The upstream protein kinases responsible for thousands of phosphorylation events in the phosphoproteome remain to be discovered. We developed a three-component chemical reaction which converts the transient non-covalent substrate-kinase complex into a covalently cross-linked product by utilizing a dialdehyde based cross-linker, 1. Unfortunately the reaction of 1 with a lysine in the kinase active site and an engineered cysteine on the substrate to form an isoindole cross-linked product could not be performed in the presence of competing cellular proteinsdue to non-specific side reactions. In order to more selectively target the cross-linker to protein kinases in cell lysates we replaced the weak, kinase-binding adenosine moiety of 1 with a potent protein kinase inhibitor scaffold. In addition, we replaced the o-phthaldialdehyde moiety in 1 with a less reactive thiophene-2,3-dicarboxaldehyde moiety. The combination of these two structural modifications provides for cross-linking of a cysteine containing substrate to its corresponding kinase in the presence of competing cellular proteins.

We have developed an efficient and highly stereocontrolled synthesis of bistramide A, a selective activator of protein kinase C isotype delta. Our synthetic strategy featured a novel bidirectional approach for spiroketal construction based on the ring-opening/cross-metathesis sequence employing a highly strained cyclopropenone acetal. The synthesis afforded the final target with the longest linear sequence of 15 steps and provided unambiguous structural determination of bistramide A, including assignment of the previously unknown C(37) stereochemistry.

Bistramide A (1) is a marine natural product with broad, potent antiproliferative effects. Bistramide A has been reported to selectively activate protein kinase C (PKC) delta, leading to the view that PKCdelta is the principal mediator of antiproliferative activity of this natural product. Contrary to this observation, we established that bistramide A binds PKCdelta with low affinity, does not activate this kinase in vitro and does not translocate GFP-PKCdelta. Furthermore, we identified actin as the cellular receptor of bistramide A. We report that bistramide A disrupts the actin cytoskeleton, inhibits actin polymerization, depolymerizes filamentous F-actin in vitro and binds directly to monomeric G-actin in a 1:1 ratio with a Kd of 7 nM. We also constructed a fully synthetic9 bistramide A-based affinity matrix and isolated actin as a specific bistramide A-binding protein. This activity provides a molecular explanation for the potent antiproliferative effects of bistramide A, identifying it as a new biochemical tool for studies of the actin cytoskeleton and as a potential lead for development of a new class of antitumor agents.

Here we report the discovery that bifunctional thiol- and amine-reactive electrophiles serve as mechanism-based covalent cross-linkers for HECT E3 ubiquitin ligase-substrate pairs. We demonstrate that these chemical cross-linkers covalently cross-link the catalytic Cys residue of the yeast HECT E3 ubiquitin ligase Rsp5 with the Lys of the ubiquitination site in the model substrate Sic60-GFP. This work represents the first example of a mechanism-based covalent cross-link of HECT E3-substrate pairs that converts transiently interacting HECT E3-substrate pairs into stable, covalently cross-linked protein complexes, thereby facilitating their subsequent isolation, identification, and study.