Ligand-Receptor Interfaces: Engineering of Important Interactions

1
Ligand-Receptor InterfacesEngineering of
Important Interactions

• Stefanie B. Bumpus
• CHEM 590A
• October 5, 2005

2
Small Molecule-Protein Interface Engineering

• Goal
• Alter enzyme substrate specificity
• Generate new ligand-receptor pairs
• Control of cellular processes
• Transcription
• Apoptosis
• Signal Transduction
• Many design strategies utilized
• Koh, JT., Engineering Selectivity and
  Discrimination into Ligand-Receptor Interfaces.
  Chemistry Biology, 917-23, 2002.

3
Overview

• Discussion of ligand-receptor interface
  engineering strategies
• Specific example of engineering the estrogen
  receptor
• Discussion Questions

4
Orthogonal Ligand-Receptor Pairs

• Orthogonal Ligand ligand able to bind altered
  receptor but not natural receptor
• Orthogonal Receptor receptor able to bind
  synthetic ligand but not natural ligand
• Functionally orthogonal pairs sufficient
• Receptor discrimination
• Ligands receptor selectivity
• Koh, JT., Engineering Selectivity and
  Discrimination into Ligand-Receptor Interfaces.
  Chemistry Biology, 917-23, 2002.

5
Defining Ligand-Receptor Interactions

• Rarely achieve absolute orthogonal interactions
• Important modified receptor be non-responsive to
  natural ligand at physiological concentrations
• Selectivity of engineered pair
• Ligands receptor selectivity (RS)
• Activity of Modified Pair/ Activity of Modified
  Ligand w/ wtR
• Receptor Discrimination Ability
• RS for natural ligand
• RS EC50 (ligand mutant)/ EC50 (ligand wtR)

Shi, Y Koh, JT. Functionally Orthogonal
Ligand-Receptor Pairs for the Selective
Regulation of Gene Expression Generated by
Manipulation of Charged Residues at the
Ligand-Receptor Interface of ERa and ERß.
Journal of the American Chemical Society,
1246921-6928, 2002.
6
Steric Complementation Bumps Holes

• Bumps ligand shape modified to be too large to
  fit in binding site
• Holes binding site modifications

• Successful method for generation of selective
  ligands
• Hole-modified receptor can retain affinity for
  natural ligand
• Dual Bump-Hole/Hole-Bump Modifications
• Complications reduction in catalytic affinity,
  maintenance of high affinity
• Koh, JT., Engineering Selectivity and
  Discrimination into Ligand-Receptor Interfaces.
  Chemistry Biology, 917-23, 2002.

7
Alterations of Polar Interactions

• Reversal of Hydrogen Bonding Patterns
• Donor/Acceptor Exchange
• Complications arise from residues involved in
  hydrogen bonding
• Manipulation of Charged Interactions
• Charged residues do not need direct interaction
  with ligand to have effect
• Koh, JT., Engineering Selectivity and
  Discrimination into Ligand-Receptor Interfaces.
  Chemistry Biology, 917-23, 2002.

8
Alterations of Polar Interactions

• Charge Reversal
• Production of orthogonal pairs possible, but see
  reduction in affinity
• Polar/Charged Group Exchange
• Only minor modifications from wild-type

• Introduction of New Ion Pairs
• Used to change substrate specificity
• Ion Pair Neutralization
• Greatly reduce affinity for natural substrate

Koh, JT., Engineering Selectivity and
Discrimination into Ligand-Receptor Interfaces.
Chemistry Biology, 917-23, 2002.
9
New Strategies in Engineering

• De Novo Binding Sites
• Engineering not restricted to existing interfaces
• Engineering Reactivity
• Many diseases associated with receptor mutations
• Design of mimics as custom pharmaceuticals

Koh, JT., Engineering Selectivity and
Discrimination into Ligand-Receptor Interfaces.
Chemistry Biology, 917-23, 2002.
10
Functionally Orthogonal PairsAn In-Depth Look

• Few examples of functionally orthogonal pairs
  able to exist completely independently of
  endogenous system
• Manipulation of Charged Residues of the estrogen
  receptor (ER)
• Rearrangement of electrostatic pairs
• Loss or gain of selectively associated with loss
  of charge
• Pairs sufficient for transcriptional regulation

Shi, Y Koh, JT. Functionally Orthogonal
Ligand-Receptor Pairs for the Selective
Regulation of Gene Expression Generated by
Manipulation of Charged Residues at the
Ligand-Receptor Interface of ERa and ERß.
Journal of the American Chemical Society,
1246921-6928, 2002.
11
The Estrogen Receptor

• Member of nuclear/steroid hormone receptor family
• Controls variety of cellular activities in
  response to estradiol-17ß (E2) and other natural
  estrogens (e.g. estrone)
• Two isoforms of receptor protein

A-Ring
Estradiol-17b
Brzozowski, A.M., et al. Nature 389753-758,
1997.
12
Basis for ER Engineering

• Mutation of a specific charged residue
• Glu353
• Involved in hydrogen bonding to A-ring
• Goal Reduced specificity for natural ligand
• Two different strategies for ligand design
• Neutral or Anionic E2 analogues

Shi, Y Koh, JT. Functionally Orthogonal
Ligand-Receptor Pairs for the Selective
Regulation of Gene Expression Generated by
Manipulation of Charged Residues at the
Ligand-Receptor Interface of ERa and ERß.
Journal of the American Chemical Society,
1246921-6928, 2002.
13
Carboxylate Funtionalized Ligands

• ES8 ligand may have RS to act as orthogonal
  ligand to both ER subtypes

Shi, Y Koh, JT. Functionally Orthogonal
Ligand-Receptor Pairs for the Selective
Regulation of Gene Expression Generated by
Manipulation of Charged Residues at the
Ligand-Receptor Interface of ERa and ERß.
Journal of the American Chemical Society,
1246921-6928, 2002.
14
Carboxylate Funtionalized Ligands

• Luciferase reporter gene assays
• ES8
• Large RS w/ ERa, agonist of ERß
• Proof of ES8 as functionally orthogonal ligand

Shi, Y Koh, JT. Functionally Orthogonal
Ligand-Receptor Pairs for the Selective
Regulation of Gene Expression Generated by
Manipulation of Charged Residues at the
Ligand-Receptor Interface of ERa and ERß.
Journal of the American Chemical Society,
1246921-6928, 2002.
15
Comparison of Structures of hERa hERb
DNA Binding Dimerization
Ligand Binding Transactivation
Hinge
Transactivation
1
180
262
302
552
595 aa
DNA Binding Dimerization
A/B
C
D
E
F
ERa
Ligand Binding Transactivation
Hinge
Transactivation
96
30
53
30
A/B
C
D
E
F
ERb
1
149
214
248
530 aa
Taken from Ogawa et al., BBRC 243122-126,1998
16
Transcriptionally Active Ligand/Receptor Pair

• Binding of LBDs of isoforms similar despite low
  homology
• Interaction of a partial agonist, genistein, in
  ERß similar to interactions of E2 in ERa
• Interactions of phenol rings with Glu and Arg in
  LBD
• Mutant ERß receptor engineering to test binding
  of ES8

Shi, Y Koh, JT. Functionally Orthogonal
Ligand-Receptor Pairs for the Selective
Regulation of Gene Expression Generated by
Manipulation of Charged Residues at the
Ligand-Receptor Interface of ERa and ERß.
Journal of the American Chemical Society,
1246921-6928, 2002.
17
Transcriptionally Active Ligand/Receptor Pair

• ERß mutant shows excellent discrimination against
  E2
• ES8 potent mutant agonist and large RS for
  modified receptor
• ES8 functionally orthogonal to both endogenous
  receptor subtypes

Shi, Y Koh, JT. Functionally Orthogonal
Ligand-Receptor Pairs for the Selective
Regulation of Gene Expression Generated by
Manipulation of Charged Residues at the
Ligand-Receptor Interface of ERa and ERß.
Journal of the American Chemical Society,
1246921-6928, 2002.
18
In vitro Ligand Binding Assays

• Binding ability of 3 modified ligands measured by
  radio-ligand displacement assays
• Differences from in vivo assays
• Important to note comparison between different
  receptors
• Association constants show only 9-fold preference
  of ERß mutant versus wt-ERa
• Enhanced binding selectivity of ES8 for ERa

Shi, Y Koh, JT. Functionally Orthogonal
Ligand-Receptor Pairs for the Selective
Regulation of Gene Expression Generated by
Manipulation of Charged Residues at the
Ligand-Receptor Interface of ERa and ERß.
Journal of the American Chemical Society,
1246921-6928, 2002.
19
Conclusions

• Need engineered pairs to have high potency and be
  orthogonal to endogenous ligand-receptor pairs
• Polar/charged interactions altered to yield pairs
  with high potency and high selectivity
• Future work in development of multiple strategies
  to complement receptor mutations

Koh, JT., Engineering Selectivity and
Discrimination into Ligand-Receptor Interfaces.
Chemistry Biology, 917-23, 2002.
Shi, Y Koh, JT. Functionally Orthogonal
Ligand-Receptor Pairs for the Selective
Regulation of Gene Expression Generated by
Manipulation of Charged Residues at the
Ligand-Receptor Interface of ERa and ERß.
Journal of the American Chemical Society,
1246921-6928, 2002.
20
References

• Koh, JT., Engineering Selectivity and
  Discrimination into Ligand-Receptor Interfaces.
  Chemistry Biology, 917-23, 2002.
• Shi, Y Koh, JT. Functionally Orthogonal
  Ligand-Receptor Pairs for the Selective
  Regulation of Gene Expression Generated by
  Manipulation of Charged Residues at the
  Ligand-Receptor Interface of ERa and ERß.
  Journal of the American Chemical Society,
  1246921-6928, 2002.
• Shi, Y and Koh, JT. Selective regulation of
  gene expression by an orthogonal estrogen
  receptor-ligand pair created by polar-group
  exchange. Chemistry Biology, 8501-510, 2001.

21
Discussion Questions

• Why is it important to examine the interactions
  of ligand-receptor by both in vitro in vivo
  assays, not in vitro binding assays alone?
• Mentioned previously was the applicability of
  this procedure to enzyme-substrate pairs. What
  would be the advantage engineering such systems?