Structure and Pharmacophore Elucidation of the HRV143C Protease Trent Bjorndahl Faculty of Pharmacy

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Structure and Pharmacophore Elucidation of the
HRV14-3C ProteaseTrent
BjorndahlFaculty of Pharmacy and Pharmaceutical
SciencesOct. 9, 2003
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Background

• Rhinovirus
• More 110 known serotypes
• polyvalent vaccine vs. Small molecule inhibitors
• 3C Protease

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Picornovirii Life Cycle
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Picornovirii Gene Products

• 3C Protease
• Lytic Activity
• RNA Binding
• Down Regulation
• NOT HAV

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HRV14-3C Specificity
1Rates of peptide hydrolysis are expressed
relative to 2C-3A and are the mean of two or more
independent determinations.

• Cordingley, M. G. et al., Cleavage of Small
  Peptides In Vitro by Human Rhinovirus 14 3C
  Protease Expressed in Escherichia coli. J.
  Virol., 1989, 63 5037-5045.

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3C Protease Backbone Dynamics
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Project

• Determine solution structures of free and
  peptidyl-inhibited HRV14-3C
• Model other enzyme-substrate complexes
• Analyze HRV14-3C pharmacophore

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Expression, Purification and Sample Conditions
OD600 0.8 30 Celsius 0.2 IPTG 12 - 14 hours
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Expression, Purification and Sample Conditions

• Q Sepharose
• Tris-HCl pH 8.8 - 9.2
• 2.5 - 5 ml/min.
• 4 Celsius

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Expression, Purification and Sample Conditions
Button test Ultracentrifugation monomer/dimer
24kDa 1mM HRV14-3C, 20mM KH2PO4, pH 6.5, 15mM
DTT, 0.5mM EDTA
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Synthesis of a peptidyl inhibitor
Modified 2C/3A cleavage site
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Inhibitor analysis
ES-MS MALDI-TOF within 5amu
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Inhibitor analysis
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NMR experiments
Backbone experiments - sequential
assignment HNCA HNCACB CBCACONH HNHA NOESY-HSQC
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NMR experiments
V47 strips ? and amide protons A. 13C
NOESY-HSQC B. 13C HCCH-TOCSY C. 15N NOESY-HSQC D.
15N TOCSY-HSQC
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Structure Determination
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Preliminary analysis
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Preliminary Analysis
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Future work

• Complete the two structures
• uninhibited 1700 NOES 1.8A
• D2O NOESY TOCSY data
• N Q side chains
• inhibited backbone assignments
• Pharmacophore analysis

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Thank You

• Inhibitor synthesis
• Lena Andrew
• Dr. Raj Jain
• Carlos Velazquez
• Praveen Rao
• NMR
• Hassan Monzavi
• Dr. Ryan McKay
• Dr. Pascal Mercier