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Substrate Identification and Determination of Binding Kinetics PowerPoint PPT Presentation


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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

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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 is 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

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, TLC, LC-MS)

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.


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Chromogenic and Flourogenic Synthetic Substrates for Culture Media


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Multi-test Identifications

with commercial

significance.

Rapid detection/identification of pathogenic

organisms in the food industry is of great

importance. This applies to ANY additive that

will be ingested. The same technology and

chemical/biochemical approaches can

be applied elsewhere (i.e. detection of cell

types and surface receptors).

See Journal of Microbiological Methods 79 (2009) 139–155


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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 poly peptide and polysacchatide

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”.

Similar to phage display technology, the beads can be washed and

sequences that “stick” can be identified or amplified.


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Identifying Inhibitors Using Phage Display


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  • Table 1. Clinical Status of Biopharmaceuticals Derived from Phage Display

  • TargetProductYear Approved/Trial StatusIndication(s)Developer(s)

    • TNFαHumira™(adalimumab)2003

    • III

    • III

    • III

    • III

    • IIIRheumatoid arthritis

    • Juvenile rheumatoid arthritis

    • Crohn’s disease

    • Ankylosing spondylitis

    • Arthritis, psoriatic

  • PsoriasisCambridge Antibody Technology Group plc, Abbott Laboratories

  • TGFβ2Trabio™(lerdelimumab)IIIScarring following glaucomasurgeryCambridge Antibody Technology Group plc

  • Neutrophil ElastaseDX-890IICystic fibrosisDyax, Debiopharm

  • EpCAMMT-201IIProstate cancerMicromet AG, Novuspharma

    • Eotaxin1BertilimumabII

    • IIAllergic rhinitis

  • ConjunctivitisCambridge Antibody Technology Group plc

  • BLySLymphoStat-B™(belimumab)IISystemic lupus erythematosusCambridge Antibody Technology Group plc

  • TGFβ1MetelimumabIIDiffuse systemic sclerosisCambridge Antibody Technology Group plc, Genzyme Corporation

    • IL-12ABT-874II

    • IIRheumatoid arthritis

  • Crohn’s diseaseCambridge Antibody Technology Group plc, Abbott Laboratories, Wyeth

    • KallikreinDX-88II

    • I/IIHereditary angioedema

    • Blood lossDyax, Genzyme

  • Dyax

  • Birch pollen allergenRBet-v-1 derivativesI/IIBirch pollen allergyUniversity of Vienna, Strasbourg Hospital

  • VEGFR2IMC-1C11IColorectal carcinomaImClone Systems

  • TRAIL-R1TRAIL-R1 MAbICancer (advanced tumors)Human Genome Sciences

  • TRAIL-R2 TRAIL-R2 MAbICancer (advanced tumors)Human Genome Sciences

  • Bacillus anthracisABthrax™IB. anthracisinfectionHuman Genome Sciences

  • CD-22BL-22IHairy cell leukemia, chronic lymphocytic leukemiaNational Cancer Institute, Enzon

    • TNFα, tumor necrosis factor alpha; TGFβ, 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 R&D Insight, Adis International; IMS R&D 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, typically 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 our first Biochemistry class that a dissociation constant is;

    [E][S]

    [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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    PDB 1A30


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    - Moreover, think about the physiological environment, is typical substrate binding driven by

    entropic contributions?


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    Surface Plasmon Resonance


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