Specific hydrophobic interactions

L1

+

• 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

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

L2

L2

+

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

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

Biotin-Streptavidin Non-Covalent Interactions koff

SA • B

SA + B

kon

Kd = o

o

[ SA ] [ B ] [ SA • B ]

• Add test compounds in varying concentrations and positive/negative controls

O

H H N

= 4 x 10-14 M

• Biotinylated peptide substrate "immobilized" to streptavidin-coated 96-well ELISA microtiter plate (ELISA = Enzyme-Linked ImmunoSorbant Assay) • Add a Tyrosine Kinase and ATP to each well, incubate, and wash

OH S

O N H

o

H

'G = 'H - T'S = - 2.303RT logKeq = - 18.3 kcal/mol

• Add anti-phosphotyrosine antibody, incubate, and wash

Biotin

• Add horse radish peroxidase (HRP)-conjugated anti-mouse IgG, incubate and wash

MW = 244.2

• 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)

Ser(-45)CH2

H

H

H

O

N

H

S

CH2Ser-88

O

O

H

H

H

HN

H

Hydrophobic pocket formed by Trp-79, -92, -108

Protein substrate

ATP

N

N

R

H

O

H

N

-

O

OH

P

P

O-

+

O

O O

O

O-

O N

P

O

O

OOH

H

HRP

N

N

CH2

H

O

O

O

O

R'

N

Asn(-23)

NH2

O

H

O

H

O

H

N O

• IC50 obtained is [drug] resulting in 50% inhibition

N OH

Ser(-27)CH2 O

Tyr-43

Asn-49

O

Binding interactions from 1STP.pdb:

OH

anti-phosphotyrosine antibody

TK

Asp-128

H

O

H

N O

S N H

H

O

H

N

O

H

O

S H

S H

OPO3-2 N

O

N H

O

H R'

O

O

N H

O

H

N

O

H

H

CH2 H

N

Binding stabilizes dipolar resonance contributors

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R

O O

signal

Y

P OO

B

-

biotinylated peptide

SA

O

H

Weber, P.C.; Ohlendorf, D.H.; Wendoloski, J.J.; Salemme, F.R. Science 1989, 243, 85.

H

N

H

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