Transcript Problem Set 2 - University of Ottawa

Problem Set 2
Advanced Chemical Biology
BPS 4129
April 9, 2010
Problem 1
A) State in your own words why the particular types of small
molecule libraries were used given that the mutant enzyme
involves the elimination of one of the amino acid side chains
that binds zinc. (/5)
• Must rescue the altered enzyme by mimicking altered
structural motifs
• Bulky hydrophobic group to replace Phe
• Aromatic nitrogen containing compounds to replace His for
Zn(II) binding
• Chose small molecules containing these two motifs
• Qunazolines, isoquinolines, benzimidazole etc.
Problem 1 continued
B) Name an experiment that could be used to independently
confirm if small molecule “hits” actually recover the zinc
finger’s ability to bind zinc. (/3)
• Reporter assay (Gal, Luc, GFP)
C) In point form, outline how it was determined the transcription
factor function is recovered in the presence of small molecule.
(/2)
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Reporter assay (GFP)
GFP reporter downstream of Zn finger recognition sequence
Zn finger mutant genes also in vector
Compared fluorescence with and without small molecule
Problem 2
A) Describe the bump-and-hole approach for studying kinase
function. (/5)
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Scheme
>500 kinases similar in structure/function
Mutate kinase of interest by introducing a “hole”
Mutated kinase must retain its function
Mutate gatekeeper residue to a smaller amino acid
Screen natural substrate analogs with “bumps” for match
Inhibitor should affect only the mutated kinase
Problem 2 continued
B) What metabolite binding site is modified? (/2)
ATP
C) What is the gatekeeper residue and why is it important? (/3)
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Ile 338
Regulates access of ATP to kinase
Can be modified without disrupting affinity for ATP
Usually mutated to Gly or Ala
Can be targeted in a kinase of interest through bump-&-hole
Problem 3
A) Propose a chemical mechanism for the following
transformation: (/3)
• Similar to derivatization of carboxylic acids with diazomethane
Methods Enzymol. 1988, 172, 288-301
Angew. Chem. Int. Ed. 2005, 44, 1328-1332
Nature Genet. 2001, 28, 317-325
Problem 3 continued
B) Describe how the modified D/RNA in “A” serves as a
photochemical switch (/2)
• Association with sugar phosphate backbone disrupts structure
• Sterics
• Perturbed electrostatic interactions
• Photolabile group (as seen before in class) can switch
transcription/translation on/off
C) What is the chemical basis for photoswitching using
azobenzene chromophores? (/2)
• UV light induces isomerization to cis isomer
• Thermal relaxation to trans isomer
Problem 3 continued
D) An example where azobenzene is used as a chemical switch.
(/3)
• Examples from class/literature
Problem 4
A) What does FRET stand for? (/2)
Fluorescence/Förster Resonance Energy Transfer
B) Describe in your own words how FRET works. (/4)
• Two chromophores in close proximity and properly oriented
• Excitation of one by light of specific wavelength
• Nonradiative transfer of energy from one to other followed by
emission of lower energy light
• Emission spectrum of first must overlap with absorption
spectrum of second for FRET
Problem 4 continued
C) Give an example of the use of FRET in chemical biology. (/4)
• I.e. Calmodulin experiment from lecture
Problem 5
A) Give an example of the use of a molecular imaging technique
in high throughput screening. (/5)
Describe a technique from class and how it’s been applied to
high throughput screens i.e. AFM,
B) Describe the advantages of the method over others. (/5)
From notes
Problem 6
A) Describe chemical force microscopy and the chemistry used
to functionalize the tip. (/5)
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Chemical interactions between surface and functionalized tip can be measured
Hydrogen bond strength, electrostatic interactions, pH
Force required on cantilever to disrupt interactions between surface and tip are
quantitated
Coat with reactive silanes
Coat with thin layer of gold, then organic thiols
Further functionalization with organic molecules
B) Discuss the use of AFM tips to perform high throughput
screening. What are the drawback/limitations of this
technique? (/5)
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Discuss DPN, DNA/DNA interactions, Protein/DNA interactions, ligand/receptor
interactions
Drawbacks: tip functionalization, resolution, surface imaging only, sample height,
scanning area limitations, slow scan rate
Problem 7
A) Identify all the functional features of the MRI probe below.
(/3)
• cNGR
• Biotin
• Diethylenetriaminepent
aacetic acid (DTPA)
Problem 7 continued
B) Describe the function of each element of the probe. (/3)
• cNGR - Asparagine, gylcine, arginine cyclic peptide
specific for CD13 aminopeptidase, overexpressed in
angiogenic endothelial cells. Site specificity
• Biotin – Affinity for avidin, tetramer provides contrast
• Diethylenetriaminepentaacetic acid (DTPA) – Chelates
gadolinium which acts as a contrast agent for MRI
C) Describe an alternative approach to add multivalency to a
molecular probe. (/4)
Superparamagnetic iron oxide
Nanotubes (gadolinium based)
+ brief description of approach