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.

SARS-CoV-2 3CL Protease and Bovine Trypsin

Protein Data Bank entries 1CE5 and 6LU7.

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

Direction Change

Warning!

Back to Irreversible Inhibition of Trypsin

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.

Pseudo First-Order Kinetics

Integrated rate expression:

ln

([E]

[E]0

)= −kappt,

[E]

[E]0= e−kappt

Standard plot:

200150100500

1.0

0.8

0.6

0.4

0.2

0

Time (min)

[E]

[E]0

Semi-logarithmic plot:

200150100500

1.0

Time (min)

[E]

[E]0 0.1