Targeting Thymidylate Kinase in Plasmodium falciparum (PfTMK)Riley Xu and Farah Abdelaziz

Thymidylate Kinase Thymidylate kinase catalyzes the

phosphotransfer from ATP to TMP

40% sequence identity to human TMK

Plasmodium

Human

Project Create a drug that disables PfTMK

Use nuclear magnetic resonance to find compounds that bind

Create allosteric inhibitor

No ligand

Ligand bound

Nuclear Magnetic Resonance Tag valines, leucines, and isoleucines

Assign peaks using mutations

Tells us how ligand binds

Mutagenesis Generate pET plasmid with PfTMK gene

Mutate wild-type gene with PCR mutagenesis

Mutations chosen to minimize structural change

Isoleucine to valine

Valine to alanine and isoleucine

Leucine to isoleucine

QuickChange Site Directed Mutagenesis

Uses complementary primer pairs that contain desired mutation

Linear amplification

Newly strands can’t be used as templates due to nicks

QuickChange Site Directed Mutagenesis (Agilent)

Failure to amplify newly synthesized strands

PfuUltra II QuickChange Mutagenesis Limited success using 50% higher Mg2+ concentration (2mM to 3mM)

and double the expected extension time

3mM Mg2+, 3:00 extension (bottom right bands)

Lower magnesium in other lanes

51 191 178 70 54

2mM Mg2+, 1:45 extension(I11V, I51V, I127V, I152V)

3mM Mg2+, 3:00 extension with new primers

55 93 55 93 55 93

PfuUltra II QuickChange Errors Primer sequence repeated upstream of mutation/annealing site

Sequence data from an I51V transformant colony (top) and an I193V transformant colony (bottom)

PfuUltra II Separated Primers Mutagenesis

Separate primers into two individual PCRs

No success

Q5 QuickChange Mutagenesis New Q5 polymerase in 2X master mix

Success for 7 mutations

Failure for 4 mutations (sequencing errors/no transformants)

191 187 184 178 176 70 51

Q5 Overlapping Primers Use overlapping primers

Exponential amplification

Can bridge nicks in newly synthesized DNA

Transformants for remaining 4 mutations. 1 sequence confirmed, 3 TBD

Liu H. and Naismith J.H. (2008)

Protein Prep Grow transformed C3013s in deuterium oxide minimal media

Add precursors and induce

Cobalt ion affinity chromatography

Future: Search for a Drug Finish purifying mutant protein and assigning NMR peaks

Go through chemical library

Compare NMR spectrums with wild-type to identify ligand binding

Synthesize allosteric inhibitor