Finding Ligand Binding Sites on a Proteome-wide Scale and its Implications

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Finding Ligand Binding Sites on a Proteome-wide
Scale and its Implications

• Philip E. Bourne
• University of California San Diego
• pbourne_at_ucsd.edu
• http//www.sdsc.edu/pb/Talks/

HUPO San Diego Feb. 2009
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Motivation

• The truth is we know very little about how the
  major drugs we take work
• We know even less about what side effects they
  might have
• Drug discovery seems to be approached in a very
  consistent and conventional way
• The cost of bringing a drug to market is huge
  800M
• The cost of failure is even higher e.g. Vioxx -
  4.85Bn

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Motivation

• The truth is we know very little about how the
  major drugs we take work receptors are unknown
• We know even less about what side effects they
  might have - receptors are unknown
• Drug discovery seems to be approached in a very
  consistent and conventional way
• The cost of bringing a drug to market is huge
  800M drug reuse is a big business
• The cost of failure is even higher e.g. Vioxx -
  4.85Bn - fail early and cheaply

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What if

• We can characterize a protein-ligand binding site
  from a 3D structure (primary site) and search for
  that site on a proteome wide scale?
• We could perhaps find alternative binding sites
  (off-targets) for existing pharmaceuticals and
  NCEs?
• We could use it for lead optimization and
  possible ADME/Tox prediction

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What Do Off-targets Tell Us?

• One of three things
• Nothing
• A possible explanation for a side-effect of a
  drug
• A possible repositioning of a drug to treat a
  completely different condition
• Today I will give you examples of both 2 and 3
  and illustrate the complexity of the problem

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Agenda

• Computational Methodology
• Side Effects - The Tamoxifen Story
• Repositioning an Existing Drug - The TB Story
• Salvaging 800M The Torcetrapib Story

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Need to Start with a 3D Drug-Receptor Complex -
The PDB Contains Many Examples
Generic Name Other Name Treatment PDBid
Lipitor Atorvastatin High cholesterol 1HWK, 1HW8
Testosterone Testosterone Osteoporosis 1AFS, 1I9J ..
Taxol Paclitaxel Cancer 1JFF, 2HXF, 2HXH
Viagra Sildenafil citrate ED, pulmonary arterial hypertension 1TBF, 1UDT, 1XOS..
Digoxin Lanoxin Congestive heart failure 1IGJ
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A Reverse Engineering Approach to Drug Discovery
Across Gene Families
Characterize ligand binding site of primary
target (Geometric Potential)
Identify off-targets by ligand binding site
similarity (Sequence order independent
profile-profile alignment)
Extract known drugs or inhibitors of the
primary and/or off-targets
Search for similar small molecules

Dock molecules to both primary and off-targets
Statistics analysis of docking score
correlations
Computational Methodology
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Characterization of the Ligand Binding Site -
The Geometric Potential

• Conceptually similar to hydrophobicity
• or electrostatic potential that is
• dependant on both global and local
• environments

• Initially assign Ca atom with a value that is the
  distance to the environmental boundary
• Update the value with those of surrounding Ca
  atoms dependent on distances and orientation
  atoms within a 10A radius define i

Xie and Bourne 2007 BMC Bioinformatics, 8(Suppl
4)S9
Computational Methodology
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Discrimination Power of the Geometric Potential

• Geometric potential can distinguish binding and
  non-binding sites

100
0
Geometric Potential Scale
Computational Methodology
Xie and Bourne 2007 BMC Bioinformatics, 8(Suppl
4)S9
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Local Sequence-order Independent Alignment with
Maximum-Weight Sub-Graph Algorithm
Structure A
Structure B
L E R
V K D L
L E R
V K D L

• Build an associated graph from the graph
  representations of two structures being compared.
  Each of the nodes is assigned with a weight from
  the similarity matrix
• The maximum-weight clique corresponds to the
  optimum alignment of the two structures

Xie and Bourne 2008 PNAS, 105(14) 5441
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Nothing in Biology including Drug Discovery
Makes Sense Except in the Light of
Evolution                                     
Theodosius Dobzhansky (1900-1975)
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Similarity Matrix of Alignment

• Chemical Similarity
• Amino acid grouping (LVIMC), (AGSTP), (FYW), and
  (EDNQKRH)
• Amino acid chemical similarity matrix
• Evolutionary Correlation
• Amino acid substitution matrix such as BLOSUM45
• Similarity score between two sequence profiles

fa, fb are the 20 amino acid target frequencies
of profile a and b, respectively Sa, Sb are the
PSSM of profile a and b, respectively
Computational Methodology
Xie and Bourne 2008 PNAS, 105(14) 5441
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Agenda

• Computational Methodology
• Repositioning an Existing Drug - The TB Story
• Side Effects - The Tamoxifen Story
• Salvaging 800M The Torcetrapib Story

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Found..

• Evolutionary linkage between
• NAD-binding Rossmann fold
• S-adenosylmethionine (SAM)-binding domain of
  SAM-dependent methyltransferases
• Catechol-O-methyl transferase (COMT) is
  SAM-dependent methyltransferase
• Entacapone and tolcapone are used as COMT
  inhibitors in Parkinsons disease treatment
• Hypothesis
• Further investigation of NAD-binding proteins may
  uncover a potential new drug target for
  entacapone and tolcapone

Repositioning an Existing Drug - The TB Story
Repositioning an Existing Drug - The TB Story
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Functional Site Similarity between COMT and ENR

• Entacapone and tolcapone docked onto 215
  NAD-binding proteins from different species
• M.tuberculosis Enoyl-acyl carrier protein
  reductase ENR (InhA) discovered as potential new
  drug target
• ENR is the primary target of many existing
  anti-TB drugs but all are very toxic
• ENR catalyses the final, rate-determining step in
  the fatty acid elongation cycle
• Alignment of the COMT and ENR binding sites
  revealed similarities ...

Repositioning an Existing Drug - The TB Story
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Summary of the TB Story

• Entacapone and tolcapone shown to have potential
  for repositioning
• Direct mechanism of action avoids M. tuberculosis
  resistance mechanisms
• Possess excellent safety profiles with few side
  effects already on the market
• In vivo support
• Assay of direct binding of entacapone and
  tolcapone to ENR reveals promising leads with no
  chemical relationship to existing drugs

Repositioning an Existing Drug - The TB Story
PLoS Comp Biol Under Review
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Agenda

• Computational Methodology
• Repositioning an Existing Drug - The TB Story
• Side Effects - The Tamoxifen Story
• Salvaging 800M The Torcetrapib Story

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Selective Estrogen Receptor Modulators (SERM)

• One of the largest classes of drugs
• Breast cancer, osteoporosis, birth control etc.
• Amine and benzine moiety

Side Effects - The Tamoxifen Story
PLoS Comp. Biol., 2007 3(11) e217
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Adverse Effects of SERMs
cardiac abnormalities
loss of calcium homeostatis
thromboembolic disorders
?????
ocular toxicities
Side Effects - The Tamoxifen Story
PLoS Comp. Biol., 3(11) e217
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Structure and Function of SERCASacroplasmic
Reticulum (SR) Ca2 ion channel ATPase

• Regulating cytosolic calcium levels in cardiac
  and skeletal muscle
• Cytosolic and transmembrane domains
• Predicted SERM binding site locates in the TM,
  inhibiting Ca2 uptake

Side Effects - The Tamoxifen Story
PLoS Comp. Biol., 3(11) e217
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The Challenge

• Design modified SERMs that bind as strongly to
  estrogen receptors but do not have strong binding
  to SERCA, yet maintain other characteristics of
  the activity profile

Side Effects - The Tamoxifen Story
PLoS Comp. Biol., 3(11) e217
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Agenda

• Computational Methodology
• Repositioning an Existing Drug - The TB Story
• Side Effects - The Tamoxifen Story
• Salvaging 800M The Torcetrapib Story

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The Torcetrapib Story
PLoS Comp Biol Under Minor Revision
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Cholesteryl Ester Transfer Protein (CETP)
CETP inhibitor
X
CETP
HDL
LDL
Bad Cholesterol
Good Cholesterol

• collects triglycerides from very low density or
  low density lipoproteins (VLDL or LDL) and
  exchanges them for cholesteryl esters from high
  density lipoproteins (and vice versa)
• A long tunnel with two major binding sites.
  Docking studies suggest that it possible that
  torcetrapib binds to both of them.
• The torcetrapib binding site is unknown. Docking
  studies show that both sites can bind to
  torcetrapib with the docking score around -8.0.

The Torcetrapib Story
PLoS Comp Biol Under Minor Revision
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Torcetrapib
Anacetrapib
JTT705
JTT705
VDR

RXR
FA

RAS
FABP
?
PPARa
PPARd
?
?
PPAR?
High blood pressure

JNK/IKK pathway JNK/NF-KB pathway
Anti-inflammatory function
Immune response to infection
PLoS Comp Biol Under Minor Revision
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Summary

• We have established a protocol to look for
  off-targets for existing therapeutics and NCEs
• Understanding these in the context of pathways
  would seem to be the next step towards a new
  understanding
• Lots of other opportunities to examine existing
  drugs

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Bioinformatics Final Examples..

• Donepezil for treating Alzheimers shows positive
  effects against other neurological disorders
• Orlistat used to treat obesity has proven
  effective against certain cancer types
• Ritonavir used to treat AIDS effective against TB
• Nelfinavir used to treat AIDS effective against
  different types of cancers

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Acknowledgements
Lei Xie
Li Xie
Jian Wang
Sarah Kinnings