UCSF

The protein kinase family represents a significant challenge in medicinal chemistry. This important class of enzymes shares a structurally conserved catalytic core, making selective inhibition of individual members difficult. Covalent inhibition offers the potential of additional selectivity based on chemical reactivity and here I have designed and synthesized a panel of electrophilic nucleosides to investigate select protein kinases. These compounds share a nucleoside scaffold that combines elements of the broad spectrum tyrosine kinase inhibitor pyrazolopyrimidine-1 (PP1) and an electrophilic nucleoside, p-fluorosulfonylbenzoyl 5' adenosine. This combination increases the scaffold's reversible binding affinity to target a subset of protein kinases. I tested the effect of several different 5' electrophiles on the activity of Src-kinase family members. A 5'-vinyl sulfonate ester reacted with a cysteine in the "glycine-rich" loop of Src. This cysteine is also found in FGFR tyrosine kinases, but is present in only three of the eleven Src-family kinases (Src, Yes, and Fgr). A pyrrolopyrimidine nucleoside bearing a 5'-vinyl sulfonate ester inhibited autophosphorylation and FGFR3 dependent cell proliferation in a cysteine dependent manner.

Kinases of the eukaryotic parasite Trypanosoma brucei were profiled using the electrophilic nucleoside 47, which targets an essential catalytic lysine found in nearly all protein kinases. This compound binds to a subset of the T. brucei kinases, five of which are essential: TbPK50 and homologues of casein kinase 1, Gsk3, NIMA, and Clk protein kinases. I used compound 47 in conjunction with quantitative mass spectrometry as a tool for chemical proteomics. Using this technique, covalently modified T. brucei kinases were profiled for binding with the reversible inhibitor PD0166326. Exchanging the 5' electrophile of 47 for an acrylate ester changes the compound's effect on T. brucei. This new electrophile still preferentially reacts with protein kinases, but with higher selectivity. One unique target of the acrylate is the T. brucei homologue of Bud32 kinase. This atypical kinase may have important functions in T. brucei biology and virulence.