Staurosporine

A tuned affinity-based staurosporine probe for in situ profiling of protein kinases†

Protein kinases tightly regulate protein function through phos- phorylation and thereby control cell signaling in eukaryotic systems.1 As these enzymes regulate essentially all cellular processes, they have emerged as a vital therapeutic target class for many human diseases.2 For instance, the BCR/Abl and Src family kinases (targets of leukemia and other types of cancers), and the EGFR (epidermal growth factor receptor receptor, a tyrosine kinase target for lung and colon cancer) have corresponding small molecule inhibitors developed into viable anticancer therapies.1–3 However, nearly all kinase inhibitors target the adenosine triphosphate (ATP) binding site, which is highly conserved, even across distantly related kinases. As a result, most small molecule kinase inhibitors suffer from broad reactivities.1 It remains a challenge to design therapeutics that are able to specifically target only the disease-causing kinase, and thus minimize toxicity and undesirable side effects. One proven strategy to enhance the selectivity of kinases inhibitors is through the development of novel kinase-targeting small molecule probes capable of interrogating endogenous kinase–drug interactions in an in vivo setting.4

Staurosporine (STS) is a natural product alkaloid isolated from Streptomyces staurosporeus, and is the most potent broad-spectrum kinase inhibitor known. It has been applied as a tool to study the binding and inhibition of various kinases. Of the approximately 518 kinases in humans, a sizeable number (at least 253) are non-covalently inhibited by staurosporine.3 We and others have sought to develop affinity-based probes (AfBPs) based on STS, which are capable of covalent interactions with kinases based on photo-crosslinking reactions.5,6 In a previous example, Fischer et al. developed a biotin- tagged STS,5 which was however too bulky and not cell-permeable. We separately designed cell-permeable, diazirine-containing STS probes capable of large-scale kinase profiling experiments in live mammalian cells.6 Due to the need for introducing a photo-reactive diazirine moiety into such probes, they have intrinsically strong background labeling toward high-abundance endogenous proteins. Herein, we report the incorporation of an electrophilic warhead into STS through a subtle modification so as to retain its biological function and activity in live cells (see STS-C1 in Fig. 1). We hypothesized that this new STS-tagging configuration may minimize non-specific labeling observed in previously developed STS probes,5,6 by preferentially gearing selectivity towards a range of kinases (or the so-called kinase cysteinome7a), which possess potentially targetable cysteine residues near the ATP-binding pocket of the enzyme. In so doing, such a strategy could prove useful in lead development, and for tuning potencies of kinase inhibitors (i.e. enhance on- and minimize off-target interactions). Similar strategies had previously been implemented successfully in the design of irreversible drugs that target different biological targets including kinases.7b

The conversion of STS into an irreversible covalent kinase probe (e.g. STS-C1) that selectively targets cysteinome commenced with modification at the secondary amine of STS (a position previously shown to be highly tolerant to structural changes8) with a small handle containing a chloroacetamide and a terminal alkyne. Many protein kinases, including c-Src, have potentially reactive cysteine residues near their ATP-binding pockets.7b In the current study, we chose c-Src as one of our intended targets due to its obvious biological importance. In addition, few irreversible inhibitors of c-Src are known. One very recent report, however, has already indicated that it is possible to irreversibly target Cys277 (a residue near the ATP pocket of c-Src; see Fig. 1) by using a aminopyrazole-based kinase inhibitor decorated with a suitable electrophilic moiety.9 We performed docking experiments to guide the design of linker size and configuration in STS-C1 (Fig. 1 bottom);10 Cys277 in c-Src was found to be located B5.2 Å from the chloroacetamide moiety of STS-C1. In addition, the alkyne handle of STS-C1 in the docked complex was shown to project toward the protein surface, thus making it accessible for subsequent click conjugation.6 The synthesis of STS-C1 is shown in Scheme 1. Briefly, Fmoc-O-tert-butyl-L-serine was coupled with propargylamine, giving 2, which was reacted with 2-chloroacetyl chloride upon Fmoc deprotection. The resulting intermediate 4 was treated with TFA to give 5. The final probe, STS-C1, was obtained by coupling 5 and 6 (obtained as previously reported6a). With the newly developed probe STS-C1 in hand, we carried out the initial experiments to establish its inhibition profiles and cellular activities (Fig. 2A and B); compared to STS, the IC50 of STS-C1 against recombinant c-Src in an in vitro kinase inhibition assay was approxi- mately 2-fold lower (Fig. 2A). Interestingly, a 4-fold increase in IC50 value was observed for STS-C1 against PKA (also a known target of STS). This suggests that while introduction of the linker moiety in STS in general negates the kinase–inhibitor interaction,6b suitable positioning of the electrophilic moiety near Cys277 in c-Src actually promotes such interactions, presumably via the formation of an irreversible covalent complex. We further evaluated the anti- proliferative activity of STS and STS-C1 in HepG2 cells (human liver cancer cells; Fig. 2B); the results showed similar toxicity profiles for both compounds at different concentrations, indicating nearly complete retention of STS cellular activities in STS-C1.
We next tested the covalent labeling of STS-C1 against recombinantly purified proteins including BSA (not a kinase) and several kinases (ERK2, CSK, Abl, PKA and c-Src). As shown in Fig. 2C, only c-Src was prominently labeled. Interestingly, even PKA was not positively labeled by STS-C1 despite a relative potent inhibition (e.g. IC50 = 267.9 + 2.1 nM). This clearly showed that in order for the probe to covalently label the target protein, a proximal reactive cysteine residue, e.g. Cys277 in c-Src, is essential. We also compared the labeling profile of STS-C1 with that of our previous developed photo-reactive probe STS-2 (Fig. S2, ESI†);6b the results further confirmed that these profiles are indeed distinctly different. We next performed labeling of c-Src in a more complex environment by using bacterial lysates that overexpress c-Src (Fig. 3D); with different amounts of STS-C1 (0 to 200 nM) being used to label 20 mg (each lane) of the lysates, it was observed that selective c-Src labeling could be readily detected with as little as 10 nM of the probe. In a separate experiment with 200 nM of STS-C1, c-Src labeling was clearly detected in as little as 1 mg of the bacterial lysate (Fig. S3B, ESI†), indicating a good detection sensitivity of the probe. Competitive labeling experi- ment was also carried out to confirm that the covalent labeling of c-Src by STS-C1 was activity-based (Fig. S3C, ESI†); the presence of excessive STS (2 mM; 10× of STS-C1) was able to significantly attenuate c-Src labeling.

Encouraged by these results, we went on to test the labeling of STS-C1 in endogenous mammalian proteomes. HepG2 liver cancer cells were used and the labeling reactions were carried out both in vitro (cell lysates) and in situ (live cells) (Fig. 3A); the results from the in-gel fluorescence scanning of the labeled proteomes showed multiple fluorescent bands, highlighting the presence of many potential cellular targets of this probe. In order to confirm that endogenous c-Src present in the HepG2 proteomes was indeed one of the labeled targets, these labeled proteomes were clicked with Rh-Biotin-N3, pulled down with avidin beads followed by Western blotting analysis with the anti-c-Src antibody (Fig. 3B); labeled c-Src was successfully detected in probe-treated proteomes, but not in negative con- trols (e.g. DMSO-treated proteomes), under both in vitro and in situ settings. We further subjected the labeled proteomes to pull-down/LC-MS/MS analysis for large-scale identification of putative labeled protein targets and the results are summarized in Fig. 3C, with select candidate proteins listed in Table 1. The full list of proteins identified from these experiments is provided in the ESI.† As should be expected, kinases formed the bulk of the most highly ranked hits. In total, 35 kinases were identified from the in vitro pull-down and 11 kinases from the in situ pull-down, with 7 kinases that overlapped across both experimental sets (Fig. 3C). Compared to proteins identified from earlier-generation probes (e.g. diazirine-containing STS probes6), STS-C1 provided a different range of targets. By focusing our analysis on the 11 kinases identified from the in situ pull-down experiments (Table 1), we found some inter- esting hits. For example, both CDK1 and CDK2 were previously predicted to possess targetable cysteine near their ATP-binding pockets,7a and they were positively pulled-down/identified from our experiments. These findings further indicate that our newly developed probe might be a suitable chemical for future identification of unknown proteins in kinase cysteinome. Finally, we tested the ability of STS-C1 as a potential small molecule cell-imaging probe (Fig. 3D); the results showed that the probe was cell-permeable and predominantly cytosolic in HepG2 cells.

In conclusion, STS was successfully converted into an irre- versible covalent probe, STC-C1, in an effort to minimize non- specific labeling from earlier-generation photoaffinity-based probes. We found that STS-C1 was able to preferentially target the protein kinase cysteinome, by using c-Src as a representative example. Through the course of our study, we have tentatively identified previously unknown proteins in the kinase cysteinome, and they will be further validated in due course.