The phosphoinositide 3-kinase p110α is an essential mediator of insulin signaling and glucose homeostasis. We interrogated the human serine, threonine, and tyrosine kinome to search for novel regulators of p110α and found that the Hippo kinases phosphorylate p110α at T1061, which inhibits its activity. This inhibitory state corresponds to a conformational change of a membrane-binding domain on p110α, which impairs its ability to engage membranes. In human primary hepatocytes, cancer cell lines, and rodent tissues, activation of the Hippo kinases MST1/2 using forskolin or epinephrine is associated with phosphorylation of T1061 and inhibition of p110α, impairment of downstream insulin signaling, and suppression of glycolysis and glycogen synthesis. These changes are abrogated when MST1/2 are genetically deleted or inhibited with small molecules or if the T1061 is mutated to alanine. Our study defines an inhibitory pathway of PI3K signaling and a link between epinephrine and insulin signaling.
Cancer presents a difficult challenge for oncologists, as there are few therapies that specifically target disease cells. Existing treatment strategies rely heavily on physical and chemical agents that nonspecifically affect DNA metabolism. To improve the effectiveness of these treatments, we have identified a new class of protein kinase inhibitor that targets a major DNA repair pathway. A representative of this class, 1-(2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone, inhibits the DNA-dependent protein kinase (DNA-PK) and differs significantly from previously studied DNA-PK inhibitors both structurally and functionally. DNA-PK participates in the cellular response to and repair of chromosomal DNA double-strand breaks (DSBs). These new selective inhibitors recapitulate the phenotype of DNA-PK defective cell lines including those from SCID mice. These compounds directly inhibit the repair of DNA DSBs and consequently enhance the cytotoxicity of physical and chemical agents that induce DSBs but not other DNA lesions. In contrast to previously studied DNA-PK inhibitors, these compounds appear benign, exhibiting no toxic effects in the absence of DSB-inducing treatments. Most importantly, 1-(2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone synergistically enhances radiation-induced tumor control in a mouse-human xenograft assay. These studies validate DNA-PK as a cancer drug target and suggest a new approach for enhancing the effects of existing cancer therapies.
An efficient three-step synthesis of chiral 3H-quinazoline-4-one derivatives from commercial materials is disclosed. The Mumm reaction of imidoyl chloride with α-amino acids followed by reductive cyclization affords enantiomerically pure (ee >93%) quinazoline-4-ones in good overall yield. A comparison with existing approaches indicates that this method is superior for hindered substrates.
ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTSynthesis and Postsynthetic Modification of Oligodeoxynucleotides Containing 4-Thio-2'-deoxyuridine (ds4U)Robert S. Coleman and Edward A. KesickiCite this: J. Am. Chem. Soc. 1994, 116, 26, 11636–11642Publication Date (Print):December 1, 1994Publication History Published online1 May 2002Published inissue 1 December 1994https://pubs.acs.org/doi/10.1021/ja00105a002https://doi.org/10.1021/ja00105a002research-articleACS PublicationsRequest reuse permissionsArticle Views616Altmetric-Citations47LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts
ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTTemplate-Directed Crosslinking of Oligonucleotides: Site-Specific Covalent Modification of dG-N7 within Duplex DNARobert S. Coleman and Edward A. KesickiCite this: J. Org. Chem. 1995, 60, 20, 6252–6253Publication Date (Print):October 1, 1995Publication History Published online1 May 2002Published inissue 1 October 1995https://pubs.acs.org/doi/10.1021/jo00125a004https://doi.org/10.1021/jo00125a004research-articleACS PublicationsRequest reuse permissionsArticle Views167Altmetric-Citations20LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts
Abstract Phosphoinositide 3-kinase alpha (PI3Kα) H1047R mutations are activating oncogenic events that occur in ~15% of advanced breast cancers. While there is one PI3Kα inhibitor FDA-approved for patients with PI3Kα-mutated breast cancer, and many others in clinical development, all of these agents inhibit wild-type PI3Kα and its mutated form with approximate equal potency. As a result, their efficacy is limited by toxicities associated with on target wild-type PI3Kα inhibition, notably hyperglycemia as well as cutaneous and GI toxicity. LOX-22783 is a highly potent, mutant-selective and brain-penetrant allosteric PI3Kα H1047R inhibitor. Here, we describe the preclinical profile of LOX-22783. H1047R selectivity was measured using biochemical kinase activity and cell-titer Glo and signal transduction assays. Tumor growth inhibition, pharmacokinetic and pharmacodynamic effects were assessed in in vivo studies using xenograft and patient-derived xenograft (PDX)-models. LOX-22783 inhibited growth and signaling responses in multiple H1047R-driven breast cancer cell lines and demonstrated high selectivity for H1047R mutated PI3Kα (EC50 values <5 nM) relative to wild-type PI3Kα (EC50 >250 nM) as well as the other wild-type PI3K isoforms (beta, gamma, and delta, all EC50 >250nM). In enzyme and cell-based assays, LOX-22783 dissociated from PI3Kα H1047R at a slower rate (3-6 hrs) compared to alpelisib (≤10 mins), potentially allowing for extended inhibition of PI3Kα H1047R by LOX-22783. LOX-22783 also normalized the EGF-stimulated membrane-localization of PI3Kα H1047R while alpelisib did not. LOX-22783 was highly kinome-selective when assayed at 3 µM, with no inhibitory activity on 17 lipid kinases or 374 protein kinases. In preclinical species, LOX-22783 demonstrated high oral bioavailability, including exposure in the CNS, a common site of metastases for patients with breast cancer. In vivo, LOX-22783 demonstrated dose-dependent tumor regression in H1047R breast cancer models without inducing hyperglycemia or other toxicities. Tumor pharmacodynamic analyses confirmed successful pathway inhibition. At doses resulting in 90% pathway inhibition, tumor regressions of ≥60% were observed. This wide therapeutic index is predicted to allow for maximizing dose intensity and efficacy in patients, without wild-type PI3Kα inhibition limiting target coverage for the H1047R mutant form. These data demonstrate that LOX-22783 potently and selectively inhibits mutant H1047R, but not wild-type PI3Kα, or other PI3K isoforms. LOX-22783 binds to an allosteric pocket distinct from the ATP binding site used by the approved and investigational PI3Kα inhibitors. We hypothesize that this profile will lead to differentiated efficacy and tolerability for patients with PI3Kα H1047R-mutated cancers, with the additional potential to address brain metastases. An IND submission is planned for 2022. Citation Format: Anke Klippel, Rui Wang, Loredana Puca, Andrew Lee Faber, Weihua Shen, Shripad V. Bhagwat, Kannan Karukurichi, Feiyu Fred Zhang, Carmen Perez, Ramon Rama, Ana Ramos, Yi Zheng, Zahid Bonday, James Thomas, Harold B. Brooks, Lisa J. Kindler, Sarah M. Bogner, Parisa Zolfaghari, Mark Hicks II, Sophie Callies, Brian Mattioni, Laurie LeBrun, Jim Durbin, Erin Anderson, Chris Mayne, Edward Kesicki, Gabrielle Kolakowski, Steven W. Andrews, Barbara J. Brandhuber. Preclinical characterization of LOX-22783, a highly potent, mutant-selective and brain-penetrant allosteric PI3Kα H1047R inhibitor [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P142.
Phenylboronic acid bioconjugates prepared from alkaline phosphatase by reaction with either 2,5-dioxopyrrolidinyl 3-{N-[3-(1,3,2-dioxaboran-2-yl)phenyl]carbamoyl}propanoate (PBA-XX-NHS) or 2,5-dioxopyrrolidinyl 6-{[3,5-di-(1,3,2-dioxaboran-2-yl)phenyl]carbonylamino}hexanoate (PDBA-X-NHS) were compared with respect to the efficiency with which they were immobilized on salicylhydroxamic acid-modified Sepharose (SHA-X-Sepharose) by boronic acid complex formation. When immobilized on moderate capacity SHA-X-Sepharose (5.4 μmol of SHA/mL of gel), PDBA−alkaline phosphatase conjugates were shown to be stable with respect to both the alkaline (pH 11.0) and acidic (pH 2.5) buffers utilized to recover anti-alkaline phosphatase during affinity chromatography. Boronic acid complex formation was compared to covalent immobilization of alkaline phosphatase on Affi-Gel 10 and Affi-Gel 15. PDBA−AP·SHA-X-Sepharose was shown to afford superior performance to both Affi-Gel 10 and Affi-Gel 15 with respect to immobilization of alkaline phosphatase, retention of anti-alkaline phosphatase and recovery of anti-alkaline phosphatase under alkaline conditions. High capacity SHA-X-Sepharose (≥7 μmol of SHA/mL of gel) was shown to afford superior performance to moderate capacity SHA-X-Sepharose (4.5 μmol of SHA/mL of gel) with respect to stability at pH 11.0 and pH 2.5 when a PDBA−αHuman IgG conjugate with a low incorporation ratio of only 1.5:1 was immobilized on SHA-X-Sepharose and subsequently utilized for affinity chromatography of Human IgG. The results are interpreted in terms of either a bivalent or trivalent interaction involving boronic acid complex formation.
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SUMMARY The phosphoinositide 3-kinase, p110α, is an essential mediator of insulin signaling and glucose homeostasis. We systematically interrogated the human serine, threonine, and tyrosine kinome to search for novel regulators of p110α and found that the Hippo kinases phosphorylate and completely inhibit its activity. This inhibitory state corresponds to a conformational change of a membrane binding domain on p110α, which impairs its ability to engage membranes. In human primary hepatocytes, cancer cell lines, and rodent tissues, activation of the Hippo kinases, MST1/2, using forskolin or epinephrine is associated with phosphorylation and inhibition of p110α, impairment of downstream insulin signaling, and suppression of glycolysis and glycogen synthesis. These changes are abrogated when MST1/2 are genetically deleted or inhibited with small molecules. Our study reveals a novel inhibitory pathway of PI3K signaling and a previously unappreciated link between epinephrine and insulin signaling.
