AMP-activated protein kinase (AMPK) plays a major role in regulating metabolism and has attracted significant attention as a therapeutic target for treating metabolic disorders. AMPK activity is ...stimulated more than 100-fold by phosphorylation of threonine 172 (Thr
). Binding of AMP to the γ subunit allosterically activates the kinase. Additionally, many small molecules, e.g. 991, have been identified that bind between the kinase domain and the carbohydrate-binding module of the β subunit, stabilising their interaction and leading to activation. It was reported recently that non-phosphorylated Thr
AMPK is activated by AMP and A769662. We present here the crystal structure of non-phosphorylated Thr
AMPK in complex with AMP and 991. This structure reveals that the activation loop, as well as the complex overall, is similar to the Thr
phosphorylated complex. We find that in the presence of AMP and 991 non-phosphorylated Thr
, AMPK is much less active than the Thr
phosphorylated enzyme. In human cells, the basal level of Thr
phosphorylation is very low (∼1%), but is increased 10-fold by treatment with 2-deoxyglucose. In cells lacking the major Thr
kinases, LKB1 and CaMKKβ, Thr
phosphorylation is almost completely abolished, and AMPK activity is virtually undetectable. Our data show that AMP and 991 binding to non-phosphorylated Thr
AMPK can induce an ordered, active-like, conformation of the activation loop explaining how AMPK activity can be measured
without Thr
phosphorylation. However, in a cellular context, phosphorylation of Thr
is critical for significant activation of AMPK.
The CRISPR-Cas9 RNA-guided endonuclease system allows precise and efficient modification of complex genomes and is continuously developed to enhance specificity, alter targeting and add new ...functional moieties. However, one area yet to be explored is the base chemistry of the associated RNA molecules. Here we show the design and optimisation of hybrid DNA-RNA CRISPR and tracr molecules based on structure-guided approaches. Through careful mapping of the ribose requirements of Cas9, we develop hybrid versions possessing minimal RNA residues, which are sufficient to direct specific nuclease activity in vitro and in vivo with reduced off-target activity. We identify critical regions within these molecules that require ribose nucleotides and show a direct correlation between binding affinity/stability and cellular activity. This is the first demonstration of a non-RNA-guided Cas9 endonuclease and first step towards eliminating the ribose dependency of Cas9 to develop a XNA-programmable endonuclease.
The DNA-encoded library (DEL) discovery platform has emerged as a powerful technology for hit identification in recent years. It has become one of the major parallel workstreams for small molecule ...drug discovery along with other strategies such as HTS and data mining. For many researchers working in the DEL field, it has become increasingly evident that many hits and leads discovered via DEL screening bind to target proteins with unique and unprecedented binding modes. This Perspective is our attempt to analyze reports of DEL screening with the purpose of providing a rigorous and useful account of the binding modes observed for DEL-derived ligands with a focus on binding mode novelty.
Excessive activity of striatal-enriched protein tyrosine phosphatase (STEP) in the brain has been detected in numerous neuropsychiatric disorders including Alzheimer’s disease. Notably, knockdown of ...STEP in an Alzheimer mouse model effected an increase in the phosphorylation levels of downstream STEP substrates and a significant reversal in the observed cognitive and memory deficits. These data point to the promising potential of STEP as a target for drug discovery in Alzheimer’s treatment. We previously reported a substrate-based approach to the development of low molecular weight STEP inhibitors with K i values as low as 7.8 μM. Herein, we disclose the first X-ray crystal structures of inhibitors bound to STEP and the surprising finding that they occupy noncoincident binding sites. Moreover, we utilize this structural information to optimize the inhibitor structure to achieve a K i of 110 nM, with 15–60-fold selectivity across a series of phosphatases.
Recent literature has claimed that inhibition of the enzyme MTH1 can eradicate cancer. MTH1 is one of the “housekeeping” enzymes that are responsible for hydrolyzing damaged nucleotides in cells and ...thus prevent them from being incorporated into DNA. We have developed orthogonal and chemically distinct tool compounds to those published in the literature to allow us to test the hypothesis that inhibition of MTH1 has wide applicability in the treatment of cancer. Here we present the work that led to the discovery of three structurally different series of MTH1 inhibitors with excellent potency, selectivity, and proven target engagement in cells. None of these compounds elicited the reported cellular phenotype, and additional siRNA and CRISPR experiments further support these observations. Critically, the difference between the responses of our highly selective inhibitors and published tool compounds suggests that the effect reported for the latter may be due to off-target cytotoxic effects. As a result, we conclude that the role of MTH1 in carcinogenesis and utility of its inhibition is yet to be established.
JAK1, JAK2, JAK3, and TYK2 belong to the JAK (Janus kinase) family. They play critical roles in cytokine signaling. Constitutive activation of JAK/STAT pathways is associated with a wide variety of ...diseases. Particularly, pSTAT3 is observed in response to the treatment with inhibitors of oncogenic signaling pathways such as EGFR, MAPK, and AKT and is associated with resistance or poorer response to agents targeting these pathways. Among the JAK family kinases, JAK1 has been shown to be the primary driver of STAT3 phosphorylation and signaling; therefore, selective JAK1 inhibition can be a viable means to overcome such treatment resistances. Herein, an account of the medicinal chemistry optimization from the promiscuous kinase screening hit 3 to the candidate drug 21 (AZD4205), a highly selective JAK1 kinase inhibitor, is reported. Compound 21 has good preclinical pharmacokinetics. Compound 21 displayed an enhanced antitumor activity in combination with an approved EGFR inhibitor, osimertinib, in a preclinical non-small-cell lung cancer (NSCLC) xenograft NCI-H1975 model.
Aberrant activity of the histone methyltransferase polycomb repressive complex 2 (PRC2) has been linked to several cancers, with small-molecule inhibitors of the catalytic subunit of the PRC2 ...enhancer of zeste homologue 2 (EZH2) being recently approved for the treatment of epithelioid sarcoma (ES) and follicular lymphoma (FL). Compounds binding to the EED subunit of PRC2 have recently emerged as allosteric inhibitors of PRC2 methyltransferase activity. In contrast to orthosteric inhibitors that target EZH2, small molecules that bind to EED retain their efficacy in EZH2 inhibitor-resistant cell lines. In this paper we disclose the discovery of potent and orally bioavailable EED ligands with good solubilities. The solubility of the EED ligands was optimized through a variety of design tactics, with the resulting compounds exhibiting in vivo efficacy in EZH2-driven tumors.
InhA is a well validated Mycobacterium tuberculosis (Mtb) target as evidenced by the clinical success of isoniazid. Translating enzyme inhibition to bacterial cidality by targeting the fatty acid ...substrate site of InhA remains a daunting challenge. The recent disclosure of a methyl-thiazole series demonstrates that bacterial cidality can be achieved with potent enzyme inhibition and appropriate physicochemical properties. In this study, we report the molecular mode of action of a lead methyl-thiazole, along with analogues with improved CYP inhibition profile. We have identified a novel mechanism of InhA inhibition characterized by a hitherto unreported “Y158-out” inhibitor-bound conformation of the protein that accommodates a neutrally charged “warhead”. An additional novel hydrophilic interaction with protein residue M98 allows the incorporation of favorable physicochemical properties for cellular activity. Notably, the methyl-thiazole prefers the NADH-bound form of the enzyme with a K d of ∼13.7 nM, as against the NAD+-bound form of the enzyme.
Transforming growth factor-β activated kinase-1 (TAK1) is a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family that regulates several signaling pathways including NF-κB ...signal transduction and p38 activation. TAK1 deregulation has been implicated in human diseases including cancer and inflammation. Here, we show that, in addition to its kinase activity, TAK1 has intrinsic ATPase activity, that (5Z)-7-Oxozeaenol irreversibly inhibits TAK1, and that sensitivity to (5Z)-7-Oxozeaenol inhibition in hematological cancer cell lines is NRAS mutation status and TAK1 pathway dependent. X-ray crystallographic and mass spectrometric studies showed that (5Z)-7-Oxozeaenol forms a covalent complex with TAK1. Detailed biochemical characterization revealed that (5Z)-7-Oxozeaenol inhibited both the kinase and the ATPase activity of TAK1 following a bi-phase kinetics, consistent with the irreversible inhibition mechanism. In DoHH2 cells, (5Z)-7-Oxozeaenol potently inhibited the p38 phosphorylation driven by TAK1, and the inhibition lasted over 6 h after withdrawal of (5Z)-7-Oxozeaenol. Profiling (5Z)-7-Oxozeaenol in a panel of hematological cancer cells showed that sensitive cell lines tended to carry NRAS mutations and that genes in TAK1 regulated pathways were enriched in sensitive cell lines. Taken together, we have elucidated the molecular mechanism of a TAK1 irreversible inhibitor and laid the foundation for designing next generation TAK1 irreversible inhibitors. The NRAS-TAK1-Wnt signaling network discerned in our study may prove to be useful in patient selection for TAK1 targeted agents in hematological cancers.
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