Stafia‐1: a STAT5a‐Selective Inhibitor Developed via Docking‐Based Screening of in Silico O‐Phosphorylated Fragments

Abstract

We present a new approach for the identification of inhibitors of phosphorylation‐dependent protein–protein interaction domains, in which phenolic fragments are adapted by in silico O‐phosphorylation before docking‐based screening. From a database of 10 369 180 compounds, we identified 85 021 natural product‐derived phenolic fragments, which were virtually O‐phosphorylated and screened for in silico binding to the STAT3 SH2 domain. Nine screening hits were then synthesized, eight of which showed a degree of in vitro inhibition of STAT3. After analysis of its selectivity profile, the most potent inhibitor was then developed to Stafia‐1, the first small molecule shown to preferentially inhibit the STAT family member STAT5a over the close homologue STAT5b. A phosphonate prodrug based on Stafia‐1 inhibited STAT5a with selectivity over STAT5b in human leukemia cells, providing the first demonstration of selective in vitro and intracellular inhibition of STAT5a by a small‐molecule inhibitor.

Direct Targeting Options for STAT3 and STAT5 in Cancer

“…The hydrophilicity of the phosphate motifs was reduced by generating a pro-drug precursor, which employs pivaloyloxymethyl esters to conceal the negatively charged phosphate groups and increase cell penetrance. Recently, Natarajan et al reported Stafia-1, a selective inhibitor for STAT5A, discovered by docking-based screening [102]. Tested applications for STAT5 inhibitors in vivo and in vitro are summarized in Table 2.…”

Asymmetrically Substituted m‐Terphenyl Phosphates Inhibit the Transcription Factor STAT5a

“…We recently presented catechol bisphosphates as the first chemical entities that inhibit STAT5b with selectivity over STAT5a [5] . Subsequently, we presented symmetrically substituted m ‐terphenyl phosphates as the first inhibitors of STAT5a which display selectivity over STAT5b [6] . The most potent and selective m ‐terphenyl phosphate‐based STAT5a inhibitor was dubbed Stafia‐1 ( 1 , Figure 1A), which inhibited STAT5a (K i =10.9±1.8 μM) with at least 9‐fold selectivity over STAT5b [6] .…”

“… A) Structure of Stafia‐1 and B) its suggested docking‐based mode of binding [6] to the X‐ray structure of murine STAT5a (PBD 1Y1U) [8] . The SH2 domain is shown in yellow, the linker domain is shown in light blue [10] .…”

“…The SH2 domain is shown in yellow, the linker domain is shown in light blue [10] . In the docking process, side chain flexibility was allowed for the STAT5a/b‐conserved amino acids Lys600 and Arg618, and for the STAT5a/b‐divergent amino acids Trp566, Asn639, and Lys644 [6] …”

STAT5A and STAT5B—Twins with Different Personalities in Hematopoiesis and Leukemia

“…Another example for a STAT5A/B SH2 domain inhibitor, IST5-002, blocked phosphorylation and nuclear translocation of STAT5A/B in BCR–ABL + in vitro and in vivo systems [164,176]. Via a virtual compound library screening approach and further structural adaptions, the first STAT5A SH2 domain inhibitor was identified, showing a modest reduction of pYSTAT5A levels in BCR–ABL + cells [177]. All compounds require further modifications to improve the bioavailability, stability, and potency.…”