covalent bonding of drug to enzyme

The Pharmacophore

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:

• Hydrogen bonds

• Hydrophobic interactions

• Electrostatic interactions

• Ion-dipole interactions

• Dipole-dipole interactions

• Charge-transfer complexes

• -cation

Solvation and intramolecular binding forces:

• Energy penalty of desolvation of drug and protein MT

• Hydrophobic collapse

• Intramolecular hydrogen bonds

- - - - - - - - - - - - - -

Aldrich catalog:

(R)-(-)-Norepinephrine; -(aminomethyl)-3,4-dihydroxybenzyl alcohol; (HO)2C6H3CH(CH2NH2)OHWentland-SC-9

HO

O

O

NH3

H

H

• •

Bioactive conformation

H

CHEM-4330 - Spring, 2004 - Module 2 1

Wentland-SC-9k

N

N

N

HN

HN

O

N

NCH3

H3C

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

Bioactive conformation of STI-571from X-ray coordinates (1IEP)

Via Protein Data Bank (http://www.rcsb.org/pdb/)

STI-571(a.k.a. imatinib or Gleevec®)


Wentland-SC-9m

Protein Structure

Primary (1o) structure:[Amino acid sequence]

NH

HN

NH

HN

NH

HN

NH

O P1'

O P2'

O

O

O

O

O P3'

P4'P1P3

P2

HN

O

P4

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

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

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


NH

HN

NH

OH

O

O

O CH3

H

HO

H3N

O

R H

O-

Wentland-SC-9v

Nearly all Natural Amino Acids have a Center of Chirality

+

20 Natural AAs have (S)- or L- absolute

configuration except Cys (R-) and Gly

L- L-glyceraldehyde (Fischer notation of

absolute configuration)

(R)- or (S)- Cahn-Ingold-Prelog notation

of absolute configuration)

The tripeptide, H-Ser-Ala-Phe-OH,

drawn in the standard "zig-zag"/

N- to C-terminus representation.


CH2-S-S-CH2

CH2

H3C

CH

H3C

CH2

O

O

NH

H2N

H2N

•••

•••

•••

•••

• • • • • •

• • • • • •

• • • • • •

• • • • • •

• • • • • •

• • • • • •

• • • • • •

• • • • • •

• • • • • •CO

2H

H2N

C O

H

N

Disulfide

Hydrophobic

-Pleated sheet

H-bond

Electrostatic

-Helix

Wentland-SC-10

Interactions that Stabilize the Secondary Structure of Proteins

• • •


Biochim. Biophys. Acta.

1974, 359, 298.

1.33

1.45Å

O

C

H

1.0Å

C

1.23Å

1.52ÅC

121.1o

N

121.9o

123.2o

115.6o

118.2o119.5o

H3N

O

R H

O-

N

O

H

NN

N

O

O

O

R

H

R''H

R'

HH

HH

N

H HCO2

-

N+

O

H

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

Primary peptide structure (transoid form)

..

Restricted rotation due to amide resonance

Amino Acid/Peptide Primer

Amino Acid

Glycine

Alanine

Valine

Leucine

Isoleucine

Serine

Threonine

Cysteine

Methionine

Phenylalanine

Tyrosine

Tryptophan

Histidine

Arginine

Lysine

Aspartic Acid

Glutamic Acid

Asparagine

Glutamine

Proline

Wentland-SC-10a

R =

H

CH3

CH(CH3)2

CH2CH(CH3)2

(S)-CH(CH3)CH2CH3

CH2OH

(R)-CH(OH)CH3

CH2SH

CH2CH2SCH3

CH2C6H5

CH2-4-C6H4OH

CH2-3-indolyl

CH2-4-imidazolyl

(CH2)3NHC(=NH)NH2

(CH2)4NH2

CH2CO2H

CH2CH2CO2H

CH2CONH2

CH2CH2CONH2

3 Letter Name

Gly

Ala

Val

Leu

Ile

Ser

Thr

Cys

Met

Phe

Tyr

Trp

His

Arg

Lys

Asp

Glu

Asn

Gln

Pro

1 Letter Name

G

A

V

L

I

S

T

C

M

F

Y

W

H

R

K

D

E

N

Q

P+


N

O

H

R

Wentland-SC-12c

N

O

H

N

H

R

N

O

H

N

O

H

R

R

O

R

R

O-

Glu

O


N

O

O

H

R

N

N

O

O H

H

N

N

O

O H

N

H

RH

R R R

N

O

O

HR

N

N

O

OH

HR

NN

O

H

HR

RR

N

HRO

N

O

O

H

R

N

N

O

O H

H

R

N

N

O

O H

N

H

RH

R R R

Wentland-SC-13c

CH3O

HThr


Wentland-SC-13d

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

N

N

N

N N

O

N

NCH3

H3C

H H

O O

CH2CH2

OCH HCH3

thr315

glu286

glu286

thr315

IC50 = 38 nM


Wentland-SC-13f

Enzyme-Ligand Non-Covalent Interactions: Competitive Inhibition

E · I E + INCC

koff

kon

Ki =

[E · I][E] [I] k

off=

kon

"Slow tight-binding" inhibitors are characterized by:

- Slow (relative to diffusion control) "on rate"

- Very slow "off rate"

- Displacement of a structured H2O from active site

- Transition state analogue

NCC (non-covalent complexes)

E · S [E · S]‡ E · PE + S H2OH

2O P + E

X-H SubstrateX-H

SubstrateInhibitor

X-H Inhibitor


Lineweaver-Burk Plots for Determination of Kiof a Competitive Inhibitor

Wentland-SC-14a

1

Km

1

Kmapp

1

Km (1 + [I]/Ki)

{

0 1 2 3 4 5 6 7 8 9 10

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

[I] = 0 M

[I]=

3XM

[I]= 2X

M

[I] =1X

M

1[S]

(mM-1)

1[v]

(min/mM)


IC50

Value and Dose Response Curves

Wentland-SC-14b

Inhibitor concentration - M

Enzy

me in

hib

ition (

%)

10

20

30

40

50

60

70

80

90

100

1.0 100.10 0.30 3.0

IC50 = 1.0 M IC50 = 10 M

30 100

Both inhibitors are equally active;

one is 10-fold more potent

IC50 = Ki 1 +[S]Km

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

IC50 = [Inhibitor] that reducesproduct formation by 50%


Enzyme-Ligand Non-Covalent Interactions: Free Energy of Binding

Wentland-SC-14c

Go = Ho - T So

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

• Entropic (S) effects:

- Unbound ligand S (translational and rotational energies)

- Bound ligand S (fewer degrees of freedom)

- Water release (ordered to disordered) S

Attributes of a competitve inhibitor:

- Active-site directed

- High affinity and specific non-covalent interactions with MT

- Does not act as alternate substrate

- "Drug-like"

Go = Go - Goproducts reactants


Drug-Protein Non-Covalent Interactions: Noncompetitive Inhibition

Inhibitor/drug binds to enzyme at a different site than substrate

Wentland-SC-14d

X-H

substrate

X-H

drug

drug

X-H

X-H substrate X-H

drug

substrate

E · S · I

Point to Ponder - Complex kinetics may

hinder quantification of activity; e.g.,

E · S · I can still be catalytically active

E · S E · I


Wentland-SC-14f

Drug-Protein Interactions: Covalent Bonding of Drug to Enzyme

E + I* E • I* E I*

NCC

Alkylation:

Br

X

+

X-H drug* drug*irreversible

N

NH

O

OH2N

O

HN

H2NNH2

Cl

S1

S2

S3

PPACK: inhibitor of human thrombinCHEM-4330 - Spring, 2004 - Module 2 15

O NH

O NH

N

O

H

N H

X

X

O NH

HO

H

OH

H

NO

CH3

HO

O

NH

N

O

H

OH H

X

X

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

• Hydrogen bond - linear non-covalent bond between a donor H (O-H or N-H) and an acceptor O, N or F.

- Stabilization: Go = H

o - T So ~ - 0.5 to -7 kcal/mol with 2.4-3.0 Å optimal

Drug-Protein Binding Forces

- Desolvation Penalty

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 !!

Drug - protein NCCSolvated drug and protein - unbound

Wentland-SC-17

....

....

....

.. ..

+ H2O....

+


O

O H NH2(CH2)4-Lys

O

O

CH2 Glu

O N

NO

NH(CH2)3-Arg

H

H

H

H

N

CH3

CH3

H

N

CH3

CH3

H N N

H

ON H

O

(CH3)3NCH2

CH2

OCOCH3

H3NN O

CN

CH2-Phe

N

Trp-84

H

CH2 TyrHO

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

o ~ -0.5 to -1.5 kcal/mol)

Drug +-

Drug +

-

··

-cation interaction between ACh and acetylcholine esterase

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


Wentland-SC-17d

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

Drug

• Electrostatic interactions ( Go ~ -5 to -10 kcal/mol)

DrugDrug

Drug Drug


C

H H

C

HH

• Enthalpic considerations - Van der Waals contacts

Wentland-SC-17g


• Hydrophobic interactions ( Go ~ - 0.5 to -1 kcal/mol)

• Entropic considerations

H2C

HC

H2C

O

OO

OO

O

+

+

-

-


O

NH

X

O

NH

X

+

Hydrophobic Interactions - Entropic Considerations

Wentland-SC-18

Ordered water molecules surrounding hydrophobic surfaces

water release = S

• The larger the surface area the greater the effect (~ 28 cal/mole/Å2)

• H2O solvation of unbound ligand may have an uncompensatable enthalpic advantage

Water release also stabilizes:

Edge-to-face

N HNH

CH2

TrpDrug

Stacking

CH2

PheDrug


O

O

OOH

OOHO

AcO

OH

O

O

PhCONH

H3C

O

• Change in conformation of a molecule bought about by dissolution in water relative to that

conformation observed in an organic environment.

• 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:

Wentland-SC-18a

Hydrophobic Collapse

NOE's observed between the 4-acetyl methyl,

2-benzoyloxy phenyl and 3'-phenyl groups in

DMSO-water solution.

Vander Velde, D.G.; Georg, G.I.; Grunewald,

G.L.; Gunn, C.W.; Mitscher, L.A. J. Amer.

Chem. Soc. 1993, 115, 11650-11651.

10

13

24

3'


Enzyme-Ligand Binding: A Closer Look

Wentland-SC-18c

Induced fit (Koshland, 1958):

Lock and key (Fisher, 1894):

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

+ L1

L1

L2

L2

+

L2


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.

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

• 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

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

Wentland-SC-18e


S

HN

NH

O

H

H

OH

OBSA

S

N

N

H

H

O

H

H

Ser(-27)CH2 O

H

OTyr-43 H

H

HN

OAsn(-23)O O

Asp-128

OH

Ser(-45)CH2

O

O

H

N

HO

CH2Ser-88

O

O

Asn-49

S

N

N

O

H

H

O

OH

H

S

N

N

O

H

H

O

O

H

H

S

N

N

O

H

H

O

OH

H

Kd =[ SA ] [ B ]

[ SA • B ]= 4 x 10-14 M

Biotin

Biotin-Streptavidin Non-Covalent Interactions

Weber, P.C.; Ohlendorf, D.H.; Wendoloski, J.J.; Salemme, F.R. Science 1989, 243, 85. Wentland-SC-20a

SA • Bkoff

kon

+

Go = H

o- T So = - 2.303RT logKeq = - 18.3 kcal/mol

Every 10-fold increase in potency (K) - 1.36 kcal/mol

MW = 244.2

Binding stabilizes dipolar resonance contributors

Hydrophobic pocket formedby Trp-79, -92, -108

Binding interactions from 1STP.pdb:

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

• Biotinylated peptide substrate "immobilized" to streptavidin-coated 96-well ELISA microtiter plate(ELISA = Enzyme-Linked ImmunoSorbant Assay)

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

• Add a Tyrosine Kinase and ATP to each well, incubate, and wash

• Add anti-phosphotyrosine antibody, incubate, and wash

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

• Develop by adding HRP substrate reagent to each well

• OD (optical density) measured by ELISA auto-reader (absorbance at 415 nm)

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

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

anti-phosphotyrosineantibody

OPO3-2

Y

SA

biotinylated peptide

HRP

B

N

N

N

O

O

R

H

H

CH2

H

H

H

OH

O

R'

H N

N

N

NH2

N

O

OHOH

OPO

O-

O

PO

O-

O

P-O

O-

O

TK

O-PO

O-

O

N

N

N

O

O

R

H

H

CH2

H

H

H

O

R'

H

ATP

signal

+

Proteinsubstrate


covalent bonding of drug to enzyme

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