Substrate Identification and Determination of Binding Kinetics

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Substrate Identification and Determination of
Binding Kinetics
AB Complex
E S
ES
Molecule A Molecule B
In the real world or massive complexity of living
organisms both the E or the S can be proteins,
DNA, RNA, carbohydrates, lipids, any number of
chemical cofactors and metabolites OR entire
cells binding to one another through a series of
interactions at cell surface receptors that are
comprised of all of the above.
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Identification of Substrates and Molecular
Complexes
How do I identify a physiologically relevant
molecular complex?
To answer this we must first begin with a
significant cellular process or metabolic
function that is of particular interest (i.e. one
that has an important role in human health or
disease). Please note that the human health is
impacted by many things for example- - Plant and
animal health in food sources (USDA) - Energy
and other environmental pollution sources (DOE,
EPA) - Technological advances in basic chemistry
and physics (NSF)
Once we know what system or function we are
interested in we can think about the second
problem of detecting and isolating important
molecular complexes. What are the important
tools of the 21st century. 1) Massive libraries
of genomic sequence information - Genomic,
proteomic, and bioinformatics tools 2)
Microarray analysis - Determine how the genome
responds to disease, or when your metabolic
pathway active. 3) Cellular imaging through
fluorescent markers that tell you when key
players are expressed and where they are going.
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General Tools for Identifying Molecular
Interactions
1- All types of chromatography. 1- Paper or
thin layer chromatography (TLC) 2- HPLC that
may or may not be followed by GC-MS analysis 3-
Affinity chromatography, Molecule A is tethered
to a bead and you go fishing for molecule B.
Similar approaches can be employed for
isolation of DNA or RNA protein complexes. 2-
If you have antibodies for molecule A then immuno
precipitation can be used to pull down or
precipitate molecule B. 3- Combination of
chromatography and electrophoresis methods that
are also coupled to MS techniques. 4- Good old
fashion biochemistry. Application of inhibitors
and or mutagenesis to catch molecular
complexes at intermediate steps. 5- Searching
for inhibitors (or lead compounds) using phage
display.
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In Living Organisms, Metabolites
Traditionally Identified by Labeling and Chemical
Analysis
Metabolite identification
Protein Identification
Cell growth in unlabeled media Induction or
stress of system and introduction of 35S for
variable defined lengths of time. Removal of
inducer or stress and growth on unlabeled
media. Cell lysis and fractionation followed by
2D gel electrophoresis and MS.
Cell growth in unlabeled media Addition of
labeled metabolite (e.x. 13C glucose) for
variable defined lengths of time (The
Pulse). Addition of excess unlabeled metabolite
(The Chase) Cell lysis and fractionation
followed by chemical and spectroscopic analysis
(e.x. NMR)
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Identifying Substrates Using Combinatorial
Chemistry
Fluorophore
Quencher
Resin or PEG Bead
Peptide or polysaccharide
The strength of the technology is in the range of
libraries that are available for screening.
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Using Combinatorial Chemistry To
Identify Inhibitors of Matrix Metalloproteases
(MMPs)
MMPs belong to a family of structurally related
zinc containing endoproteinases. Their primary
function is degradation of a variety of
extracellular matrix components. They are known
to participate in various pathological conditions
such as arthritis, cancer and osteoporosis, hence
inhibition of MMPs may be very important
in clinical treatment The design of many MMP
inhibitors has focused on finding a zinc binding
motif, which can chelate the active-site
zinc(II)ion effectively, a backbone which can
provide hydrogen bond interactions with
the enzyme, and one or more side chains which can
have effective van der Waals interaction with
MMP subsites.
One such example PDB ID 1SMP Baumann et al. JMB
1995 248 653-661
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Identifying Inhibitors Using Combinatorial
Chemistry
Fluorophore
Quencher
Resin or PEG Bead
Substrate
Inhibitor
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Building a Library
The general structure for the library was
H-XX-azole-XX-N2
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Building a Library
Made a library of 240,000 and got 184 potential
hits or dark beads.
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Identifying Inhibitors Using Phage Display
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Table 1. Clinical Status of Biopharmaceuticals
Derived from Phage Display Target Product Year
Approved/Trial Status Indication(s) Developer(s)
TNFa Humira(adalimumab) 2003 III III III III II
I Rheumatoid arthritis Juvenile rheumatoid
arthritis Crohns disease Ankylosing
spondylitis Arthritis, psoriatic Psoriasis Cambrid
ge Antibody Technology Group plc, Abbott
Laboratories TGFß2 Trabio(lerdelimumab) III Sca
rring following glaucomasurgery Cambridge
Antibody Technology Group plc Neutrophil
Elastase DX-890 II Cystic fibrosis Dyax,
Debiopharm EpCAM MT-201 II Prostate
cancer Micromet AG, Novuspharma Eotaxin1 Bertilim
umab II II Allergic rhinitis Conjunctivitis Cambri
dge Antibody Technology Group plc BLyS LymphoStat
-B(belimumab) II Systemic lupus
erythematosus Cambridge Antibody Technology Group
plc TGFß1 Metelimumab II Diffuse systemic
sclerosis Cambridge Antibody Technology Group
plc, Genzyme Corporation IL-12 ABT-874 II II Rheu
matoid arthritis Crohns disease Cambridge
Antibody Technology Group plc, Abbott
Laboratories, Wyeth Kallikrein DX-88 II I/II Here
ditary angioedema Blood loss Dyax,
Genzyme Dyax Birch pollen allergen RBet-v-1
derivatives I/II Birch pollen allergy University
of Vienna, Strasbourg Hospital VEGFR2 IMC-1C11 I
Colorectal carcinoma ImClone Systems TRAIL-R1 TRA
IL-R1 MAb I Cancer (advanced tumors) Human Genome
Sciences TRAIL-R2 TRAIL-R2 MAb I Cancer
(advanced tumors) Human Genome Sciences Bacillus
anthracis ABthrax I B. anthracisinfection Human
Genome Sciences CD-22 BL-22 I Hairy cell
leukemia, chronic lymphocytic leukemia National
Cancer Institute, Enzon TNFa, tumor necrosis
factor alpha TGFß, transforming growth factor
beta BlyS, B lymphocyte stimulator VEGFR2,
vascular endothelial growth factor receptor
2. Debiopharm, Lausanne, Switzerland
Novuspharma, Bresso (MI), Italy ImClone Systems,
New York, NY Enzon, Bridgewater, NJ. Source
Adis RD Insight, Adis International IMS RD
Focus Drug Updates, IMS Health Pharmaprojects,
PJB Publications Ltd. IDdb3, Current Drugs
Ltd.
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Lysozyme Epitopes Identified by Phage Display
PDB Entrys 1VFB C chain 3HFL Y chain 3HFM
Y chain All have residues 1-129
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Kinetic Characterization of Substrate Binding
The basics

• How tight is the binding?
• How many binding sites?
• Is there any cooperation between binding sites?

You need to know the basics, do the kinetic
constants make physiological sense.
Alternatively, for an inhibitor or drug, will an
effective dose be achievable?
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Old Fashion (Low Budget) Binding Experiments
What are the requirements? - Ability to get a
significant amount of protein and substrate,
typical several mg. - The ability to detect and
accurately measure both the protein and the
substrate in solution. - A gel filtration
column that is compatible with the protein and
substrate as well as fraction collector What is
the protocol? - Equilibrate column with low
concentration of substrate/inhibitor and pass the
protein over the column in that buffer. -
Collect fraction prior to protein elution AND
fraction with protein. - Measure protein and
substrate in both fractions and compare to
determine the amount bound by the protein.
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Old Fashion (Low Budget) Binding Experiments
We all remember from 8010 that a dissociation
constant is
ES
E S
ES
KD
ES
In general, binding can be represented by
Michaelis Menton kinetics where V is replaced
with ES
or
n
n
Where n is referred to a cooperativity coefficient
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Isothermal Titration Calorimetry (ITC)
Most biochemical reactions, including binding,
involve a small change in heat. This is what ITC
measures.
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Isothermal Titration Calorimetry (ITC)
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Isothermal Titration Calorimetry (ITC)
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Isothermal Titration Calorimetry (ITC)
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Isothermal Titration Calorimetry (ITC)
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Isothermal Titration Calorimetry (ITC)
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Isothermal Titration Calorimetry (ITC)
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What if the Heat Change is Small?
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Surface Plasmon Resonance
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