Graphical AbstractHighlights d Structure-guided design and optimization yield potent FTO inhibitors d mRNA m 6 A acts as the major effector of the inhibitor/FTO axis in AML cells d FTO inhibitor FB23-2 displays therapeutic effects in PDX AML models d Targeting epitranscriptomic RNA methylation holds potential to treat AML SUMMARY FTO, an mRNA N 6 -methyladenosine (m 6 A) demethylase, was reported to promote leukemogenesis. Using structure-based rational design, we have developed two promising FTO inhibitors, namely FB23 and FB23-2, which directly bind to FTO and selectively inhibit FTO's m 6 A demethylase activity. Mimicking FTO depletion, FB23-2 dramatically suppresses proliferation and promotes the differentiation/apoptosis of human acute myeloid leukemia (AML) cell line cells and primary blast AML cells in vitro. Moreover, FB23-2 significantly inhibits the progression of human AML cell lines and primary cells in xeno-transplanted mice. Collectively, our data suggest that FTO is a druggable target and that targeting FTO by small-molecule inhibitors holds potential to treat AML. SignificanceAs the most abundant internal mRNA modification, m 6 A impacts various biological processes. As a major m 6 A demethylase, FTO is overexpressed in certain subtypes of AMLs and promotes leukemogenesis. Thus, the development of effective inhibitors to target FTO's aberrant m 6 A demethylase activity is urgently needed for leukemia therapy. Here we report two selective FTO inhibitors that efficiently reverse/suppress FTO-mediated aberrant epitranscriptome in AML cells and significantly inhibit AML progression in vivo. Our studies provide the proof-of-concept evidence demonstrating that small-molecule inhibitors targeting oncogenic FTO represent a promising targeted therapeutic strategy for the effective treatment of AML. Moreover, given the overexpression of FTO in various cancers, our work may have a broad impact on cancer therapy by targeting the FTO-mediated epitranscriptome.

ATP-dependent Clp protease (ClpP), a highly conserved serine protease in vast bacteria, could be converted into a noncontrollable enzyme capable of degrading mature proteins in the presence of acyldepsipeptides (ADEPs). Here, we design such a gain-of-function mutant of Staphylococcus aureus ClpP (SaClpP) capable of triggering the same level of dysfunctional activity that occurs upon ADEPs treatment. The SaClpPY63A mutant degrades FtsZ in vivo and inhibits staphylococcal growth. The crystal structure of SaClpPY63A indicates that Asn42 would be an important domino to fall for further activation of ClpP. Indeed, the SaClpPN42AY63A mutant demonstrates promoted self-activated proteolysis, which is a result of an enlarged entrance pore as observed in cryo-electron microscopy images. In addition, the expression of the engineered clpP allele phenocopies treatment with ADEPs; inhibition of cell division occurs as does showing sterilizing with rifampicin antibiotics. Collectively, we show that the gain-of-function SaClpPN42AY63A mutant becomes a fairly nonspecific protease and kills persisters by degrading over 500 proteins, thus providing new insights into the structure of the ClpP protease.

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