420 results on '"Shokat KM"'
Search Results
2. Using hydrogen deuterium exchange mass spectrometry to engineer optimized constructs for crystallization of protein complexes: Case study of PI4KIII with Rab11
- Author
-
Shokat, Kevan, Fowler, ML, McPhail, JA, Jenkins, ML, Masson, GR, Rutaganira, FU, Shokat, KM, Williams, RL, and Burke, JE
- Published
- 2016
3. Differential genetic interactions of yeast stress response MAPK pathways
- Author
-
Krogan, Nevan, Shokat, Kevan, Lim, Wendell, Martin, H, Shales, M, Fernandez-Piñar, P, Wei, P, Molina, M, Fiedler, D, Shokat, KM, Beltrao, P, and Krogan, NJ
- Abstract
© 2015 The Authors.Abstract Genetic interaction screens have been applied with great success in several organisms to study gene function and the genetic architecture of the cell. However, most studies have been performed under optimal growth conditions eve
- Published
- 2015
4. Structure of the human autophagy initiating kinase ULK1 in complex with potent inhibitors
- Author
-
Shokat, Kevan, Lazarus, MB, Novotny, CJ, and Shokat, KM
- Abstract
© 2014 American Chemical Society.Autophagy is a conserved cellular process that involves the degradation of cellular components for energy maintenance and cytoplasmic quality control that has recently gained interest as a novel target for a variety of huma
- Published
- 2015
5. Incomplete inhibition of phosphorylation of 4E-BP1 as a mechanism of primary resistance to ATP-competitive mTOR inhibitors.
- Author
-
Ducker, GS, Atreya, CE, Simko, JP, Hom, YK, Matli, MR, Benes, CH, Hann, B, Nakakura, EK, Bergsland, EK, Donner, DB, Settleman, J, Shokat, KM, and Warren, RS
- Subjects
Cell Line ,Tumor ,Animals ,Humans ,Mice ,Colonic Neoplasms ,Indoles ,Purines ,ras Proteins ,Adaptor Proteins ,Signal Transducing ,Carrier Proteins ,Cell Cycle Proteins ,Proto-Oncogene Proteins ,Phosphoproteins ,Eukaryotic Initiation Factors ,Ribosomal Protein S6 ,Adenosine Triphosphate ,Antineoplastic Agents ,Xenograft Model Antitumor Assays ,MAP Kinase Signaling System ,Binding ,Competitive ,Phosphorylation ,Drug Resistance ,Neoplasm ,Mutation ,Female ,Proto-Oncogene Proteins p21(ras) ,Phosphatidylinositol 3-Kinases ,Class I Phosphatidylinositol 3-Kinases ,TOR Serine-Threonine Kinases ,mTOR ,colon cancer ,PIK3CA ,KRAS ,xenograft ,PP242 ,Biotechnology ,Genetics ,Colo-Rectal Cancer ,Digestive Diseases ,Cancer ,5.1 Pharmaceuticals ,Development of treatments and therapeutic interventions ,Clinical Sciences ,Oncology and Carcinogenesis ,Oncology & Carcinogenesis - Abstract
The mammalian target of rapamycin (mTOR) regulates cell growth by integrating nutrient and growth factor signaling and is strongly implicated in cancer. But mTOR is not an oncogene, and which tumors will be resistant or sensitive to new adenosine triphosphate (ATP) competitive mTOR inhibitors now in clinical trials remains unknown. We screened a panel of over 600 human cancer cell lines to identify markers of resistance and sensitivity to the mTOR inhibitor PP242. RAS and phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) mutations were the most significant genetic markers for resistance and sensitivity to PP242, respectively; colon origin was the most significant marker for resistance based on tissue type. Among colon cancer cell lines, those with KRAS mutations were most resistant to PP242, whereas those without KRAS mutations most sensitive. Surprisingly, cell lines with co-mutation of PIK3CA and KRAS had intermediate sensitivity. Immunoblot analysis of the signaling targets downstream of mTOR revealed that the degree of cellular growth inhibition induced by PP242 was correlated with inhibition of phosphorylation of the translational repressor eIF4E-binding protein 1 (4E-BP1), but not ribosomal protein S6 (rpS6). In a tumor growth inhibition trial of PP242 in patient-derived colon cancer xenografts, resistance to PP242-induced inhibition of 4E-BP1 phosphorylation and xenograft growth was again observed in KRAS mutant tumors without PIK3CA co-mutation, compared with KRAS wild-type controls. We show that, in the absence of PIK3CA co-mutation, KRAS mutations are associated with resistance to PP242 and that this is specifically linked to changes in the level of phosphorylation of 4E-BP1.
- Published
- 2014
6. Inhibition of the kinase Csk in thymocytes reveals a requirement for actin remodeling in the initiation of full TCR signaling
- Author
-
Shokat, Kevan, Weiss, Arthur, Tan, YX, Manz, BN, Freedman, TS, Zhang, C, and Shokat, KM
- Abstract
Signaling via the T cell antigen receptor (TCR) is initiated by Src-family kinases (SFKs). To understand how the kinase Csk, a negative regulator of SFKs, controls the basal state and the initiation of TCR signaling, we generated mice that express a Csk va
- Published
- 2014
7. Quantitative and temporal requirements revealed for Zap70 catalytic activity during T cell development
- Author
-
Zikherman, Julie, Shokat, Kevan, Weiss, Arthur, Au-Yeung, BB, Melichar, HJ, Ross, JO, Cheng, DA, Shokat, KM, and Robey, EA
- Abstract
The catalytic activity of Zap70 is crucial for T cell antigen receptor (TCR) signaling, but the quantitative and temporal requirements for its function in thymocyte development are not known. Using a chemical-genetic system to selectively and reversibly in
- Published
- 2014
8. PTEN expression is consistent in colorectal cancer primaries and metastases and associates with patient survival
- Author
-
Warren, Robert, Shokat, Kevan, Atreya, CE, Sangale, Z, Xu, N, Matli, MR, Tikishvili, E, Welbourn, W, Stone, S, Shokat, KM, and Warren, RS
- Abstract
Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) negatively regulates the phosphoinositide-3-kinase (PI3K) signaling pathway. In colorectal cancer (CRC), observed frequencies of loss of PTEN expression, concordant expression in primary tumo
- Published
- 2013
9. Cyclin-dependent kinase control of the initiation-to-elongation switch of RNA polymerase II
- Author
-
Shokat, Kevan, Larochelle, S, Amat, R, Glover-Cutter, K, Sansó, M, Zhang, C, Allen, JJ, Shokat, KM, Bentley, DL, and Fisher, RP
- Abstract
Promoter-proximal pausing by RNA polymerase II (Pol II) ensures gene-specific regulation and RNA quality control. Structural considerations suggested a requirement for initiation-factor eviction in elongation-factor engagement and pausing of transcription
- Published
- 2012
10. Chemical Genetics Reveals a Specific Requirement for Cdk2 Activity in the DNA Damage Response and Identifies Nbs1 as a Cdk2 Substrate in Human Cells
- Author
-
Shokat, Kevan, Wohlbold, L, Merrick, KA, De, S, Amat, R, Kim, JH, Larochelle, S, Allen, JJ, Zhang, C, Shokat, KM, and Petrini, JHJ
- Abstract
The cyclin-dependent kinases (CDKs) that promote cell-cycle progression are targets for negative regulation by signals from damaged or unreplicated DNA, but also play active roles in response to DNA lesions. The requirement for activity in the face of DNA
- Published
- 2012
11. A Positive Feedback Loop Links Opposing Functions of P-TEFb/Cdk9 and Histone H2B Ubiquitylation to Regulate Transcript Elongation in Fission Yeast
- Author
-
Shokat, Kevan, Sansó, M, Lee, KM, Viladevall, L, Jacques, PE, Pagé, V, Nagy, S, Racine, A, St, CV, Zhang, C, and Shokat, KM
- Abstract
Transcript elongation by RNA polymerase II (RNAPII) is accompanied by conserved patterns of histone modification. Whereas histone modifications have established roles in transcription initiation, their functions during elongation are not understood. Mono-u
- Published
- 2012
12. In vivo conditions to identify Prkci phosphorylation targets using the analog-sensitive kinase method in zebrafish
- Author
-
Shokat, Kevan, Uhalte, E, Kirchner, M, Hellwig, N, Allen, JJ, Donat, S, Shokat, KM, Selbach, M, and Abdelilah-Seyfried, S
- Abstract
Protein kinase C iota is required for various cell biological processes including epithelial tissue polarity and organ morphogenesis. To gain mechanistic insight into different roles of this kinase, it is essential to identify specific substrate proteins i
- Published
- 2012
13. Orm protein phosphoregulation mediates transient sphingolipid biosynthesis response to heat stress via the Pkh-Ypk and Cdc55-PP2A pathways
- Author
-
Shokat, Kevan, Sun, Y, Miao, Y, Yamane, Y, Zhang, C, Shokat, KM, Takematsu, H, Kozutsumi, Y, and Drubin, DG
- Abstract
Sphingoid intermediates accumulate in response to a variety of stresses, including heat, and trigger cellular responses. However, the mechanism by which stress affects sphingolipid biosynthesis has yet to be identified. Recent studies in yeast suggest that
- Published
- 2012
14. Cofactor-mediated conformational control in the bifunctional kinase/RNase Ire1
- Author
-
Korennykh, AV, Egea, PF, Korostelev, AA, Finer-Moore, J, Stroud, RM, Zhang, C, Shokat, KM, and Walter, P
- Abstract
Background: Ire1 is a signal transduction protein in the endoplasmic reticulum (ER) membrane that serves to adjust the protein-folding capacity of the ER according to the needs of the cell. Ire1 signals, in a transcriptional program, the unfolded protein response (UPR) via the coordinated action of its protein kinase and RNase domains. In this study, we investigated how the binding of cofactors to the kinase domain of Ire1 modulates its RNase activity.Results: Our results suggest that the kinase domain of Ire1 initially binds cofactors without activation of the RNase domain. RNase is activated upon a subsequent conformational rearrangement of Ire1 governed by the chemical properties of bound cofactors. The conformational step can be selectively inhibited by chemical perturbations of cofactors. Substitution of a single oxygen atom in the terminal β-phosphate group of a potent cofactor ADP by sulfur results in ADPβS, a cofactor that binds to Ire1 as well as to ADP but does not activate RNase. RNase activity can be rescued by thiophilic metal ions such as Mn2+and Cd2+, revealing a functional metal ion-phosphate interaction which controls the conformation and RNase activity of the Ire1 ADP complex. Mutagenesis of the kinase domain suggests that this rearrangement involves movement of the αC-helix, which is generally conserved among protein kinases. Using X-ray crystallography, we show that oligomerization of Ire1 is sufficient for placing the αC-helix in the active, cofactor-bound-like conformation, even in the absence of cofactors.Conclusions: Our structural and biochemical evidence converges on a model that the cofactor-induced conformational change in Ire1 is coupled to oligomerization of the receptor, which, in turn, activates RNase. The data reveal that cofactor-Ire1 interactions occur in two independent steps: binding of a cofactor to Ire1 and subsequent rearrangement of Ire1 resulting in its self-association. The pronounced allosteric effect of cofactors on protein-protein interactions involving Ire1's kinase domain suggests that protein kinases and pseudokinases encoded in metazoan genomes may use ATP pocket-binding ligands similarly to exert signaling roles other than phosphoryl transfer. © 2011 Korennykh et al; licensee BioMed Central Ltd.
- Published
- 2011
15. Structural and functional basis for RNA cleavage by Ire1
- Author
-
Korennykh, AV, Korostelev, AA, Egea, PF, Finer-Moore, J, Stroud, RM, Zhang, C, Shokat, KM, and Walter, P
- Abstract
Background: The unfolded protein response (UPR) controls the protein folding capacity of the endoplasmic reticulum (ER). Central to this signaling pathway is the ER-resident bifunctional transmembrane kinase/endoribonuclease Ire1. The endoribonuclease (RNase) domain of Ire1 initiates a non-conventional mRNA splicing reaction, leading to the production of a transcription factor that controls UPR target genes. The mRNA splicing reaction is an obligatory step of Ire1 signaling, yet its mechanism has remained poorly understood due to the absence of substrate-bound crystal structures of Ire1, the lack of structural similarity between Ire1 and other RNases, and a scarcity of quantitative enzymological data. Here, we experimentally define the active site of Ire1 RNase and quantitatively evaluate the contribution of the key active site residues to catalysis.Results: This analysis and two new crystal structures suggest that Ire1 RNase uses histidine H1061 and tyrosine Y1043 as the general acid-general base pair contributing ≥ 7.6 kcal/mol and 1.4 kcal/mol to transition state stabilization, respectively, and asparagine N1057 and arginine R1056 for coordination of the scissile phosphate. Investigation of the stem-loop recognition revealed that additionally to the stem-loops derived from the classic Ire1 substrates HAC1 and Xbp1 mRNA, Ire1 can site-specifically and rapidly cleave anticodon stem-loop (ASL) of unmodified tRNAPhe, extending known substrate specificity of Ire1 RNase.Conclusions: Our data define the catalytic center of Ire1 RNase and suggest a mechanism of RNA cleavage: each RNase monomer apparently contains a separate catalytic apparatus for RNA cleavage, whereas two RNase subunits contribute to RNA stem-loop docking. Conservation of the key residues among Ire1 homologues suggests that the mechanism elucidated here for yeast Ire1 applies to Ire1 in metazoan cells, and to the only known Ire1 homologue RNase L. © 2011 Korennykh et al; licensee BioMed Central Ltd.
- Published
- 2011
16. Immunoglobulin signal transduction guides the specificity of B cell-T cell interactions and is blocked in tolerant self-reactive B cells.
- Author
-
Cooke, MP, Heath, AW, Shokat, KM, Zeng, Y, Finkelman, FD, Linsley, PS, Howard, M, and Goodnow, CC
- Subjects
Emerging Infectious Diseases ,Vaccine Related ,HIV/AIDS ,Biodefense ,Prevention ,1.1 Normal biological development and functioning ,Underpinning research ,Inflammatory and immune system ,Animals ,Antigens ,B-Lymphocytes ,Cell Communication ,Cell Membrane ,Cells ,Cultured ,Immune Tolerance ,Immunoglobulin D ,Immunoglobulin M ,Mice ,Mice ,Inbred C57BL ,Mice ,Transgenic ,Muramidase ,Receptors ,Antigen ,B-Cell ,Signal Transduction ,T-Lymphocytes ,Medical and Health Sciences ,Immunology - Abstract
The specificity of antibody (Ab) responses depends on focusing helper T (Th) lymphocyte signals to suitable B lymphocytes capable of binding foreign antigens (Ags), and away from nonspecific or self-reactive B cells. To investigate the molecular mechanisms that prevent the activation of self-reactive B lymphocytes, the activation requirements of B cells specific for the Ag hen egg lysozyme (HEL) obtained from immunoglobulin (Ig)-transgenic mice were compared with those of functionally tolerant B cells isolated from Ig-transgenic mice which also express soluble HEL. To eliminate the need for surface (s)Ig-mediated Ag uptake and presentation and allow the effects of sIg signaling to be studied in isolation, we assessed the ability of allogeneic T cells from bm12 strain mice to provide in vivo help to C57BL/6 strain-transgenic B cells. Interestingly, non-tolerant Ig-transgenic B cells required both allogeneic Th cells and binding of soluble HEL for efficient activation and Ab production. By contrast, tolerant self-reactive B cells from Ig/HEL double transgenic mice responded poorly to the same combination of allogeneic T cells and soluble HEL. The tolerant B cells were nevertheless normally responsive to stimulation with interleukin 4 and anti-CD40 Abs in vitro, suggesting that they retained the capacity to respond to mediators of T cell help. However, the tolerant B cells exhibited a proximal block in the sIg signaling pathway which prevented activation of receptor-associated tyrosine kinases in response to the binding of soluble HEL. The functional significance of this sIg signaling defect was confirmed by using a more potent membrane-bound form of HEL capable of triggering sIg signaling in tolerant B cells, which markedly restored their ability to collaborate with allogeneic Th cells and produce Ab. These findings indicate that Ag-specific B cells require two signals for mounting a T cell-dependent Ab response and identify regulation of sIg signaling as a mechanism for controlling self-reactive B cells.
- Published
- 1994
17. N-Myc Drives Neuroendocrine Prostate Cancer Initiated from Human Prostate Epithelial Cells
- Author
-
Lee, JK, Phillips, JW, Smith, BA, Park, JW, Stoyanova, T, McCaffrey, EF, Baertsch, R, Sokolov, A, Meyerowitz, JG, Mathis, C, Cheng, D, Stuart, JM, Shokat, KM, Gustafson, WC, Huang, J, and Witte, ON
- Subjects
Male ,Aging ,Cell Transformation ,Mice ,2.1 Biological and endogenous factors ,Exome ,Molecular Targeted Therapy ,Neoplasm Metastasis ,Aetiology ,Aurora Kinase A ,Cancer ,Tumor ,Prostate Cancer ,Azepines ,myc ,Neoplasm Proteins ,Neuroendocrine Tumors ,5.1 Pharmaceuticals ,Neoplastic Stem Cells ,Development of treatments and therapeutic interventions ,Biotechnology ,Urologic Diseases ,Recombinant Fusion Proteins ,Oncology and Carcinogenesis ,Antineoplastic Agents ,Laser Capture Microdissection ,Adenocarcinoma ,SCID ,Cell Line ,Proto-Oncogene Proteins c-myc ,Transduction ,Genetic ,Animals ,Humans ,Neoplasm Invasiveness ,Oncology & Carcinogenesis ,Protein Kinase Inhibitors ,Neoplastic ,Phenylurea Compounds ,Neurosciences ,Prostatic Neoplasms ,Epithelial Cells ,Xenograft Model Antitumor Assays ,Enzyme Activation ,Pyrimidines ,Gene Expression Regulation ,Genes ,Inbred NOD ,Orchiectomy ,Proto-Oncogene Proteins c-akt - Abstract
© 2016 Elsevier Inc. MYCN amplification and overexpression are common in neuroendocrine prostate cancer (NEPC). However, the impact of aberrant N-Myc expression in prostate tumorigenesis and the cellular origin of NEPC have not been established. We define N-Myc and activated AKT1 as oncogenic components sufficient to transform human prostate epithelial cells to prostate adenocarcinoma and NEPC with phenotypic and molecular features of aggressive, late-stage human disease. We directly show that prostate adenocarcinoma and NEPC can arise from a common epithelial clone. Further, N-Myc is required for tumor maintenance, and destabilization of N-Myc through Aurora A kinase inhibition reduces tumor burden. Our findings establish N-Myc as a driver of NEPC and a target for therapeutic intervention.
- Published
- 2016
18. Integrin α9β1in airway smooth muscle suppresses exaggerated airway narrowing
- Author
-
Chen, C, Kudo, M, Rutaganira, F, Takano, H, Lee, C, Atakilit, A, Robinett, KS, Uede, T, Wolters, PJ, Shokat, KM, Huang, X, and Sheppard, D
- Subjects
respiratory system ,respiratory tract diseases - Abstract
Exaggerated contraction of airway smooth muscle is the major cause of symptoms in asthma, but the mechanisms that prevent exaggerated contraction are incompletely understood. Here, we showed that integrin α9β1on airway smooth muscle localizes the polyamine catabolizing enzyme spermidine/spermine N1-acetyltransferase (SSAT) in close proximity to the lipid kinase PIP5K1γ. As PIP5K1γ is the major source of PIP2 in airway smooth muscle and its activity is regulated by higher-order polyamines, this interaction inhibited IP3-dependent airway smooth muscle contraction. Mice lacking integrin α9β1in smooth muscle had increased airway responsiveness in vivo, and loss or inhibition of integrin α9β1increased in vitro airway narrowing and airway smooth muscle contraction in murine and human airways. Contraction was enhanced in control airways by the higher-order polyamine spermine or by cell-permeable PIP2, but these interventions had no effect on airways lacking integrin α9β1or treated with integrin α9β1-blocking antibodies. Enhancement of SSAT activity or knockdown of PIP5K1γ inhibited airway contraction, but only in the presence of functional integrin α9β1. Therefore, integrin α9β1appears to serve as a brake on airway smooth muscle contraction by recruiting SSAT, which facilitates local catabolism of polyamines and thereby inhibits PIP5K1γ. Targeting key components of this pathway could thus lead to new treatment strategies for asthma.
- Published
- 2012
19. IL-8 Inhibits cAMP-Stimulated Alveolar Epithelial Fluid Transport Via a GRK2/PI3K-Dependent Mechanism.
- Author
-
Roux, J, primary, Carles, M, additional, Koh, H, additional, Goolaerts, A, additional, Howard, M, additional, Houseman, BT, additional, Shokat, KM, additional, Paquet, AC, additional, Matthay, MA, additional, and Pittet, JF, additional
- Published
- 2009
- Full Text
- View/download PDF
20. Role of the Different Isoforms of PI3K in Modulating the Regulation of β2AR Signaling in Alveolar Epithelial Cells: AnIn SilicoApproach to Acute Lung Injury.
- Author
-
Roux, J, primary, Arkin, AP, additional, Shokat, KM, additional, and Pittet, JF, additional
- Published
- 2009
- Full Text
- View/download PDF
21. Entry of B cell receptor into signaling domains is inhibited in tolerant B cells.
- Author
-
Weintraub, BC, Weintraub, BC, Jun, JE, Bishop, AC, Shokat, KM, Thomas, ML, Goodnow, CC, Weintraub, BC, Weintraub, BC, Jun, JE, Bishop, AC, Shokat, KM, Thomas, ML, and Goodnow, CC
- Abstract
Signal transduction through the B cell antigen receptor (BCR) is altered in B cells that express a receptor that recognizes self-antigen. To understand the molecular basis for the change in signaling in autoreactive B cells, a transgenic model was used to isolate a homogeneous population of tolerant B lymphocytes. These cells were compared with a similar population of naive B lymphocytes. We show that the BCR from naive B cells enters a detergent-insoluble domain of the cell within 6 s after antigen binding, before a detectable increase in BCR phosphorylation. This fraction appears to be important for signaling because it is enriched for lyn kinase but lacks CD45 tyrosine phosphatase and because the BCR that moves into this domain becomes more highly phosphorylated. Partitioning of the BCR into this fraction is unaffected by src family kinase inhibition. Tolerant B cells do not efficiently partition the BCR into the detergent-insoluble domain, providing an explanation for their reduced tyrosine kinase activation and calcium flux in response to antigen. These results identify an early, regulated step in antigen receptor signaling and self-tolerance.
- Published
- 2000
22. Integrin α9β1 in airway smooth muscle suppresses exaggerated airway narrowing.
- Author
-
Chen C, Kudo M, Rutaganira F, Takano H, Lee C, Atakilit A, Robinett KS, Uede T, Wolters PJ, Shokat KM, Huang X, Sheppard D, Chen, Chun, Kudo, Makoto, Rutaganira, Florentine, Takano, Hiromi, Lee, Candace, Atakilit, Amha, Robinett, Kathryn S, and Uede, Toshimitsu
- Abstract
Exaggerated contraction of airway smooth muscle is the major cause of symptoms in asthma, but the mechanisms that prevent exaggerated contraction are incompletely understood. Here, we showed that integrin α9β1 on airway smooth muscle localizes the polyamine catabolizing enzyme spermidine/spermine N1-acetyltransferase (SSAT) in close proximity to the lipid kinase PIP5K1γ. As PIP5K1γ is the major source of PIP2 in airway smooth muscle and its activity is regulated by higher-order polyamines, this interaction inhibited IP3-dependent airway smooth muscle contraction. Mice lacking integrin α9β1 in smooth muscle had increased airway responsiveness in vivo, and loss or inhibition of integrin α9β1 increased in vitro airway narrowing and airway smooth muscle contraction in murine and human airways. Contraction was enhanced in control airways by the higher-order polyamine spermine or by cell-permeable PIP2, but these interventions had no effect on airways lacking integrin α9β1 or treated with integrin α9β1-blocking antibodies. Enhancement of SSAT activity or knockdown of PIP5K1γ inhibited airway contraction, but only in the presence of functional integrin α9β1. Therefore, integrin α9β1 appears to serve as a brake on airway smooth muscle contraction by recruiting SSAT, which facilitates local catabolism of polyamines and thereby inhibits PIP5K1γ. Targeting key components of this pathway could thus lead to new treatment strategies for asthma. [ABSTRACT FROM AUTHOR]
- Published
- 2012
- Full Text
- View/download PDF
23. Subcellular activation of β-adrenergic receptors using a spatially restricted antagonist.
- Author
-
Liccardo F, Morstein J, Lin TY, Pampel J, Lang D, Shokat KM, and Irannejad R
- Subjects
- Humans, Animals, Signal Transduction drug effects, Myocytes, Cardiac metabolism, Myocytes, Cardiac drug effects, HEK293 Cells, Adrenergic beta-Antagonists pharmacology, Receptors, Adrenergic, beta metabolism, Calcium metabolism, Golgi Apparatus metabolism, Golgi Apparatus drug effects, Rats, Cell Membrane metabolism, Cell Membrane drug effects, Receptors, Adrenergic, beta-1 metabolism
- Abstract
Gprotein-coupled receptors (GPCRs) regulate several physiological and pathological processes and represent the target of approximately 30% of Food and Drug Administration-approved drugs. GPCR-mediated signaling was thought to occur exclusively at the plasma membrane. However, recent studies have unveiled their presence and function at subcellular membrane compartments. There is a growing interest in studying compartmentalized signaling of GPCRs. This requires development of tools to separate GPCR signaling at the plasma membrane from the ones initiated at intracellular compartments. We leveraged the structural and pharmacological information available for β-adrenergic receptors (βARs) and focused on β1AR as exemplary GPCR that functions at subcellular compartments, and rationally designed spatially restricted antagonists. We generated a cell-impermeable βAR antagonist by conjugating a suitable pharmacophore to a sulfonate-containing fluorophore. This cell-impermeable antagonist only inhibited β1AR on the plasma membrane. In contrast, a cell-permeable βAR antagonist containing a nonsulfonated fluorophore efficiently inhibited both the plasma membrane and Golgi pools of β1ARs. Furthermore, the cell-impermeable antagonist selectively inhibited the phosphorylation of PKA downstream effectors near the plasma membrane, which regulate sarcoplasmic reticulum (SR) Ca
2+ release in adult cardiomyocytes, while the β1AR Golgi pool remained active. Our tools offer promising avenues for investigating compartmentalized βAR signaling in various contexts, potentially advancing our understanding of βAR-mediated cellular responses in health and disease. They also offer a general strategy to study compartmentalized signaling for other GPCRs in various biological systems., Competing Interests: Competing interests statement:K.M.S. has consulting agreements for the following companies, which involve monetary and/or stock compensation: Revolution Medicines, Black Diamond Therapeutics, BridGene Biosciences, Denali Therapeutics, Dice Molecules, eFFECTOR Therapeutics, Erasca, Genentech/Roche, Janssen Pharmaceuticals, Kumquat Biosciences, Kura Oncology, Mitokinin, Nested, Type6 Therapeutics, Venthera, Wellspring Biosciences (Araxes Pharma), Turning Point, Ikena, Initial Therapeutics, Vevo and BioTheryX.- Published
- 2024
- Full Text
- View/download PDF
24. Direct RAS inhibitors turn 10.
- Author
-
Ostrem JML, Peters U, and Shokat KM
- Subjects
- Humans, Enzyme Inhibitors pharmacology, Enzyme Inhibitors chemistry, Antineoplastic Agents pharmacology, Antineoplastic Agents chemistry, ras Proteins antagonists & inhibitors, ras Proteins metabolism
- Published
- 2024
- Full Text
- View/download PDF
25. Targeting Ras-, Rho-, and Rab-family GTPases via a conserved cryptic pocket.
- Author
-
Morstein J, Bowcut V, Fernando M, Yang Y, Zhu L, Jenkins ML, Evans JT, Guiley KZ, Peacock DM, Krahnke S, Lin Z, Taran KA, Huang BJ, Stephen AG, Burke JE, Lightstone FC, and Shokat KM
- Abstract
The family of Ras-like GTPases consists of over 150 different members, regulated by an even larger number of guanine exchange factors (GEFs) and GTPase-activating proteins (GAPs) that comprise cellular switch networks that govern cell motility, growth, polarity, protein trafficking, and gene expression. Efforts to develop selective small molecule probes and drugs for these proteins have been hampered by the high affinity of guanosine triphosphate (GTP) and lack of allosteric regulatory sites. This paradigm was recently challenged by the discovery of a cryptic allosteric pocket in the switch II region of K-Ras. Here, we ask whether similar pockets are present in GTPases beyond K-Ras. We systematically surveyed members of the Ras, Rho, and Rab family of GTPases and found that many GTPases exhibit targetable switch II pockets. Notable differences in the composition and conservation of key residues offer potential for the development of optimized inhibitors for many members of this previously undruggable family., Competing Interests: Declaration of interests K.M.S., J.M., and L.Z. are inventors on patents owned by University of California, San Francisco, covering GTPase-targeting small molecules. K.M.S. has consulting agreements for the following companies, which involve monetary and/or stock compensation: AperTOR, BioTheryX, BridGene Biosciences, Erasca, Exai, G Protein Therapeutics, Genentech, Initial Therapeutics, Kumquat Biosciences, Kura Oncology, Lyterian, Merck, Montara Therapeutics, Nested, Nextech, Revolution Medicines, Rezo, Totus, Type6 Therapeutics, Vevo, Vicinitas, and Wellspring Biosciences (Araxes Pharma). J.E.B. has consulting agreements for the following companies, which involve monetary and/or stock compensation: Reactive Biosciences, Scorpion Therapeutics, and Olema Oncology., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2024
- Full Text
- View/download PDF
26. Strain-release alkylation of Asp12 enables mutant selective targeting of K-Ras-G12D.
- Author
-
Zheng Q, Zhang Z, Guiley KZ, and Shokat KM
- Subjects
- Humans, Animals, Alkylation, Mice, Cell Line, Tumor, Antineoplastic Agents pharmacology, Antineoplastic Agents chemistry, Antineoplastic Agents chemical synthesis, Crystallography, X-Ray, Models, Molecular, Aspartic Acid chemistry, Mutation, Proto-Oncogene Proteins p21(ras) genetics, Proto-Oncogene Proteins p21(ras) antagonists & inhibitors, Proto-Oncogene Proteins p21(ras) metabolism, Proto-Oncogene Proteins p21(ras) chemistry, Cell Proliferation drug effects
- Abstract
K-Ras is the most commonly mutated oncogene in human cancer. The recently approved non-small cell lung cancer drugs sotorasib and adagrasib covalently capture an acquired cysteine in K-Ras-G12C mutation and lock it in a signaling-incompetent state. However, covalent inhibition of G12D, the most frequent K-Ras mutation particularly prevalent in pancreatic ductal adenocarcinoma, has remained elusive due to the lack of aspartate-targeting chemistry. Here we present a set of malolactone-based electrophiles that exploit ring strain to crosslink K-Ras-G12D at the mutant aspartate to form stable covalent complexes. Structural insights from X-ray crystallography and exploitation of the stereoelectronic requirements for attack of the electrophile allowed development of a substituted malolactone that resisted attack by aqueous buffer but rapidly crosslinked with the aspartate-12 of K-Ras in both GDP and GTP state. The GTP-state targeting allowed effective suppression of downstream signaling, and selective inhibition of K-Ras-G12D-driven cancer cell proliferation in vitro and xenograft growth in mice., (© 2024. The Author(s).)
- Published
- 2024
- Full Text
- View/download PDF
27. Absence of ABL1 exon 2-encoded SH3 residues in BCR::ABL1 destabilizes the autoinhibited kinase conformation and confers resistance to asciminib.
- Author
-
Leyte-Vidal A, DeFilippis R, Outhwaite IR, Kwan I, Lee JY, Leavitt C, Miller KB, Rea D, Rangwala AM, Lou K, Patel S, Alvarez A, Shokat KM, Bahar I, Seeliger MA, and Shah NP
- Subjects
- Humans, Protein Kinase Inhibitors pharmacology, Protein Kinase Inhibitors therapeutic use, Leukemia, Myelogenous, Chronic, BCR-ABL Positive drug therapy, Leukemia, Myelogenous, Chronic, BCR-ABL Positive genetics, Leukemia, Myelogenous, Chronic, BCR-ABL Positive pathology, src Homology Domains, Proto-Oncogene Proteins c-abl genetics, Protein Conformation, Pyrazoles pharmacology, Pyrazoles therapeutic use, Niacinamide analogs & derivatives, Fusion Proteins, bcr-abl genetics, Drug Resistance, Neoplasm genetics, Exons genetics
- Published
- 2024
- Full Text
- View/download PDF
28. Nanomolar Protein Thermal Profiling with Modified Cyanine Dyes.
- Author
-
Malakoutikhah M, Mahran R, Gooran N, Masoumi A, Lundell K, Liljeblad A, Guiley K, Dai S, Zheng Q, Zhu L, Shokat KM, Kopra K, and Härmä H
- Subjects
- Carbocyanines chemistry, Luminescence, Fluorescent Dyes chemistry, Coloring Agents, Peptides chemistry
- Abstract
Protein properties and interactions have been widely investigated by using external labels. However, the micromolar sensitivity of the current dyes limits their applicability due to the high material consumption and assay cost. In response to this challenge, we synthesized a series of cyanine5 (Cy5) dye-based quencher molecules to develop an external dye technique to probe proteins at the nanomolar protein level in a high-throughput one-step assay format. Several families of Cy5 dye-based quenchers with ring and/or side-chain modifications were designed and synthesized by introducing organic small molecules or peptides. Our results showed that steric hindrance and electrostatic interactions are more important than hydrophobicity in the interaction between the luminescent negatively charged europium-chelate-labeled peptide (Eu-probe) and the quencher molecules. The presence of substituents on the quencher indolenine rings reduces their quenching property, whereas the increased positive charge on the indolenine side chain improved the interaction between the quenchers and the luminescent compound. The designed quencher structures entirely altered the dynamics of the Eu-probe (protein-probe) for studying protein stability and interactions, as we were able to reduce the quencher concentration 100-fold. Moreover, the new quencher molecules allowed us to conduct the experiments using neutral buffer conditions, known as the peptide-probe assay. These improvements enabled us to apply the method in a one-step format for nanomolar protein-ligand interaction and protein profiling studies instead of the previously developed two-step protocol. These improvements provide a faster and simpler method with lower material consumption.
- Published
- 2023
- Full Text
- View/download PDF
29. Protocol for performing and optimizing differential scanning fluorimetry experiments.
- Author
-
Wu T, Hornsby M, Zhu L, Yu JC, Shokat KM, and Gestwicki JE
- Subjects
- Calorimetry, Differential Scanning, Temperature, Fluorometry methods, Proteins, Amines
- Abstract
Differential scanning fluorimetry (DSF) is a widely used technique for determining the apparent melting temperature (Tma) of a purified protein. Here, we present a protocol for performing and optimizing DSF experiments. We describe steps for designing and performing the experiment, analyzing data, and optimization. We provide benchmarks for typical Tmas and ΔTmas, standard assay conditions, and upper and lower limits of commonly altered experimental variables. We also detail common pitfalls of DSF and ways to avoid, identify, and overcome them., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2023
- Full Text
- View/download PDF
30. Selective activation of intracellular β1AR using a spatially restricted antagonist.
- Author
-
Liccardo F, Morstein J, Lin TY, Pampel J, Shokat KM, and Irannejad R
- Abstract
G-protein-coupled receptors (GPCRs) regulate several physiological and pathological processes and represent the target of approximately 30% of FDA-approved drugs. GPCR-mediated signaling was thought to occur exclusively at the plasma membrane. However, recent studies have unveiled their presence and function at subcellular membrane compartments. There is a growing interest in studying compartmentalized signaling of GPCRs. This requires development of novel tools to separate GPCRs signaling at the plasma membrane from the ones initiated at intracellular compartments. We took advantage of the structural and pharmacological information available for β1-adrenergic receptor (β1AR), an exemplary GPCR that functions at subcellular compartments, and rationally designed spatially restricted antagonists. We generated a cell impermeable β1AR antagonist by conjugating a suitable pharmacophore to a sulfonate-containing fluorophore. This cell-impermeable antagonist only inhibited β1AR on the plasma membrane. In contrast, a cell permeable β1AR agonist containing a non-sulfonated fluorophore, efficiently inhibited both the plasma membrane and Golgi pools of β1ARs. Furthermore, the cell impermeable antagonist selectively inhibited the phosphorylation of downstream effectors of PKA proximal to the plasma membrane in adult cardiomyocytes while β1AR intracellular pool remained active. Our tools offer promising avenues for investigating compartmentalized β1AR signaling in various context, potentially advancing our understanding of β1AR-mediated cellular responses in health and disease. They also offer a general strategy to study compartmentalized signaling for other GPCRs in various biological systems., Competing Interests: Competing interests K.M.S. has consulting agreements for the following companies, which involve monetary and/or stock compensation: Revolution Medicines, Black Diamond Therapeutics, BridGene Biosciences, Denali Therapeutics, Dice Molecules, eFFECTOR Therapeutics, Erasca, Genentech/Roche, Janssen Pharmaceuticals, Kumquat Biosciences, Kura Oncology, Mitokinin, Nested, Type6 Therapeutics, Venthera, Wellspring Biosciences (Araxes Pharma), Turning Point, Ikena, Initial Therapeutics, Vevo and BioTheryX.
- Published
- 2023
- Full Text
- View/download PDF
31. Proteomic and genetic analyses of influenza A viruses identify pan-viral host targets.
- Author
-
Haas KM, McGregor MJ, Bouhaddou M, Polacco BJ, Kim EY, Nguyen TT, Newton BW, Urbanowski M, Kim H, Williams MAP, Rezelj VV, Hardy A, Fossati A, Stevenson EJ, Sukerman E, Kim T, Penugonda S, Moreno E, Braberg H, Zhou Y, Metreveli G, Harjai B, Tummino TA, Melnyk JE, Soucheray M, Batra J, Pache L, Martin-Sancho L, Carlson-Stevermer J, Jureka AS, Basler CF, Shokat KM, Shoichet BK, Shriver LP, Johnson JR, Shaw ML, Chanda SK, Roden DM, Carter TC, Kottyan LC, Chisholm RL, Pacheco JA, Smith ME, Schrodi SJ, Albrecht RA, Vignuzzi M, Zuliani-Alvarez L, Swaney DL, Eckhardt M, Wolinsky SM, White KM, Hultquist JF, Kaake RM, García-Sastre A, and Krogan NJ
- Subjects
- Humans, Influenza A Virus, H3N2 Subtype metabolism, Proteomics, Virus Replication genetics, SARS-CoV-2, Antiviral Agents metabolism, Host-Pathogen Interactions genetics, Influenza A virus genetics, Influenza, Human genetics, Influenza A Virus, H5N1 Subtype genetics, COVID-19
- Abstract
Influenza A Virus (IAV) is a recurring respiratory virus with limited availability of antiviral therapies. Understanding host proteins essential for IAV infection can identify targets for alternative host-directed therapies (HDTs). Using affinity purification-mass spectrometry and global phosphoproteomic and protein abundance analyses using three IAV strains (pH1N1, H3N2, H5N1) in three human cell types (A549, NHBE, THP-1), we map 332 IAV-human protein-protein interactions and identify 13 IAV-modulated kinases. Whole exome sequencing of patients who experienced severe influenza reveals several genes, including scaffold protein AHNAK, with predicted loss-of-function variants that are also identified in our proteomic analyses. Of our identified host factors, 54 significantly alter IAV infection upon siRNA knockdown, and two factors, AHNAK and coatomer subunit COPB1, are also essential for productive infection by SARS-CoV-2. Finally, 16 compounds targeting our identified host factors suppress IAV replication, with two targeting CDK2 and FLT3 showing pan-antiviral activity across influenza and coronavirus families. This study provides a comprehensive network model of IAV infection in human cells, identifying functional host targets for pan-viral HDT., (© 2023. Springer Nature Limited.)
- Published
- 2023
- Full Text
- View/download PDF
32. Direct Modulators of K-Ras-Membrane Interactions.
- Author
-
Morstein J, Shrestha R, Van QN, López CA, Arora N, Tonelli M, Liang H, Chen, Zhou Y, Hancock JF, Stephen AG, Turbyville TJ, and Shokat KM
- Subjects
- Cell Membrane metabolism, Molecular Dynamics Simulation, Lipids, Proto-Oncogene Proteins p21(ras) genetics, ras Proteins metabolism, Signal Transduction
- Abstract
Protein-membrane interactions (PMIs) are ubiquitous in cellular signaling. Initial steps of signal transduction cascades often rely on transient and dynamic interactions with the inner plasma membrane leaflet to populate and regulate signaling hotspots. Methods to target and modulate these interactions could yield attractive tool compounds and drug candidates. Here, we demonstrate that the conjugation of a medium-chain lipid tail to the covalent K-Ras(G12C) binder MRTX849 at a solvent-exposed site enables such direct modulation of PMIs. The conjugated lipid tail interacts with the tethered membrane and changes the relative membrane orientation and conformation of K-Ras(G12C), as shown by molecular dynamics (MD) simulation-supported NMR studies. In cells, this PMI modulation restricts the lateral mobility of K-Ras(G12C) and disrupts nanoclusters. The described strategy could be broadly applicable to selectively modulate transient PMIs.
- Published
- 2023
- Full Text
- View/download PDF
33. mTOR inhibition reprograms cellular proteostasis by regulating eIF3D-mediated selective mRNA translation and promotes cell phenotype switching.
- Author
-
Shin S, Han MJ, Jedrychowski MP, Zhang Z, Shokat KM, Plas DR, Dephoure N, and Yoon SO
- Subjects
- RNA, Messenger metabolism, TOR Serine-Threonine Kinases metabolism, Sirolimus, Protein Biosynthesis, Proteostasis, Receptor, Insulin metabolism
- Abstract
Cells maintain and dynamically change their proteomes according to the environment and their needs. Mechanistic target of rapamycin (mTOR) is a key regulator of proteostasis, homeostasis of the proteome. Thus, dysregulation of mTOR leads to changes in proteostasis and the consequent progression of diseases, including cancer. Based on the physiological and clinical importance of mTOR signaling, we investigated mTOR feedback signaling, proteostasis, and cell fate. Here, we reveal that mTOR targeting inhibits eIF4E-mediated cap-dependent translation, but feedback signaling activates a translation initiation factor, eukaryotic translation initiation factor 3D (eIF3D), to sustain alternative non-canonical translation mechanisms. Importantly, eIF3D-mediated protein synthesis enables cell phenotype switching from proliferative to more migratory. eIF3D cooperates with mRNA-binding proteins such as heterogeneous nuclear ribonucleoprotein F (hnRNPF), heterogeneous nuclear ribonucleoprotein K (hnRNPK), and Sjogren syndrome antigen B (SSB) to support selective mRNA translation following mTOR inhibition, which upregulates and activates proteins involved in insulin receptor (INSR)/insulin-like growth factor 1 receptor (IGF1R)/insulin receptor substrate (IRS) and interleukin 6 signal transducer (IL-6ST)/Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling. Our study highlights the mechanisms by which cells establish the dynamic change of proteostasis and the resulting phenotype switch., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2023
- Full Text
- View/download PDF
34. Evolutionarily divergent mTOR remodels translatome for tissue regeneration.
- Author
-
Zhulyn O, Rosenblatt HD, Shokat L, Dai S, Kuzuoglu-Öztürk D, Zhang Z, Ruggero D, Shokat KM, and Barna M
- Subjects
- Animals, Humans, Mice, Amino Acid Sequence, Extremities physiology, RNA, Messenger genetics, RNA, Messenger metabolism, Wound Healing, Mechanistic Target of Rapamycin Complex 1 metabolism, Species Specificity, Antioxidants metabolism, Nutrients metabolism, Polyribosomes genetics, Polyribosomes metabolism, Ambystoma mexicanum physiology, Regeneration physiology, TOR Serine-Threonine Kinases metabolism, Biological Evolution, Protein Biosynthesis
- Abstract
An outstanding mystery in biology is why some species, such as the axolotl, can regenerate tissues whereas mammals cannot
1 . Here, we demonstrate that rapid activation of protein synthesis is a unique feature of the injury response critical for limb regeneration in the axolotl (Ambystoma mexicanum). By applying polysome sequencing, we identify hundreds of transcripts, including antioxidants and ribosome components that are selectively activated at the level of translation from pre-existing messenger RNAs in response to injury. By contrast, protein synthesis is not activated in response to non-regenerative digit amputation in the mouse. We identify the mTORC1 pathway as a key upstream signal that mediates tissue regeneration and translational control in the axolotl. We discover unique expansions in mTOR protein sequence among urodele amphibians. By engineering an axolotl mTOR (axmTOR) in human cells, we show that these changes create a hypersensitive kinase that allows axolotls to maintain this pathway in a highly labile state primed for rapid activation. This change renders axolotl mTOR more sensitive to nutrient sensing, and inhibition of amino acid transport is sufficient to inhibit tissue regeneration. Together, these findings highlight the unanticipated impact of the translatome on orchestrating the early steps of wound healing in a highly regenerative species and provide a missing link in our understanding of vertebrate regenerative potential., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)- Published
- 2023
- Full Text
- View/download PDF
35. Chemical Genetic Identification of PKC Epsilon Substrates in Mouse Brain.
- Author
-
Dugan MP, Ferguson LB, Hertz NT, Chalkley RJ, Burlingame AL, Shokat KM, Parker PJ, and Messing RO
- Subjects
- Mice, Animals, Ethanol, Alcohol Drinking genetics, Brain metabolism, Protein Kinase C-epsilon genetics, Protein Kinase C-epsilon metabolism, Signal Transduction
- Abstract
PKC epsilon (PKCε) plays important roles in behavioral responses to alcohol and in anxiety-like behavior in rodents, making it a potential drug target for reducing alcohol consumption and anxiety. Identifying signals downstream of PKCε could reveal additional targets and strategies for interfering with PKCε signaling. We used a chemical genetic screen combined with mass spectrometry to identify direct substrates of PKCε in mouse brain and validated findings for 39 of them using peptide arrays and in vitro kinase assays. Prioritizing substrates with several public databases such as LINCS-L1000, STRING, GeneFriends, and GeneMAINA predicted interactions between these putative substrates and PKCε and identified substrates associated with alcohol-related behaviors, actions of benzodiazepines, and chronic stress. The 39 substrates could be broadly classified in three functional categories: cytoskeletal regulation, morphogenesis, and synaptic function. These results provide a list of brain PKCε substrates, many of which are novel, for future investigation to determine the role of PKCε signaling in alcohol responses, anxiety, responses to stress, and other related behaviors., Competing Interests: Conflicts of Interest The authors with the exception of K. M. S. and N. T. H declare that there are no competing interests associated with the manuscript. K. M. S. is an inventor on patents covering the analog sensitive and analog specific kinase engineering owned by Princeton University. K. M. S. has consulting agreements for the following companies, which involve monetary and/or stock compensation: Revolution Medicines, Black Diamond Therapeutics, BridGene Biosciences, Denali Therapeutics, Dice Molecules, eFFECTOR Therapeutics, Erasca, Genentech/Roche, Janssen Pharmaceuticals, Kumquat Biosciences, Kura Oncology, Mitokinin Inc, Nested, Type6 Therapeutics, Venthera, Wellspring Biosciences (Araxes Pharma), Turning Point, Ikena, Initial Therapeutics, Vevo, and BioTheryX. N. T. H owns shares and is an employee of Mitokinin Inc., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2023
- Full Text
- View/download PDF
36. A Small Molecule Reacts with the p53 Somatic Mutant Y220C to Rescue Wild-type Thermal Stability.
- Author
-
Guiley KZ and Shokat KM
- Subjects
- Humans, Cysteine genetics, Mutation, Tumor Suppressor Protein p53 genetics, Tumor Suppressor Protein p53 metabolism, Neoplasms drug therapy, Neoplasms genetics, Neoplasms metabolism
- Abstract
The transcription factor and tumor suppressor protein p53 is the most frequently mutated and inactivated gene in cancer. Mutations in p53 result in deregulated cell proliferation and genomic instability, both hallmarks of cancer. There are currently no therapies available that directly target mutant p53 to rescue wild-type function. In this study, we identify covalent compsounds that selectively react with the p53 somatic mutant cysteine Y220C and restore wild-type thermal stability., Significance: The tumor suppressor p53 is the most mutated gene in cancer, and yet no therapeutics to date directly target the mutated protein to rescue wild-type function. In this study, we identify the first allele-specific compound that selectively reacts with the cysteine p53 Y220C to rescue wild-type thermal stability and gene activation. See related commentary by Lane and Verma, p. 14. This article is highlighted in the In This Issue feature, p. 1., (©2022 The Authors; Published by the American Association for Cancer Research.)
- Published
- 2023
- Full Text
- View/download PDF
37. IFITM proteins assist cellular uptake of diverse linked chemotypes.
- Author
-
Lou K, Wassarman DR, Yang T, Paung Y, Zhang Z, O'Loughlin TA, Moore MK, Egan RK, Greninger P, Benes CH, Seeliger MA, Taunton J, Gilbert LA, and Shokat KM
- Abstract
The search for cell-permeable drugs has conventionally focused on low-molecular weight (MW), nonpolar, rigid chemical structures. However, emerging therapeutic strategies break traditional drug design rules by employing flexibly linked chemical entities composed of more than one ligand. Using complementary genome-scale chemical-genetic approaches we identified an endogenous chemical uptake pathway involving interferon-induced transmembrane proteins (IFITMs) that modulates the cell permeability of a prototypical biopic inhibitor of MTOR (RapaLink-1, MW: 1784 g/mol). We devised additional linked inhibitors targeting BCR-ABL1 (DasatiLink-1, MW: 1518 g/mol) and EIF4A1 (BisRoc-1, MW: 1466 g/mol), uptake of which was facilitated by IFITMs. We also found that IFITMs moderately assisted some proteolysis-targeting chimeras and examined the physicochemical requirements for involvement of this uptake pathway.
- Published
- 2022
- Full Text
- View/download PDF
38. Chemical acylation of an acquired serine suppresses oncogenic signaling of K-Ras(G12S).
- Author
-
Zhang Z, Guiley KZ, and Shokat KM
- Subjects
- Humans, Proto-Oncogene Proteins p21(ras) genetics, Proto-Oncogene Proteins p21(ras) metabolism, Mutation, Oncogenes, Acylation, Cysteine metabolism, Serine metabolism
- Abstract
Drugs that directly impede the function of driver oncogenes offer exceptional efficacy and a therapeutic window. The recently approved mutant selective small-molecule cysteine-reactive covalent inhibitor of the G12C mutant of K-Ras, sotorasib, provides a case in point. KRAS is the most frequently mutated proto-oncogene in human cancer, yet despite success targeting the G12C allele, targeted therapy for other hotspot mutants of KRAS has not been described. Here we report the discovery of small molecules that covalently target a G12S somatic mutation in K-Ras and suppress its oncogenic signaling. We show that these molecules are active in cells expressing K-Ras(G12S) but spare the wild-type protein. Our results provide a path to targeting a second somatic mutation in the oncogene KRAS by overcoming the weak nucleophilicity of an acquired serine residue. The chemistry we describe may serve as a basis for the selective targeting of other unactivated serines., (© 2022. The Author(s).)
- Published
- 2022
- Full Text
- View/download PDF
39. Probing the KRas Switch II Groove by Fluorine NMR Spectroscopy.
- Author
-
Peacock DM, Kelly MJS, and Shokat KM
- Subjects
- Ligands, Magnetic Resonance Spectroscopy, Nucleotides, Proto-Oncogene Proteins p21(ras) genetics, Mutation, Fluorine, Genes, ras
- Abstract
While there has been recent success in the development of KRas
G12C inhibitors, unmet needs for selective inhibitors of KRasG12D and the remaining oncogenic KRas proteins remain. Here, we applied trifluoromethyl-containing ligands of KRas proteins as competitive probe ligands to assay the occupancy of the switch II pocket by19 F NMR spectroscopy. Structure-activity-relationship studies of probe ligands increased the sensitivity of the assay and identified structures that differentially detected each nucleotide state of KRasG12D . These differences in selectivity, combined with the high resolution of19 F NMR spectroscopy, enabled this method to be expanded to assay both nucleotide states of the protein simultaneously.- Published
- 2022
- Full Text
- View/download PDF
40. State-selective modulation of heterotrimeric Gαs signaling with macrocyclic peptides.
- Author
-
Dai SA, Hu Q, Gao R, Blythe EE, Touhara KK, Peacock H, Zhang Z, von Zastrow M, Suga H, and Shokat KM
- Subjects
- GTP Phosphohydrolases, Guanine Nucleotides, Nucleotides, Peptides, Cyclic pharmacology, Peptides chemistry, Receptors, G-Protein-Coupled
- Abstract
The G protein-coupled receptor cascade leading to production of the second messenger cAMP is replete with pharmacologically targetable proteins, with the exception of the Gα subunit, Gαs. GTPases remain largely undruggable given the difficulty of displacing high-affinity guanine nucleotides and the lack of other drug binding sites. We explored a chemical library of 10
12 cyclic peptides to expand the chemical search for inhibitors of this enzyme class. We identified two macrocyclic peptides, GN13 and GD20, that antagonize the active and inactive states of Gαs, respectively. Both macrocyclic peptides fine-tune Gαs activity with high nucleotide-binding-state selectivity and G protein class-specificity. Co-crystal structures reveal that GN13 and GD20 distinguish the conformational differences within the switch II/α3 pocket. Cell-permeable analogs of GN13 and GD20 modulate Gαs/Gβγ signaling in cells through binding to crystallographically defined pockets. The discovery of cyclic peptide inhibitors targeting Gαs provides a path for further development of state-dependent GTPase inhibitors., Competing Interests: Declaration of interests S.A.D., Q.H., R.W., H.P., H.S., and K.M.S. are inventors on patent applications jointly owned by University of Tokyo and UCSF. S.A.D., Q.H., R.W., H.P., H.S., and K.M.S. own shares in G-Protein Therapeutics, a subsidiary of Bridge Bio. K.M.S. has consulting agreements with the following companies, which involve monetary and/or stock compensation: Revolution Medicines, Black Diamond Therapeutics, BridGene Biosciences, Denali Therapeutics, Dice Molecules, eFFECTOR Therapeutics, Erasca, Genentech/Roche, G-Protein Therapeutics, Janssen Pharmaceuticals, Kumquat Biosciences, Kura Oncology, Mitokinin, Nested, Type6 Therapeutics, Venthera, Wellspring Biosciences (Araxes Pharma), Turning Point, Ikena, Initial Therapeutics, Vevo, Rezo, and BioTheryX., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)- Published
- 2022
- Full Text
- View/download PDF
41. Tissue-restricted inhibition of mTOR using chemical genetics.
- Author
-
Wassarman DR, Bankapalli K, Pallanck LJ, and Shokat KM
- Subjects
- Humans, Organ Specificity, Phosphorylation, Signal Transduction, Tacrolimus Binding Protein 1A genetics, Tacrolimus Binding Protein 1A metabolism, Protein Kinase Inhibitors pharmacology, Sirolimus analogs & derivatives, Sirolimus pharmacology, TOR Serine-Threonine Kinases antagonists & inhibitors, TOR Serine-Threonine Kinases genetics
- Abstract
Mammalian target of rapamycin (mTOR) is a highly conserved eukaryotic protein kinase that coordinates cell growth and metabolism, and plays a critical role in cancer, immunity, and aging. It remains unclear how mTOR signaling in individual tissues contributes to whole-organism processes because mTOR inhibitors, like the natural product rapamycin, are administered systemically and target multiple tissues simultaneously. We developed a chemical-genetic system, termed selecTOR, that restricts the activity of a rapamycin analog to specific cell populations through targeted expression of a mutant FKBP12 protein. This analog has reduced affinity for its obligate binding partner FKBP12, which reduces its ability to inhibit mTOR in wild-type cells and tissues. Expression of the mutant FKBP12, which contains an expanded binding pocket, rescues the activity of this rapamycin analog. Using this system, we show that selective mTOR inhibition can be achieved in Saccharomyces cerevisiae and human cells, and we validate the utility of our system in an intact metazoan model organism by identifying the tissues responsible for a rapamycin-induced developmental delay in Drosophila .
- Published
- 2022
- Full Text
- View/download PDF
42. Cold shock domain-containing protein E1 is a posttranscriptional regulator of the LDL receptor.
- Author
-
Smith GA, Padmanabhan A, Lau BH, Pampana A, Li L, Lee CY, Pelonero A, Nishino T, Sadagopan N, Xia VQ, Jain R, Natarajan P, Wu RS, Black BL, Srivastava D, Shokat KM, and Chorba JS
- Subjects
- Animals, Humans, Mice, RNA, Messenger genetics, Receptors, LDL genetics, Receptors, LDL metabolism, Transcription, Genetic, Cold-Shock Response, DNA-Binding Proteins metabolism, Proprotein Convertase 9 genetics, Proprotein Convertase 9 metabolism, RNA-Binding Proteins metabolism
- Abstract
The low-density lipoprotein receptor (LDLR) controls cellular delivery of cholesterol and clears LDL from the bloodstream, protecting against atherosclerotic heart disease, the leading cause of death in the United States. We therefore sought to identify regulators of the LDLR beyond the targets of current therapies and known causes of familial hypercholesterolemia. We found that cold shock domain-containing protein E1 (CSDE1) enhanced hepatic LDLR messenger RNA (mRNA) decay via its 3' untranslated region and regulated atherogenic lipoproteins in vivo. Using parallel phenotypic genome-wide CRISPR interference screens in a tissue culture model, we identified 40 specific regulators of the LDLR that were not previously identified by observational human genetic studies. Among these, we demonstrated that, in HepG2 cells, CSDE1 regulated the LDLR at least as strongly as statins and proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors. In addition, we showed that hepatic gene silencing of Csde1 treated diet-induced dyslipidemia in mice to a similar degree as Pcsk9 silencing. These results suggest the therapeutic potential of targeting CSDE1 to manipulate the posttranscriptional regulation of the LDLR mRNA for the prevention of cardiovascular disease. Our approach of modeling a clinically relevant phenotype in a forward genetic screen, followed by mechanistic pharmacologic dissection and in vivo validation, may serve as a generalizable template for the identification of therapeutic targets in other human disease states.
- Published
- 2022
- Full Text
- View/download PDF
43. A covalent inhibitor of K-Ras(G12C) induces MHC class I presentation of haptenated peptide neoepitopes targetable by immunotherapy.
- Author
-
Zhang Z, Rohweder PJ, Ongpipattanakul C, Basu K, Bohn MF, Dugan EJ, Steri V, Hann B, Shokat KM, and Craik CS
- Subjects
- Antibodies, Humans, Immunologic Factors, Immunotherapy, Peptides pharmacology, Proto-Oncogene Proteins p21(ras) genetics, Antineoplastic Agents pharmacology, Histocompatibility Antigens Class I immunology, Neoplasms, Proto-Oncogene Proteins p21(ras) antagonists & inhibitors
- Abstract
Immunotargeting of tumor-specific antigens is a powerful therapeutic strategy. Immunotherapies directed at MHC-I complexes have expanded the scope of antigens and enabled the direct targeting of intracellular oncoproteins at the cell surface. We asked whether covalent drugs that alkylate mutated residues on oncoproteins could act as haptens to generate unique MHC-I-restricted neoantigens. Here, we report that KRAS G12C mutant cells treated with the covalent inhibitor ARS1620 present ARS1620-modified peptides in MHC-I complexes. Using ARS1620-specific antibodies identified by phage display, we show that these haptenated MHC-I complexes can serve as tumor-specific neoantigens and that a bispecific T cell engager construct based on a hapten-specific antibody elicits a cytotoxic T cell response against KRAS G12C cells, including those resistant to direct KRAS G12C inhibition. With multiple K-RAS G12C inhibitors in clinical use or undergoing clinical trials, our results present a strategy to enhance their efficacy and overcome the rapidly arising tumor resistance., Competing Interests: Declaration of interests C.S.C., K.S.M., Z.Z., and P.J.R. are inventors on a provisional patent application covering this work and owned by the University of California, San Francisco (UCSF). K.M.S. is an inventor on patent applications related to this technology owned by UCSF. K.M.S. is an inventor on patents covering covalent inhibitors of K-Ras(G12C) owned by UCSF and licensed to Wellspring Biosciences. K.M.S. is a consultant to and shareholder in the following companies: Revolution Medicines, Black Diamond Therapeutics, BridGene Biosciences, Denali Therapeutics, Dice Molecules, eFFECTOR Therapeutics, Erasca, Genentech/Roche, Janssen Pharmaceuticals, Kumquat Biosciences, Kura Oncology, Mitokinin, Nested, Type6 Therapeutics, Venthera, Wellspring Biosciences (Araxes Pharma), Nextech, Radd, Totus, Vicinitas, Turning Point, Ikena, Initial Therapeutics, Vevo, and BioTheryX., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2022
- Full Text
- View/download PDF
44. Chemoselective Covalent Modification of K-Ras(G12R) with a Small Molecule Electrophile.
- Author
-
Zhang Z, Morstein J, Ecker AK, Guiley KZ, and Shokat KM
- Subjects
- Arginine, Genes, ras, Humans, Ligands, Mutation, Neoplasms, Proto-Oncogene Proteins p21(ras) genetics
- Abstract
KRAS mutations are one of the most common oncogenic drivers in human cancer. While small molecule inhibitors for the G12C mutant have been successfully developed, allele-specific inhibition for other KRAS hotspot mutants remains challenging. Here we report the discovery of covalent chemical ligands for the common oncogenic mutant K-Ras(G12R). These ligands bind in the Switch II pocket and irreversibly react with the mutant arginine residue. An X-ray crystal structure reveals an imidazolium condensation product formed between the α,β-diketoamide ligand and the ε- and η-nitrogens of arginine 12. Our results show that arginine residues can be selectively targeted with small molecule electrophiles despite their weak nucleophilicity and provide the basis for the development of mutant-specific therapies for K-Ras(G12R)-driven cancer.
- Published
- 2022
- Full Text
- View/download PDF
45. Brain-restricted mTOR inhibition with binary pharmacology.
- Author
-
Zhang Z, Fan Q, Luo X, Lou K, Weiss WA, and Shokat KM
- Subjects
- Humans, Drug Therapy, Combination, Glioblastoma drug therapy, Ligands, Tacrolimus Binding Protein 1A metabolism, Xenograft Model Antitumor Assays, Brain drug effects, Brain metabolism, MTOR Inhibitors metabolism, MTOR Inhibitors pharmacokinetics, MTOR Inhibitors pharmacology, Sirolimus analogs & derivatives, TOR Serine-Threonine Kinases antagonists & inhibitors, TOR Serine-Threonine Kinases metabolism
- Abstract
On-target-off-tissue drug engagement is an important source of adverse effects that constrains the therapeutic window of drug candidates
1,2 . In diseases of the central nervous system, drugs with brain-restricted pharmacology are highly desirable. Here we report a strategy to achieve inhibition of mammalian target of rapamycin (mTOR) while sparing mTOR activity elsewhere through the use of the brain-permeable mTOR inhibitor RapaLink-1 and the brain-impermeable FKBP12 ligand RapaBlock. We show that this drug combination mitigates the systemic effects of mTOR inhibitors but retains the efficacy of RapaLink-1 in glioblastoma xenografts. We further present a general method to design cell-permeable, FKBP12-dependent kinase inhibitors from known drug scaffolds. These inhibitors are sensitive to deactivation by RapaBlock, enabling the brain-restricted inhibition of their respective kinase targets., (© 2022. The Author(s).)- Published
- 2022
- Full Text
- View/download PDF
46. KRAS is vulnerable to reversible switch-II pocket engagement in cells.
- Author
-
Vasta JD, Peacock DM, Zheng Q, Walker JA, Zhang Z, Zimprich CA, Thomas MR, Beck MT, Binkowski BF, Corona CR, Robers MB, and Shokat KM
- Subjects
- Humans, Ligands, Mutation, Multiple Myeloma, Proto-Oncogene Proteins p21(ras) genetics
- Abstract
Current small-molecule inhibitors of KRAS(G12C) bind irreversibly in the switch-II pocket (SII-P), exploiting the strong nucleophilicity of the acquired cysteine as well as the preponderance of the GDP-bound form of this mutant. Nevertheless, many oncogenic KRAS mutants lack these two features, and it remains unknown whether targeting the SII-P is a practical therapeutic approach for KRAS mutants beyond G12C. Here we use NMR spectroscopy and a cellular KRAS engagement assay to address this question by examining a collection of SII-P ligands from the literature and from our own laboratory. We show that the SII-Ps of many KRAS hotspot (G12, G13, Q61) mutants are accessible using noncovalent ligands, and that this accessibility is not necessarily coupled to the GDP state of KRAS. The results we describe here emphasize the SII-P as a privileged drug-binding site on KRAS and unveil new therapeutic opportunities in RAS-driven cancer., (© 2022. The Author(s).)
- Published
- 2022
- Full Text
- View/download PDF
47. CD74-NRG1 Fusions Are Oncogenic In Vivo and Induce Therapeutically Tractable ERBB2:ERBB3 Heterodimerization.
- Author
-
Werr L, Plenker D, Dammert MA, Lorenz C, Brägelmann J, Tumbrink HL, Klein S, Schmitt A, Büttner R, Persigehl T, Shokat KM, Wunderlich FT, Schram AM, Peifer M, Sos ML, Reinhardt HC, and Thomas RK
- Subjects
- Animals, Carcinogenesis genetics, Humans, Mice, Neuregulin-1 genetics, Oncogenes, Receptor, ErbB-2 genetics, Receptor, ErbB-3 genetics, Adenocarcinoma of Lung, Lung Neoplasms
- Abstract
NRG1 fusions are recurrent somatic genome alterations occurring across several tumor types, including invasive mucinous lung adenocarcinomas and pancreatic ductal adenocarcinomas and are potentially actionable genetic alterations in these cancers. We initially discovered CD74-NRG1 as the first NRG1 fusion in lung adenocarcinomas, and many additional fusion partners have since been identified. Here, we present the first CD74-NRG1 transgenic mouse model and provide evidence that ubiquitous expression of the CD74-NRG1 fusion protein in vivo leads to tumor development at high frequency. Furthermore, we show that ERBB2:ERBB3 heterodimerization is a mechanistic event in transformation by CD74-NRG1 binding physically to ERBB3 and that CD74-NRG1-expressing cells proliferate independent of supplemented NRG1 ligand. Thus, NRG1 gene fusions are recurrent driver oncogenes that cause oncogene dependency. Consistent with these findings, patients with NRG1 fusion-positive cancers respond to therapy targeting the ERBB2:ERBB3 receptors., (©2022 The Authors; Published by the American Association for Cancer Research.)
- Published
- 2022
- Full Text
- View/download PDF
48. Targeting a splicing-mediated drug resistance mechanism in prostate cancer by inhibiting transcriptional regulation by PKCβ1.
- Author
-
Melnyk JE, Steri V, Nguyen HG, Hwang YC, Gordan JD, Hann B, Feng FY, and Shokat KM
- Subjects
- Androgen Antagonists pharmacology, Androgen Antagonists therapeutic use, Drug Resistance, Gene Expression Regulation, Neoplastic, Humans, Male, RNA Splicing genetics, Receptors, Androgen genetics, Receptors, Androgen metabolism, Prostatic Neoplasms drug therapy, Prostatic Neoplasms genetics, Prostatic Neoplasms metabolism, Prostatic Neoplasms, Castration-Resistant genetics, Protein Kinase C beta metabolism
- Abstract
The androgen receptor (AR) is a central driver of aggressive prostate cancer. After initial treatment with androgen receptor signaling inhibitors (ARSi), reactivation of AR signaling leads to resistance. Alternative splicing of AR mRNA yields the AR-V7 splice variant, which is currently an undruggable mechanism of ARSi resistance: AR-V7 lacks a ligand binding domain, where hormones and anti-androgen antagonists act, but still activates AR signaling. We reveal PKCβ as a druggable regulator of transcription and splicing at the AR genomic locus. We identify a clinical PKCβ inhibitor in combination with an FDA-approved anti-androgen as an approach for repressing AR genomic locus expression, including expression of AR-V7, while antagonizing full-length AR. PKCβ inhibition reduces total AR gene expression, thus reducing AR-V7 protein levels and sensitizing prostate cancer cells to current anti-androgen therapies. We demonstrate that this combination may be a viable therapeutic strategy for AR-V7-positive prostate cancer., (© 2022. The Author(s).)
- Published
- 2022
- Full Text
- View/download PDF
49. Drugging the Next Undruggable KRAS Allele-Gly12Asp.
- Author
-
Zheng Q, Peacock DM, and Shokat KM
- Subjects
- Alleles, Animals, Drug Discovery, Gene Expression Regulation, Neoplastic, Humans, Mutation, Antineoplastic Agents pharmacology, Pancreatic Neoplasms drug therapy, Pancreatic Neoplasms genetics, Proto-Oncogene Proteins p21(ras) drug effects, Proto-Oncogene Proteins p21(ras) genetics
- Abstract
Since its discovery as the first human oncogene in 1983, the small GTPase KRAS has been a major target of cancer drug discovery. The paper reported in this issue describes a long-awaited small molecule drug candidate of the oncogenic KRAS (G12D) mutant for the treatment of currently incurable pancreatic cancer.
- Published
- 2022
- Full Text
- View/download PDF
50. Dissecting the biology of mTORC1 beyond rapamycin.
- Author
-
Yang G, Francis D, Krycer JR, Larance M, Zhang Z, Novotny CJ, Diaz-Vegas A, Shokat KM, and James DE
- Subjects
- Biology, Mechanistic Target of Rapamycin Complex 1, Sirolimus pharmacology, TOR Serine-Threonine Kinases
- Abstract
Rapamycin extends maximal life span and increases resistance to starvation in many organisms. The beneficial effects of rapamycin are thought to be mediated by its inhibitory effects on the mechanistic target of rapamycin complex 1 (mTORC1), although it only partially inhibits the kinase activity of mTORC1. Other mTOR kinase inhibitors have been developed, such as Torin-1, but these readily cross-react with mTORC2. Here, we report the distinct characteristics of a third-generation mTOR inhibitor called RapaLink1. We found that low doses of RapaLink1 inhibited the phosphorylation of all mTORC1 substrates tested, including those whose phosphorylation is sensitive or resistant to inhibition by rapamycin, without affecting mTORC2 activity even after prolonged treatment. Compared with rapamycin, RapaLink1 showed better efficacy for inhibiting mTORC1 and potently blocked cell proliferation and induced autophagy. Moreover, using RapaLink1, we demonstrated that mTORC1 and mTORC2 exerted differential effects on cell glycolysis and glucose uptake. Last, we found that RapaLink1 and rapamycin had opposing effects on starvation resistance in Drosophila . Consistent with the effects of RapaLink1, genetic blockade of mTORC1 activity made flies more sensitive to starvation, reflecting the complexity of the mTORC1 network that extends beyond effects that can be inhibited by rapamycin. These findings extend our understanding of mTOR biology and provide insights into some of the beneficial effects of rapamycin.
- Published
- 2021
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.