Design and Synthesis of AX7574:  A Microcystin-Derived, Fluorescent Probe for Serine/Threonine Phosphatases

Abstract

The design and synthesis of AX7574, a microcystin-derived probe for serine/threonine phosphatases, is described. A key step in the synthesis was the conjugation under basic conditions of a tetramethylrhodamine 1,3-diketone derivative to the arginine side chain present in microcystin-LR. The resulting conjugate specifically labeled the active site of protein phosphatases 1 (PP-1) with a 1:1 stoichiometry and IC50 of 4.0 nM. AX7574 was used to isolate and identify PP-1, PP-2A, PP-4, and PP-6 in Jurkat cells. Finally, AX7574 was able to record changes in the phosphatase activity levels of calyculin A treated Jurkat cells versus untreated control cells.

Strategies for Discovering and Derisking Covalent, Irreversible Enzyme Inhibitors

“…MS platforms, such as ABPP-MudPIT, enable the enrichment and identification of probe-labeled proteins from a complex proteome [64–66]. ABPs (1–12) have been developed for a number of enzyme classes [62], including serine hydrolases [67–69], cysteine proteases [70–72], serine/threonine [73] and tyrosine phosphatases [74], glycosidases [75,76], ubiquitin-conjugating/-hydrolyzing enzymes [77–79], proteasomes [80], oxidoreductases [81,82], ATP-binding enzymes (e.g., kinases) [83–85] and cytochrome P450s (Figure 4) [86,87]. …”

Mechanism-Based Profiling of Enzyme Families

“…155,156 Taking advantage of extensive SAR studies, Shreder and coworkers prepared a microcystin derivative with an appended fluorophore positioned to preserve the reactivity of the parent natural product for its known phosphatase targets. 157 On examination of the global reactivity of this ABP in soluble Jurkat lysates, two previously unappreciated phosphatase targets of microcystin, PP-4 and PP-6, were identified. Of perhaps more general significance, these studies provide a prototype example of an ABP that targets a noncatalytic residue in enzyme active sites to achieve classselective labeling and inhibition in complex proteomes.…”

Activity‐Based Protein Profiling: Recent Advances in Probe Development and Applications

“…Therefore, many of the best examples of ABPs have been designed to target hydrolases, such as fluorophosphonate probes (1) for serine hydrolases [10] and epoxide and AOMK probes for cysteine proteases (2, 3), [11] which have a reactive nucleophilic residue in the active site and possess a distinct catalytic mechanism. [12] Other such directed ABPs includes probes targeting cathepsins, legumains, [13] caspases, [14] protein arginine methyltransferases (4), [15] proteasomal proteases (5), [16] kinases (6), [17] tyrosine phosphatase, [18] serine/threonine phosphatase, [19] and glycosidases. [20] It may be expected that the design of ABPs for enzyme classes with known covalent inhibitors is, at least in concept, straightforward.…”

“…[69] Similarly, a microcystin-derived fluorescent probe, AX7574 (36), was synthesized to target serine/threonine phosphatases in cell extracts. [19] Most recently, Sieber and other groups further advanced a "label-free" design by adding a short alkyne/azide group rather than a bulky fluorescent reporter to the natural product. As discussed before, such a design eliminated the adverse effect of the bulky fluorescent group on binding specificity and bioavailability of the natural product derivatives.…”