lecture 18: coronaviruses and their proteases and kinetics ......covid-19 in a bit of context...
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Biological Chemistry LaboratoryBiology 3515/Chemistry 3515
Spring 2020
Lecture 18:
Coronaviruses and Their Proteases
and
Kinetics of Irreversible Inhibition
5 March 2020c©David P. Goldenberg
University of Utahgoldenberg@biology.utah.edu
How do we know? What do we do with it?
Information
Observations
Experiments
Data
Knowledge
Organized information
Theories
Predictions
Wisdom ?
Applications
Coronaviruses
Name comes from corona of spike proteinssurrounding the surface.
Positive-strand RNA virus
Very common among mammals and birds,typically causing mild respiratory illness.
Two previous major outbreaks with severesymptoms.• SARS (severe acute respiratory syndrome),
2002-2004
• MERS Middle East respiratory syndrome,2012-2016
Current outbreak:• Disease: COVID-19
• Virus: SARS-CoV-2
Images from https://en.wikipedia.org/wiki/Coronavirus and Centers for Disease Control.
COVID-19 in a Bit of Context
COVID-192002-2004
SARS2012–2016
MERS
1918Pandemicinfluenza
Seasonalinfluenza
R0 2.2 3 1.9–3.9 1.4–2.8 0.9–2.1
Total cases 95,124 8,906 2,494 500 million 7,780,000
Deaths 3,254 744 858 50 million 389,000
Fatality rate (%) 2–3 8 34 10 0.05
R0 Transmission ratio, the number of new cases that can develop from 1 confirmed case.
Table adapted from: Yee, J., Unger, L., Zadravecz, F., Cariello, P., Seibert, A., Johnson, M. A. & Fuller, M. J.(2020). Novel coronavirus 2019 (COVID-19): Emergence and implications for emergency care. JACEP Open,2020, 1–7. https://doi.org/10.1002/emp2.12034
COVID-19 data updated on 4 March 2020, from:https://www.arcgis.com/apps/opsdashboard/index.html#/bda7594740fd40299423467b48e9ecf6
The Coronavirus Lifecycle
Illustration from: Morse, J. S., Lalonde, T., Xu, S. & Liu, W. R. (2020). Learning from the past: Possible urgentprevention and treatment options for severe acute respiratory infections caused by 2019-nCoV. ChemBioChem,21. https://doi.org/10.1002/cbic.202000047
Possible Strategies for Preventing and Treating Coronavirus Infections
Prevention
• Public health measures to reduce transmission.
• Personal health measures to reduce transmission.
• Vaccines: Likely to take about a year to develop and distribute.
Treatment
• Antibodies to spike protein, natural or engineered.
• Other molecules that interfere with spike-protein binding to receptors.
• Inhibitors of RNA-dependent RNA polymerase.
• Inhibitors to proteases: 3C-like protease (3CLpro) and papain-like protease (PLpro)
The RNA-dependent RNA Polymerase
Kirchdoerfer, R. N. & Ward, A. B. (2019). Structure of the SARS-CoV nsp12 polymerase bound to nsp7 and nsp8co-factors. Nature Comm., 10, 2342. https://doi.org/10.1038/s41467-019-10280-3
Remdesivir: A Promising Inhibitor of Coronavirus RNA Polymerases
Remdesivir
GS-441524-triphosphate
Developed by Gilead Sciences as atreatment for Ebola and Marburg viruses.
Emergency use for Ebola demonstratedhuman safety, but low efficacy.
Effective against other RNA viruses,including SARS and MERS.
GS-4415244 inhibits feline coronavirus.
Blocks extension of RNA after beingincorporated.
Currently being tested for COVID-19, on alimited scale.
Gordon, C. J., Tchesnokov, E. P., Feng, J. Y., Porter, D. P. & Gotte, M. (2020). The antiviral compound remdesivirpotently inhibits RNA-dependent RNA polymerase from Middle East respiratory syndrome coronavirus. J. Biol.Chem... http://doi.org/10.1074/jbc.AC120.013056
Crystal Structure of SARS-CoV-2 3CL Protease
Protein Data Bank entry 6LU7, deposited 26 January 2020. X. Liu, B. Zhang, Z. Jin, H. Yang and Z. Rao.
An Inhibitor for Coronavirus 3CL Protease based on Michael Addition
Yang, H., Xie, W., Xue, X., Yang, K., Ma, J., Liang, W.,Zhao, Q., Zhou, Z., Pei, D., Ziebuhr, J., Hilgenfeld, R.,Yuen, K. Y., Wong, L., Gao, G., Chen, S., Chen, Z., Ma,D., Bartlam, M. & Rao, Z. (2005).Design of wide-spectrum inhibitors targetingcoronavirus main proteases. PLoS Biol., 3, e428.https://doi.org/10.1371/journal.pbio.0030324
SARS-CoV-2 3CL and Inhibitor N3
Protein Data Bank entry 6LU7, deposited 26 January 2020. X. Liu, B. Zhang, Z. Jin, H. Yang and Z. Rao.
SARS-CoV-2 3CL and Inhibitor N3
Protein Data Bank entry 6LU7, deposited 26 January 2020. X. Liu, B. Zhang, Z. Jin, H. Yang and Z. Rao.
HIV Protease with Ritonavir:
Currently in Limited Clinical Trials for COVID-19
Protein Data Bank entry 1RL8, Rezacova, P., Brynda, J., Sedlacek, J., Konvalinka, J., Fabry, M. and Horejsi, M.
More Information about COVID-19
At the U:https://dps.utah.edu/coronavirus/
World Health Organization:https://www.who.int/emergencies/diseases/novel-coronavirus-2019
Updated statistics from Johns Hopkins University:https://www.arcgis.com/apps/opsdashboard/index.html#
/bda7594740fd40299423467b48e9ecf6
Protein Data Bank:http:
//www.rcsb.org/news?year=2020&article=5e3c4bcba5007a04a313edcc
Irreversible Inhibition of Trypsin by AEBSF
4-(2-aminoethyl)-benzenesulfonyl fluoride
+
-Ser- AEBSF
Reaction is specific for the catalytic Ser residue.
Reaction is irreversible.
Experimental Protocol for Studying Irreversible Inhibition
Follow the reaction by measuring enzymatic activity at increasing times aftermixing enzyme and inhibitor.
E I
timet = 0
+Substrate
time
A
Assay
For each sample withdrawn, measure reaction velocity.
V ∝ concentration of uninhibited enzyme.
Time for assay must be short relative to time of inactivation.
Clicker Question #1
How does the concentration of active enzyme change with time?
0
1
0
A
[E]
[E0]= 1 − kt
0
1
0
B
[E]
[E0]=
1
kt
0
1
0
C
[E]
[E0]= e−kt
All answers count for now.
Kinetics of Irreversible Inactivation
E + I→ E-I
Second-order kinetics:
d [E]
dt=
d [I]
dt= −k2[I][E]
If initial concentrations of enzyme andinhibitor are equal:
Time
[E]
This is not an exponential decay function!
Both [I] and [E] decrease with time, and bothdecreases contribute to reduced rate withtime.
Pseudo First-Order Kinetics
If [I]� [E], [I] will remain approximately constant during the reaction.
d [E]
dt= − k2[I]︸︷︷︸
constant
[E]
Define a pseudo first-order rate constant: kapp = k2[I]
d [E]
dt= −kapp[E]
Rearrange and integrate the rate expression:∫ [E][E]0
d [E]
[E]=
∫ tt=0−kappdt
[E]0 = Initial enzyme concentration.
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